TY - JOUR TI - Gene banks for pig genetic resources: optimisation criteria AU - Gandini, G. AU - Pizzi, F. AU - Maltecca, C. AU - Heinzl, E. AU - Pagnacco, G. T2 - Zootecnica E Nutrizione Animale DA - 2001/// PY - 2001/// VL - 27 IS - 6 SP - 285–294 ER - TY - CONF TI - Creation of the semen bank of Italian pig genetic resources AU - Gandini, G. AU - Maltecca, C. AU - Heinzl, E. AU - Pizzi, F. T2 - Associazione per la Scienza e le Produzioni Animali (ASPA) Congress C2 - 2001/// C3 - Proceedings of the Associazione per la Scienza e le Produzioni Animali (ASPA) Congress DA - 2001/// PY - 2001/// ER - TY - CONF TI - Comparison of milking speed in Reggiana and Italian Holstein cattle [Emilia-Romagna AU - Bagnato, A. AU - Gandini, G.C. AU - Maltecca, C. AU - Orlandini, P. AU - Pizzi, F. T2 - Associazione per la Scienza e le Produzioni Animali (ASPA) Congress C2 - 2001/// C3 - Proceedings of the ASPA Congress - Recent Progress in Animal Production Science (Italy) DA - 2001/// PY - 2001/// VL - 2 SP - 329-221 ER - TY - JOUR TI - Hydroxylation of camphor by reduced oxy-cytochrome p450cam: Mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes AU - Davydov, R. AU - Makris, T.M. AU - Kofman, V. AU - Werst, D.E. AU - Sligar, S.G. AU - Hoffman, B.M. T2 - Journal of the American Chemical Society AB - We have employed gamma-irradiation at cryogenic temperatures (77 K and also approximately 6 K) of the ternary complexes of camphor, dioxygen, and ferro-cytochrome P450cam to inject the "second" electron of the catalytic process. We have used EPR and ENDOR spectroscopies to characterize the primary product of reduction as well as subsequent states created by annealing reduced oxyP450, both the WT enzyme and the D251N and T252A mutants, at progressively higher temperatures. (i) The primary product upon reduction of oxyP450 4 is the end-on, "H-bonded peroxo" intermediate 5A. (ii) This converts even at cryogenic temperatures to the hydroperoxo-ferriheme species, 5B, in a step that is sensitive to these mutations. Yields of 5B are as high as 40%. (iii) In WT and D251N P450s, brief annealing in a narrow temperature range around 200 K causes 5B to convert to a product state, 7A, in which the product 5-exo-hydroxycamphor is coordinated to the ferriheme in a nonequilibrium configuration. Chemical and EPR quantitations indicate the reaction pathway involving 5B yields 5-exo-hydroxycamphor quantitatively. Analogous (but less extensive) results are seen for the alternate substrate, adamantane. (iv) Although the T252A mutation does not interfere with the formation of 5B, the cryoreduced oxyT252A does not yield product, which suggests that 5B is a key intermediate at or near the branch-point that leads either to product formation or to nonproductive "uncoupling" and H(2)O(2) production. The D251N mutation appears to perturb multiple stages in the catalytic cycle. (v) There is no spectroscopic evidence for the buildup of a high-valence oxyferryl/porphyrin pi-cation radical intermediate, 6. However, ENDOR spectroscopy of 7A in H(2)O and D(2)O buffers shows that 7A contains hydroxycamphor, rather than water, bound to Fe(3+), and that the proton removed from the C(5) carbon of substrate during hydroxylation is trapped as the hydroxyl proton. This demonstrates that hydroxylation of substrates by P450cam in fact occurs by the formation and reaction of 6. (vi) Annealing at > or = 220 K converts the initial product state 7A to the equilibrium product state 7, with the transition occurring via a second nonequilibrium product state, 7B, in the D251N mutant; in states 7B and 7 the hydroxycamphor hydroxyl proton no longer is trapped. (vii) The present results are discussed in the context of other efforts to detect intermediates in the P450 catalytic cycle. DA - 2001/// PY - 2001/// DO - 10.1021/ja003583l VL - 123 IS - 7 SP - 1403-1415 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035925164&partnerID=MN8TOARS ER - TY - JOUR TI - Cryotrapped Reaction Intermediates of Cytochrome P450 Studied by Radiolytic Reduction with Phosphorus-32 AU - Denisov, I.G. AU - Makris, T.M. AU - Sligar, S.G. T2 - Journal of Biological Chemistry AB - Unstable reaction intermediates of the cytochrome P450 catalytic cycle have been prepared at cryogenic temperatures using radiolytic one-electron reduction of the oxy-P450 CYP101 complex. Since a rate-limiting step in the catalytic cycle of the enzyme is the reduction of the ferrous oxygenated heme protein, subsequent reaction intermediates do not normally accumulate. Using 60Co γ-irradiation, the primary reduced oxy-P450 species at 77 K has been identified as a superoxo- or hydroperoxo-Fe3+-heme complex (Davydov, R., Macdonald, I. D. G., Makris, T. M., Sligar, S. G., and Hoffman, B. M. (1999)J. Am. Chem. Soc. 121, 10654–10655). The electronic absorption spectroscopy is an essential tool to characterize cytochrome P450 intermediates and complements paramagnetic methods, which are blind to important diamagnetic or antiferromagnetically coupled states. We report a method of trapping unstable states of redox enzymes using phosphorus-32 as an internal source of electrons. We determine the UV-visible optical spectra of the reduced oxygenated state of CYP101 and show that the primary intermediate, a hydroperoxo-P450, is stable below 180 K and converts smoothly to the product complex at ∼195 K. In the course of the thermal annealing, no spectral changes indicating the presence of oxoferryl species (the so-called compound I type spectrum) was observed. Unstable reaction intermediates of the cytochrome P450 catalytic cycle have been prepared at cryogenic temperatures using radiolytic one-electron reduction of the oxy-P450 CYP101 complex. Since a rate-limiting step in the catalytic cycle of the enzyme is the reduction of the ferrous oxygenated heme protein, subsequent reaction intermediates do not normally accumulate. Using 60Co γ-irradiation, the primary reduced oxy-P450 species at 77 K has been identified as a superoxo- or hydroperoxo-Fe3+-heme complex (Davydov, R., Macdonald, I. D. G., Makris, T. M., Sligar, S. G., and Hoffman, B. M. (1999)J. Am. Chem. Soc. 121, 10654–10655). The electronic absorption spectroscopy is an essential tool to characterize cytochrome P450 intermediates and complements paramagnetic methods, which are blind to important diamagnetic or antiferromagnetically coupled states. We report a method of trapping unstable states of redox enzymes using phosphorus-32 as an internal source of electrons. We determine the UV-visible optical spectra of the reduced oxygenated state of CYP101 and show that the primary intermediate, a hydroperoxo-P450, is stable below 180 K and converts smoothly to the product complex at ∼195 K. In the course of the thermal annealing, no spectral changes indicating the presence of oxoferryl species (the so-called compound I type spectrum) was observed. Detailed information about the intermediates in complex chemical and biochemical reactions is of vital importance in mechanistic studies. Typical relaxation methods can often monitor the progress of only one kinetically limiting step of the reaction if there is a dominance of a slow step in the catalytic cycle. It is much more difficult, however, to obtain information about the subsequent (fast) stages of the reaction and the properties of corresponding intermediate species, since they are not accumulated at ambient conditions in the course of the reaction, and their concentrations are nominally very small. Ideally, one would like to stop the reaction at each step to follow the reaction progress and collect information about each intermediate compound involved in the reaction path. Given a suitable set of activation barriers and enthalpies, cryogenic trapping of unstable intermediates allows such dissection of the reaction cycle, provided a method of preparation of initial nonequilibrium reactive complexes is available. The field of matrix isolation chemistry is based on this approach, where the active complexes are trapped in host matrix, usually solid inert gases at cryogenic temperatures (1Turner J.J. Pimentel G.C. Science. 1963; 140: 974-975Crossref PubMed Scopus (113) Google Scholar). Such methods help to create and study unstable reaction states that rapidly decompose at ambient conditions (2Khriachtchev L. Pettersson M. Runeberg N. Lundell J. Rasanen M. Nature. 2000; 406: 874-876Crossref PubMed Scopus (518) Google Scholar). Recently, the same approach was developed in field of structural and mechanistic enzymology of the redox active enzyme cytochrome P450 (3Davydov R. Macdonald I.D.G. Makris T.M. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10654-10655Crossref Scopus (133) Google Scholar, 4Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1189) Google Scholar, 5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar, 6Davydov R. Kappl R. Hutterman R. Peterson J. FEBS Lett. 1991; 295: 113-115Crossref PubMed Scopus (67) Google Scholar). The cytochromes P450 are heme-containing metalloproteins involved in numerous biochemical reactions including xenobiotic metabolism and steroid biosynthesis (7Ortiz de Montellano, P. R. (ed) (1995) Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd Ed., Plenum Press, New YorkGoogle Scholar). The reaction cycle of these enzymes involves two one-electron reductions, interspersed by the binding of dioxygen. Cleavage of a putative peroxo or reduced oxydioxygen bond is thought to lead to the generation of a high valent “ferryl” intermediate analogous to the compound I state of the peroxidases. The proposed sequential steps of the P450 reaction cycle are depicted in Scheme I,Fe3+(P)+e−→Fe2+(P)Fe2+(P)+O2→Fe3+(P)−O2−Fe3+(P)−O2−+e−→Fe3+(P)−O22−Fe3+(P)−O22−+H+→Fe3+(P)−(OOH)−Fe3+(P)−(OOH)−+H+→Fe4+(P+*)=O+H2OSCHEMEIwhere (P) represents the porphyrin macrocycle, and (P+*) represents the porphyrin π-cation radical. The sequential one-electron reductions of the heme iron at the active site are provided by a protein redox partner. Alternatively, the same one-electron reduction can be reached by reaction with solvated electrons generated by pulsed radiolysis of water (8Kobayashi K. Amano M. Kanbara Y. Hayashi K. J. Biol. Chem. 1987; 262: 5445-5447Abstract Full Text PDF PubMed Google Scholar). When radiolytic reduction is performed at low temperature (at 77 K), the system is effectively immobilized, and the reactive complexes can be accumulated and studied (3Davydov R. Macdonald I.D.G. Makris T.M. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10654-10655Crossref Scopus (133) Google Scholar, 4Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1189) Google Scholar, 5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar, 6Davydov R. Kappl R. Hutterman R. Peterson J. FEBS Lett. 1991; 295: 113-115Crossref PubMed Scopus (67) Google Scholar). While these techniques have proved to be important for understanding the detailed chemistry of many redox enzymes (3Davydov R. Macdonald I.D.G. Makris T.M. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10654-10655Crossref Scopus (133) Google Scholar, 4Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1189) Google Scholar, 5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar, 6Davydov R. Kappl R. Hutterman R. Peterson J. FEBS Lett. 1991; 295: 113-115Crossref PubMed Scopus (67) Google Scholar, 9Davydov R.M. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10656-10657Crossref Scopus (144) Google Scholar, 10Brugna M. Rodgers S. Schricker A. Montoya G. Kazmeier M. Nitschke W. Sinning I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2069-2074Crossref PubMed Scopus (68) Google Scholar, 11Karlsson A. Parales J.V. Parales R.E. Gibson D.T. Eklund H. Ramaswamy S. J. Inorg. Biochem. 2000; 78: 83-87Crossref PubMed Scopus (35) Google Scholar, 12Davydov R. Kuprin S. Graslund A. Ehrenberg A. J. Am. Chem. Soc. 1994; 1994: 11120-11128Crossref Scopus (91) Google Scholar), the cryogenic radiolytic reduction remains technically demanding. The high doses (2–6 megarads) that are necessary to obtain sufficient yield of reducing equivalents in frozen solution are usually introduced with powerful 60Co γ-sources or with synchrotron radiation. These radiation sources are not commonly available to biochemical laboratories. Radiolysis at low temperatures (77 K or even 4 K) creates more problems, particularly due to the difficulties in monitoring the progress of the cryoradiolytic reduction in the course of irradiation using these types of sources. In this paper, we describe how another source of ionizing radiation, phosphorus-32, can be a convenient means for generating unstable active compounds through one-electron radiolytic reduction of the reagents trapped in aqueous/organic glass at 77 K. We show that using commercially available 32P-enriched phosphate it is possible to generate sufficient concentrations of intermediates (radicals or active complexes) of different biological molecules in frozen aqueous/glycerol solutions at 77 K. Although quite unstable at ambient temperatures, many such reaction intermediates are effectively stabilized below the glass transition temperature of solvent and can be accumulated and stored for months (6Davydov R. Kappl R. Hutterman R. Peterson J. FEBS Lett. 1991; 295: 113-115Crossref PubMed Scopus (67) Google Scholar, 13Davydov R.M. Blyumenfeld L.A. Arutyunyan A.M. Sharonov Yu A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 1037-1042PubMed Google Scholar, 14Blumenfeld L.A. Davydov R.M. Biochim. Biophys. Acta. 1979; 549: 255-280Crossref PubMed Scopus (19) Google Scholar, 15Magonov S.N. Davydov R.M. Blyumenfeld L.A. Vilu R.O. Arutyunyan A.M. Sharonov Yu A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 913-919Google Scholar). Different spectroscopic methods can be used to study the structure of these intermediates and to monitor the progress of subsequent chemical reactions after gradual warming of the system (16Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar, 17Gasyna Z. Biochim. Biophys. Acta. 1979; 577: 207-216Crossref PubMed Scopus (23) Google Scholar, 18Greschner S. Davydov R.M. Janig G.-R. Ruckpaul K. Blumenfeld L.A. Acta Biol. Med. Germ. 1979; 38: 443-448PubMed Google Scholar, 19Greschner S. Biophys. Struct. Mech. 1982; 9: 29-34Crossref PubMed Scopus (4) Google Scholar, 20Magonov S.N. Davydov R.M. Blyumenfeld L.A. Arutyunyan A.M. Sharonov Yu. A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 919-924Google Scholar, 21Magonov S.N. Davydov R.M. Blyumenfeld L.A. Vilu R.O. Arutyunyan A.M. Sharonov Yu. A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 725-733PubMed Google Scholar, 22Oshtrakh M.I. Semionkin V.A. Kopelyan E.A. Milder O.B. Radiat. Phys. Chem. 1999; 55: 549-554Crossref Scopus (8) Google Scholar, 23Prusakov V.E. Stukan R.A. Davydov R.M. Gersonde K. FEBS Lett. 1985; 186: 158-162Crossref Scopus (11) Google Scholar, 24Fessenden R.W. Schuler R.H. Burton M. Magee J.L. Advances in Radiation Chemistry. Wiley and Sons, 1970: 1-176Google Scholar). Using the technique of in situ radiolytic reduction at cryogenic temperature, we present the first determination of the optical spectra of a two electron reduced dioxygen intermediate, the iron-hydroperoxo state, in cytochrome P450cam (CYP101). 32P-enriched aqueous solution of orthophosphoric acid (activity 50 mCi/ml) was purchased from Amersham Pharmacia Biotech. Activity was measured by the manufacturer and tested using the Fricke dosimeter method (25Schuler R.H. Allen A.O. J. Chem. Phys. 1956; 24: 56-59Crossref Scopus (46) Google Scholar). Cytochrome P450 CYP101 fromPseudomonas putida was expressed and purified as described (26Unger B.P. Gunsalus I.C. Sligar S.G. J. Biol. Chem. 1986; 261: 1158-1163Abstract Full Text PDF PubMed Google Scholar) and stored in concentrated frozen aqueous solutions at 200 K in the presence of camphor in the ferric form. All other chemicals were of spectrophotometric grade and used without additional purification. Sodium dithionite (Na2S2O4) from Sigma was stored and used in an anaerobic chamber. Cytochrome P450 CYP101 was reduced by adding several small crystals of dithionite in an anaerobic chamber to a concentrated solution of ferric protein. The products of reaction and the remaining dithionite were removed by passing the solution through a small G-25 column using deoxygenated 0.1 m potassium phosphate buffer, pH 8.0, containing 1 mm camphor. Solutions of reduced cytochrome P450 were then concentrated using microcentricons inside the anaerobic chamber and used within 1 h after preparation. Samples for incubation with radioactive phosphate were prepared on ice to minimize autoxidation. To prepare the sample, 0.75 ml of the glycerol/phosphate buffer solvent (final camphor concentration 1 mm) was mixed with 0.25 ml of 32P-enriched orthophosphoric acid, and 60 μl of catalase solution (∼1200 units final activity) was added to consume hydrogen peroxide produced due to water radiolysis in radioactive solution occurring during transportation and storage (concentration estimated to be about 1 mm based on radiolytic yield (27Spinks J.W.T. Woods R.J. An Introduction to Radiation Chemistry. Wiley and Sons, New York1990Google Scholar)). After incubation of the sample solution for 45 min to eliminate all H2O2, the anaerobic solution of reduced ferrous cytochrome P450 CYP101 (120 μl) was added and stirred for 90 s to ensure the homogeneous mixing with the viscous glycerol solution at 4 °C. The final concentration of the enzyme was ∼30 μm, and it was completely oxygenated in aerobic glycerol/ buffer solvent. The sample was transferred into a Dewar and cooled to 200 K within 3–4 min to form a clear transparent glass and then to 77 K within 25–30 min. Several spectra in the range 300–900 nm were taken during cooling to control the state of the sample and the absence of significant autoxidation. All samples were kept fully immersed in liquid nitrogen at 77 K when not in use. Cobalt-60 irradiation was used in control experiments. Samples for γ-irradiation were prepared similarly with the exception of addition of 32P-enriched phosphate. Oxygenation of the reduced ferrous cytochrome P450 CYP101 was accomplished either by simple mixing of the concentrated deoxygenated solution of the enzyme with aerobic aqueous/glycerol buffer or by bubbling the oxygen gas through the deoxygenated solution of the enzyme prepared in the final glycerol/water mixture at the anaerobic chamber. Both methods gave identical results. The final fraction of autoxidized P450 CYP101 was estimated as less than 5% from absorbance value at 646 nm. The samples were kept in liquid nitrogen and irradiated in a silvered Dewar flask containing liquid nitrogen. A typical γ-irradiation using 60Co was for 4 h (at the measured on site dose rate of 21 kilorads/min for a total dose of ∼5 megarads), and the irradiation Dewar flask was refilled after 2 h to maintain the samples at 77 K. The similar samples with ferric cytochrome P450, carbon monoxide complex of ferrous cytochrome P450, horseradish peroxidase, and riboflavin were also prepared and irradiated in identical conditions to check the radiolytic reduction yield and compare our results with earlier data (16Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar, 17Gasyna Z. Biochim. Biophys. Acta. 1979; 577: 207-216Crossref PubMed Scopus (23) Google Scholar, 20Magonov S.N. Davydov R.M. Blyumenfeld L.A. Arutyunyan A.M. Sharonov Yu. A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 919-924Google Scholar, 21Magonov S.N. Davydov R.M. Blyumenfeld L.A. Vilu R.O. Arutyunyan A.M. Sharonov Yu. A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 725-733PubMed Google Scholar). Optical spectra at low temperatures were obtained using a Cary 3 UV-visible spectrophotometer (Varian Instruments) and a homemade cryostat with liquid nitrogen used as a cooling agent. The samples were mixed directly in the disposable methacrylate cells (UV-enhanced semimicro cells from Fisher, total sample volume ∼1 ml, 4.3-mm path length, 15–30 μm final concentration of the protein) and mounted on the holder of the cryostat. To obtain the good optically transparent frozen solutions without significant turbidity, the 1:1 and 3:1 (v/v) glycerol/ buffer solutions were cooled without direct contact with liquid nitrogen to prevent cracking (glycerol and ethylene glycol do not change the catalytic mechanism of CYP 101, although the turnover rate is slowed (28Di Primo C. Sligar S.G. Hui Bon Hoa G. Douzou P. FEBS Lett. 1992; 312: 252-254Crossref PubMed Scopus (25) Google Scholar)). As was noted previously (29Shibata Y. Kurita A. Kushida T. Biochemistry. 1999; 38: 1789-1801Crossref PubMed Scopus (22) Google Scholar), the 3:1 glycerol/water mixture is stable at all studied temperatures, while 1:1 mixtures undergo phase separation and water crystallization when incubated for some time at 190–230 K, making further optical measurements in the visible region impossible. Below 190 K, both solvents could be used for optical spectroscopy at UV-visible range for prolonged measurements. The temperature was controlled by a calibrated thermocouple fixed in a thermal contact with a brass sample holder close to the light beam position. All spectra were measured in a single beam mode in the 300–900-nm range with 2.0-nm resolution; data points were taken every 1 nm with a scan speed of 60–100 nm/min. Background subtraction, differentiation, singular value decomposition analysis, and all other calculations were accomplished using MATLAB (MathWorks, Natick, MA). In a typical annealing experiment, the sample was warmed stepwise, 4–8 K at a time, and then several spectra were taken while keeping the sample at the temperature of annealing or 3–5 K below to check the thermal equilibration and reproducibility of the measurements. It was observed that all changes in the spectra of (nonequilibrium) cryoreduced species were irreversible and that partially annealed samples could be cooled again to 77 K and were stable at this temperature for many weeks. Thus, it was possible to work with one sample for several days and to accumulate the products of cryoradiolysis, keeping the sample immersed in liquid nitrogen. The protein integrity after irradiation was tested by UV-visible spectroscopy at the end of the experiment at room temperature. We always obtained a pure CO-bound P450 spectrum with no sign of an inactive form of the protein, P420, being produced. The carbonmonoxy-bound ferrous cytochrome P450 was formed, because the large amount of reducing species and CO formed during radiolysis of aqueous/organic solvent can react with the enzyme when the sample is thawed, as has been observed in previous radiolytic studies (13Davydov R.M. Blyumenfeld L.A. Arutyunyan A.M. Sharonov Yu A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 1037-1042PubMed Google Scholar, 15Magonov S.N. Davydov R.M. Blyumenfeld L.A. Vilu R.O. Arutyunyan A.M. Sharonov Yu A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 913-919Google Scholar, 21Magonov S.N. Davydov R.M. Blyumenfeld L.A. Vilu R.O. Arutyunyan A.M. Sharonov Yu. A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 725-733PubMed Google Scholar). With an increase of the absorbed dose, we observed the appearance and increase of a strong and very broad absorption band with a maximum at about 560 nm, a clear indication of the accumulation of the trapped electrons in the frozen at 77 K aqueous/alcohol solvent (27Spinks J.W.T. Woods R.J. An Introduction to Radiation Chemistry. Wiley and Sons, New York1990Google Scholar,30Kolodziejski M. Abramczyk H. J. Mol. Struct. 1997; 436–437: 543-553Crossref Scopus (6) Google Scholar). The initial rate of absorption increase at maximum was ∼0.07/h with 10 mCi/ml 32P used. This rate is in good agreement with an estimated radiolytic yield of solvated electrons at a given dose rate (27Spinks J.W.T. Woods R.J. An Introduction to Radiation Chemistry. Wiley and Sons, New York1990Google Scholar) and typical values of molar absorption of electron solvated in water (17 mm−1cm−1 at 715 nm) and alcohols (13 mm−1 cm−1at 520 nm) (see Ref. 27Spinks J.W.T. Woods R.J. An Introduction to Radiation Chemistry. Wiley and Sons, New York1990Google Scholar and references therein). This absorption could be completely eliminated by the illumination of the sample with a regulated Oriel tungsten-halogen lamp with a typical illumination time of 12 min. The cut-off filter (λ > 450 nm) was used to prevent possible degradation of photosensitive intermediates. The sample was fully immersed in liquid nitrogen during illumination to prevent heating. In the separate experiment, it was shown that even with the filter with cut-off λ >600 nm it was possible to eliminate almost all absorption originating from the solvated (trapped) electrons, although in this case it took longer than 2 h. The possibility of photobleaching this broad background absorption band at the visible region using the light of different spectral composition also confirms its origin as a result of trapped electron accumulation (15Magonov S.N. Davydov R.M. Blyumenfeld L.A. Vilu R.O. Arutyunyan A.M. Sharonov Yu A. Molek. Biol. ( Eng. Transl. ). 1978; 12: 913-919Google Scholar, 16Gasyna Z. FEBS Lett. 1979; 106: 213-218Crossref PubMed Scopus (46) Google Scholar, 27Spinks J.W.T. Woods R.J. An Introduction to Radiation Chemistry. Wiley and Sons, New York1990Google Scholar). These electrons are photolyzed by the visible light and disappear through recombination via numerous chemical reactions with other products of radiolysis as well as with the original components of the solution. Radioactive 32P-enriched phosphate is very well suited for use as an internal radiation source in aqueous and organic solutions. Phosphate, as well as sulfate, the product of β-decay of phosphate, are natural components of many buffer systems and do not interfere with most of reactions. The commercially available orthophosphoric acid (as an aqueous or dilute HCl solutions) with32P activity up to 50 mCi/ml makes it possible to reach radiation doses of 30 megarads or more. Accumulation of side radiolysis products, however, usually limits the radiolytic dose to about 5 megarads (24Fessenden R.W. Schuler R.H. Burton M. Magee J.L. Advances in Radiation Chemistry. Wiley and Sons, 1970: 1-176Google Scholar). Since the half-life of 32P is 14.31 days, 2–3 megarads can be generated in a 2-week incubation. This makes easy the monitoring of the progress of radiolytic reduction and accumulation of the primary reaction intermediates, using spectroscopic or other noninvasive methods. A decided advantage of the low temperature, radiolytic trapping of reactive enzyme states is the ability to follow the reaction coordinate of the system by selectively annealing the sample at higher temperatures. To measure the dose rate generated by 32P-enriched phosphate, we used the reaction of aerobic radiolytic oxidation of ferrous sulfate in aqueous sulfuric acid known as the Fricke dosimeter (27Spinks J.W.T. Woods R.J. An Introduction to Radiation Chemistry. Wiley and Sons, New York1990Google Scholar). The time course of Fe2+ oxidation is monitored by absorption growth at 304 nm (Fig. 1) (25Schuler R.H. Allen A.O. J. Chem. Phys. 1956; 24: 56-59Crossref Scopus (46) Google Scholar). The agreement between the theoretical curve calculated using the mean energy of electrons generated in β decay of 32P (0.7 MeV) and reported activity of the commercial sample and the experimentally measured dose rate is excellent. These results show that32P can indeed be used as an easy and readily available source of ionizing radiation, successfully replacing 60Co γ-sources for the radiochemical generation of biological samplesin situ. As has been shown (6Davydov R. Kappl R. Hutterman R. Peterson J. FEBS Lett. 1991; 295: 113-115Crossref PubMed Scopus (67) Google Scholar, 14Blumenfeld L.A. Davydov R.M. Biochim. Biophys. Acta. 1979; 549: 255-280Crossref PubMed Scopus (19) Google Scholar, 31Symons M.C.R. Petersen R.L. Proc. R. Soc. Lond. B. 1978; 201: 285-300Crossref PubMed Scopus (82) Google Scholar, 32Symons M.C.R. Taiwo F.A. J. Chem. Soc. Faraday Trans. I. 1989; 85: 2427-2433Crossref Google Scholar), using γ-irradiation from the60Co source, it is possible to accumulate the reduced hemoproteins stabilized at 77 K without their conformational or chemical relaxation. Electronic paramagnetic resonance (EPR) (3Davydov R. Macdonald I.D.G. Makris T.M. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10654-10655Crossref Scopus (133) Google Scholar,5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar, 6Davydov R. Kappl R. Hutterman R. Peterson J. FEBS Lett. 1991; 295: 113-115Crossref PubMed Scopus (67) Google Scholar, 12Davydov R. Kuprin S. Graslund A. Ehrenberg A. J. Am. Chem. Soc. 1994; 1994: 11120-11128Crossref Scopus (91) Google Scholar, 14Blumenfeld L.A. Davydov R.M. Biochim. Biophys. 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Biol. ( Eng. Transl. ). 1978; 12: 725-733PubMed Google Scholar) spectroscopy have been used to study the structure of primary intermediates obtained through radiolytic one-electron reduction of several metalloproteins and the details of subsequent reactions after annealing of the sample at and above the glass transition temperature of the solvent. Here we compare the products of γ-irradiation at 77 K and of cryoradiolysis using 32P as an internal source and show that both methods give identical results, with each having its own technical advantages. The radiolytic reduction of cytochrome P450 CYP101 was followed at low temperatures (77 K) in glycerol/water (1:1 or 3:1 (v/v), molar fraction of glycerol x m = 0.2 or 0.43, respectively) frozen solutions containing 32P-enriched phosphate containing an activity of 10 mCi/ml, which corresponds to a total dose of ∼7 megarads, or 3.5 megarads during the first half-life of32P. Fig. 2 A shows the spectra of the oxy form of cytochrome P450 CYP101 immediately after preparation of a sample containing 32P (10 mCi/ml) at 77 K and after incubation at low temperature for different periods of time. The absorbance at 417 nm (maximum of the oxy-P450 spectrum at low temperatures) decreases, and the new peak at ∼440 nm appears with time following the increase in absorbed dose. These changes show the progress of one-electron reduction of oxy-P450 and formation of the reduced oxy-P450 complex. To our knowledge, this is a first report of UV-visible spectra of this unstable reaction intermediate of cytochrome P450. This result complements the recent EPR studies of the same system (3Davydov R. Macdonald I.D.G. Makris T.M. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 1999; 121: 10654-10655Crossref Scopus (133) Google Scholar, 5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar). In the EPR measurement, the direct conversion of oxy-P450 to reduced oxy-P450 cannot be observed, since the oxy-P450 is EPR-silent, and only the products of radiolytic reduction can be detected. The spectra in Fig. 2 A were used to estimate the spectrum of the pure reduced oxy-P450 intermediate. Concentration of the remaining oxy-P450 was estimated from these spectra by means of comparing the second derivatives of these spectra. The derivative peaks corresponding to the maxima at 417 and 440 nm are better resolved (results not shown), and hence the area of the former peak was used to calculate the decay in the oxy-P450 fraction with increase in dose. The remainder was assigned to the reduced oxy-P450 intermediate, the spectrum of the latter was then calculated using the experimental spectra at Fig.2 A by subtracting corresponding fractions of the oxy-P450 spectrum. The calculated spectrum of the pure reduced oxy-P450 intermediate is shown in Fig. 2 B together with the spectrum of pure oxy-P450 shown for comparison. Similar spectra were obtained with the samples of oxy-P450 prepared in 50 and 75% glycerol/water solutions and irradiated with γ-rays from60Co source with a 4–5-megarad total dose in several independent experiments. This suggests that the radiolytic reduction in frozen solution with solvated electrons acting as reducing species does not depend on the source of the primary radiolytic electrons, γ-photons, or high energy electrons from 32P radioactive decay. The observed similarity is not surprising, since32P, 3H, 35S, and other β-active isotopes have been shown to produce the same products of radiolysis in the aqueous solution as do γ-rays (33Hardwick T.J. Can. J. Chem. 1952; 30: 39-46Crossref Google Scholar, 34Peisach M. Steyn J. Nature. 1960; 187: 58-59Crossref Scopus (7) Google Scholar, 35Yamamoto O. Int. J. Radiat. Biol. 1982; 42: 661-665Google Scholar, 36Berezin I.V. Martinek K. Russian J. Phys. Chem. ( Eng. Transl. ). 1964; 38: 1468-1469Google Scholar). These results give a solid foundation for generating stable one-electron reduced intermediates at cryogenic temperatures in frozen solutions containing easily obtained β-emitting isotopes. The spectrum of the reduced oxy-P450 shown in Fig. 2 B is in excellent agreement with that calculated by Harris et al.(37Harris D. Loew G. Waskell L. J. Am. Chem. Soc. 1998; 120: 4308-4318Crossref Scopus (86) Google Scholar) for the same system. The pronounced split Soret band and 30-nm red shift were the main features of their calculated spectrum. Our experimentally obtained red shift is 23 nm, and the split Soret shape of the spectrum is remarkably similar to the theoretical results. Optical changes have also been observed using pulse radiolysis of oxycomplex of the deuteroheme-substituted cytochrome P450 CYP101 (8Kobayashi K. Amano M. Kanbara Y. Hayashi K. J. Biol. Chem. 1987; 262: 5445-5447Abstract Full Text PDF PubMed Google Scholar), which are nearly identical to our results on the native metalloprotein containing protoporphyrin IX as a prosthetic group. The irreversible evolution of the reduced oxy-P450 reaction intermediate with temperature increase was monitored by the spectra shown in Fig. 3. The gradual annealing of the samples reduced radiolytically at 77 K results in the sequence of conformational relaxations and chemical transformations. At the same time, the various organic radicals, which appear as the products of glycerol radiolysis, gradually recombine and decay. These latter processes result in continuous changes of the background absorbance in the optical spectra in the visible and near UV range. To subtract this background, the spectra of the 75% glycerol/buffer solution without added cytochrome P450 were obtained. The reference sample was irradiated in identical conditions (total dose 5 megarads) and carefully annealed from 77 up to 240 K. In these experiments, spectra were taken every 4 K at temperatures above 140 K, where significant optical changes begin to occur. The resulting spectral array was used for background subtraction in the analysis of the enzyme spectra at different temperatures with the actual base line at each temperature calculated using linear interpolation with respect to the temperature. The calculated spectra of pure reduced oxy-P450 with subtracted base line as described are shown in Fig. 3 A in a three-dimensional representation. The simple visual inspection of these spectra shows only one main process, the decay of the primary reaction intermediate with a Soret maximum at 443 nm and concomitant increase in absorbance at 392 nm, a characteristic of the high spin ferric cytochrome P450. More careful analysis reveals two minor temperature-dependent spectral processes. The presence of only four spectrally distinguishable components was confirmed by singular value decomposition analysis (data not shown). All spectral changes are observed in the temperature interval 192–205 K and are presented in the more convenient form in Fig. 3 B. The first process is the red shift of the maximum from 440 to 443 nm in the narrow temperature interval 192–194 K, which is not accompanied by notable optical changes in other regions of the spectrum. This small shift could be due to protonation of superoxide anion bound to iron of the heme (5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar), to thermal relaxation of the heme-ligand reduced complex, or to other relatively minor perturbation of the chromophore. At the same temperature, the beginning of the product formation is observed with the active reduced oxy-P450 intermediate decay. The amplitude of the peak at 443 nm decreases, the new peak appears at 417 nm, and the absorbance at 392 nm begins to increase. The Soret peak at 417 nm is characteristic of the low spin ferric cytochrome P450, the state at which the heme iron is hexacoordinated with the weak sixth ligand. In the CYP101 system, the appearance of this maximum can be assigned to the formation of the product-bound ferric cytochrome P450, the oxygen of the hydroxyl group in 5-exo-hydroxycamphor being coordinated with heme iron as a sixth ligand. The formation of this complex as an intermediate step before the product release, obtained at the same temperature in aqueous/glycerol solution was also determined by EPR spectroscopy (5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar), and our optical data confirm this observation. The longer annealing at slightly higher temperatures results in the disappearance of the signal from this low spin product complex and an increase of absorbance at 392 nm from high spin ferric cytochrome P450. The stabilization and detection of the main putative active intermediate in cytochrome P450, proposed to have the main features of the “compound I” state of peroxidases, the oxoferryl porphyrin π-cation radical (38Groves J.T. Han Y. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. Plenum Press, New York1995: 3-48Crossref Google Scholar, 39Watanabe Y. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. Academic Press, Inc., New York2000: 97-117Google Scholar), remains a subject of debate. In radiolytic reduction and annealing experiments, we did not obtain any spectral evidence for the existence of this state. The only new intermediate observed is the peroxo (hydroperoxo) complex. The known features of a compound I-type spectra, namely a broad Soret maximum between 370 and 410 nm with relatively low amplitude and an increase of absorbance at 650–700 nm (40Cunningham I.D. Danks T.N. O'Connel K.T.A. Scott P.W. J. Chem. Soc. Perkin Trans. 1999; 2: 2133-2139Crossref Scopus (29) Google Scholar), were not observed. This may indicate that even at 200 K the active intermediate has high activity and is not accumulated at sufficient concentrations. Alternatively, the lack of the aforementioned features in visible spectra can be the result of the different electronic structure of the active intermediate in cytochrome P450 compared with the known analogs in chloroperoxidase, horseradish peroxidase, and model systems. Such a difference may involve, but certainly not be limited to, the different distribution of unpaired electron density due to the presence of thiolate proximal ligand (39Watanabe Y. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. Academic Press, Inc., New York2000: 97-117Google Scholar,41Champion P.M. J. Am. Chem. Soc. 1989; 111: 3433-3434Crossref Scopus (46) Google Scholar, 42Urano Y. Higuchi T. Hirobe M. Nagano T. J. Am. Chem. Soc. 1997; 119: 12008-12009Crossref Scopus (73) Google Scholar, 43Green M.T. J. Am. Chem. Soc. 1999; 121: 7939-7940Crossref Scopus (175) Google Scholar). An alternative explanation involving a different active hydroxylating compound has been proposed by Newcomb and Toy (44Newcomb M. Toy P.H. Acc. Chem. Res. 2000; 33: 449-455Crossref PubMed Scopus (249) Google Scholar). Recently, much more direct mechanistic evidences in favor of oxoferryl hydrogen abstraction and the subsequent oxygen rebound mechanism were obtained by means of EPR and ENDOR analysis of H/D exchangeable protons in all reaction intermediates (5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar), although the direct observation of compound I was not achieved. However, using x-ray cryocrystallography, Schlichting et al. (4Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1189) Google Scholar) observed evidence for the transient formation of a single oxygen-containing intermediate in CYP101. Such an observation may be due to further stabilization of highly reactive intermediates by the crystal lattice or subtle differences in radiation chemistry in the tracks. In summary, we describe here a new application of radiation chemistry at cryogenic temperatures, the use of radioactive 32P as an internal source for generation of unstable intermediates of the cytochrome P450 CYP101 enzymatic cycle. The one-electron reduced oxy-P450 is stable at low temperatures (77–190 K) and undergoes a series of irreversible transformations resulting in formation of ferric P450 and the product (5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar). The optical spectra of the reduced oxy-P450cam are in a good agreement with theoretical calculations. Thermal annealing monitored by optical spectroscopy confirms the results obtained on the same system using EPR (5Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (401) Google Scholar). We gratefully appreciate constant help and support provided by Dr. J. Bentley while using the 60Co source in the Notre Dame Radiation Laboratory (Notre Dame University) and discussions with Professor R. H. Schuler. Irradiations were conducted at the Notre Dame Radiation Laboratory, which is a facility of the U. S. Department of Energy, Office of Basic Energy Sciences. Useful discussions with Drs. S. Balashov and J. Brandon are acknowledged. DA - 2001/// PY - 2001/// DO - 10.1074/jbc.M010219200 VL - 276 IS - 15 SP - 11648-11652 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035853766&partnerID=MN8TOARS ER - TY - BOOK TI - Introduction to Biochemical Toxicology DA - 2001/// PY - 2001/// ET - 3rd PB - J. Wiley and Sons ER - TY - JOUR TI - Method of treating alopecia AU - Smart, R. C. AU - Oh, H.-S. DA - 2001/// PY - 2001/// VL - 6,204,258 IS - 2001 Mar. 20 ER - TY - BOOK TI - Introduction to Biochemical Toxicology A3 - Hodgson, E. A3 - Smart, R.C. DA - 2001/// PY - 2001/// ET - 3rd PB - J. Wiley and Sons ER - TY - CHAP TI - Carcinogenesis AU - Smart, R.C. AU - Akunda, J.K. T2 - Introduction to Biochemical Toxicology A2 - Hodgson, E. A2 - Smart, R.C. PY - 2001/// ET - 3rd SP - 343–398 PB - J. Wiley and Sons ER - TY - CHAP TI - Molecular Techniques in Toxicology AU - Smart, R.C. T2 - Introduction to Biochemical Toxicology A2 - Hodgson, E. A2 - Smart, R.C. PY - 2001/// ET - 3rd SP - 11–32 PB - J. Wiley and Sons ER - TY - CHAP TI - Biochemical Toxicology: Definition and Scope AU - Hodgson, E. AU - Smart, R.C. T2 - Introduction to Biochemical Toxicology A2 - Hodgson, E. A2 - Smart, R.C. PY - 2001/// SP - 1–10 PB - J. Wiley and Sons ER - TY - JOUR TI - Comparison of dietary sources of long-chain polyunsaturated fatty acids (LCPUFA) in the neonatal piglet: Impact on intestinal and liver parameters AU - Mathews, S.A. AU - Harrell, R.J. AU - Oliver, W.T. AU - Odle, J. AU - Diersen-Schade, D.A. T2 - FASEB Journal DA - 2001/// PY - 2001/// VL - 15 ER - TY - JOUR TI - Comparison of dietary sources of long-chain polyunsaturated fatty acids (LCPUFA) in the neonatal piglet: Assessment of clinical parameters AU - Mathews, S.A. AU - Harrell, R.J. AU - Oliver, W.T. AU - Odle, J. AU - Diersen-Schade, D.A. T2 - FASEB Journal DA - 2001/// PY - 2001/// VL - 15 SP - A643 ER - TY - JOUR TI - Conjugated linoleic acid (CLA) supplementation increases belly weight in lean-genotype gilts AU - Averette, L.A. AU - See, M.T. AU - Odle, J. T2 - Journal of Animal Science DA - 2001/// PY - 2001/// IS - Suppl. 1 ER - TY - MGZN TI - Liquid-fed pigs outperform dry AU - Odle, J. T2 - Pork Magazine DA - 2001/8// PY - 2001/8// SP - 32 ER - TY - MGZN TI - Liquid diets accelerate growth of SEW pigs AU - Odle, J. T2 - National Hog Farmer DA - 2001/12// PY - 2001/12// SP - 16–18 ER - TY - CONF TI - Nutritional and growth factor effects on gastrointestinal growth AU - Harrell, R.J. AU - Odle, J. T2 - Cornell Nutrition Conference C2 - 2001/// C3 - Proceedings of the Cornell Nutrition Conference DA - 2001/// PY - 2001/// SP - 123–131 ER - TY - CONF TI - Liquid diets for early-weaned pigs – A “solution” for post-weaning morbidity AU - Odle, J. AU - Harrell, R. T2 - Cornell Nutrition Conference C2 - 2001/// C3 - Proceedings of the Cornell Nutrition Conference DA - 2001/// PY - 2001/// SP - 14–24 ER - TY - RPRT TI - Effect of L-carnitine and medium-chain triglyceride on plasma and urinary in newborn piglets AU - Heo, K. AU - Odle, J. AU - Lin, X. AU - van Kempen, T. AU - Han, I.K. DA - 2001/// PY - 2001/// M3 - Swine Reports ER - TY - RPRT TI - Kinetics of carnitine palmitoyltrasferase-I in liver and skeletal muscle of young pigs AU - Heo, K. AU - Lin, X. AU - Odle, J. AU - Han, I.K. DA - 2001/// PY - 2001/// M3 - Swine Reports ER - TY - RPRT TI - Effects of emulsification on amino acid and lipid digestibility in finishing pigs AU - Averette, L.A. AU - See, M.T. AU - Odle, J. DA - 2001/// PY - 2001/// M3 - Swine Reports ER - TY - RPRT TI - Emulsification of dietary fat for finishing pigs AU - Averette, L.A. AU - See, M.T. AU - Odle, J. DA - 2001/// PY - 2001/// M3 - Swine Reports ER - TY - SOUND TI - Nutritional and growth factor effects on gastrointestinal growth AU - Odle, J. DA - 2001/// PY - 2001/// M3 - Seminar ER - TY - SOUND TI - Liquid diets for early-weaned pigs – A “solution” for post-weaning morbidity AU - Odle, J. DA - 2001/// PY - 2001/// M3 - Seminar ER - TY - SOUND TI - Accelerating young pig growth via liquid diets AU - Odle, J. DA - 2001/// PY - 2001/// M3 - Seminar ER - TY - SOUND TI - Long-chain polyunsaturated fatty acid supplementation of infant formulas: insights from a piglet model AU - Odle, J. DA - 2001/// PY - 2001/// M3 - Professional testimony ER - TY - SOUND TI - New Adventures in Swine Nutrition: A Research Program Overview AU - Odle, J. DA - 2001/// PY - 2001/// M3 - Seminar ER - TY - CONF TI - Conjugated linoleic acid supplementation increases belly weight in lean-genotype gilts AU - Averette, L.A. AU - See, M.T. AU - Odle, J. T2 - Midwestern Section Animal Science meetings C2 - 2001/// C3 - Midwestern Section Animal Science meetings CY - Des Moines, IA DA - 2001/// PY - 2001/3/19/ SP - 49 ER - TY - SOUND TI - Piglet studies addressing the safety and efficacy of AA and DHA supplementation to infant formulas AU - Odle, J. DA - 2001/// PY - 2001/// M3 - Professional testimony ER - TY - CONF TI - Effects of tallow and conjugated linoleic acid on swine adipose tissue quality and delta-9 desaturase activity AU - Averette Gatlin, L. AU - Odle, J. AU - See, M.T. T2 - NC Institute of Nutrition annual meeting C2 - 2001/// DA - 2001/// PY - 2001/10/12/ ER - TY - SOUND TI - Enhancing intestinal growth: challenges and opportunities AU - Odle, J. DA - 2001/2/20/ PY - 2001/2/20/ M3 - Seminar ER - TY - CONF TI - Development of a porcine model for caliciviral enteritis AU - McPhatter, L.A. AU - Green, S.R. AU - Moe, C.L. AU - Odle, J. T2 - FASEB summer conference: Diet and gene expression C2 - 2001/// DA - 2001/// PY - 2001/8/21/ ER - TY - JOUR TI - Descriptive flavor analysis of bacon and pork loin from lean-genotype gilts fed conjugated linoleic acid AU - Averette Gatlin, L.A. AU - Larick, D.K. AU - See, M.T. AU - Odle, J. T2 - Journal of Animal Science DA - 2001/// PY - 2001/// VL - 79 IS - Suppl. 1 SP - 183 ER - TY - JOUR TI - Effects of long-chain polyunsaturated fatty acids (LCPUFA) on body composition and tissue accretion in the neonatal pig AU - Mathews, S.A. AU - Harrell, R.J. AU - Oliver, W.T. AU - Brown, J.A. AU - Phillips, O. AU - Lin, X. AU - Odle, J. AU - Diersen-Schade, D.A. T2 - Journal of Animal Science DA - 2001/// PY - 2001/// VL - 79 IS - Suppl. 1 SP - 398 ER - TY - CONF TI - Development of a porcine model for the study of caliciviral enteritis: preliminary data AU - McPhatter, L.A. AU - Green, S.R. AU - Moe, C.L. AU - Odle, J. T2 - Annual meeting of the Center for Gastrointestinal Biology and Disease. C2 - 2001/// CY - University of North Carolina - Chapel Hill DA - 2001/// PY - 2001/4/21/ ER - TY - JOUR TI - Genetic diversity of Bradyrhizobium strains isolated from Arachis hypogaea T2 - Canadian Journal of Microbiology AB - Rhizobia are used exclusively in agricultural systems for enhancing the ability of legumes to fix atmospheric nitrogen. Knowledge about the indigenous population is necessary for the selection and application of inoculant strains. In this study, we have assessed the genetic diversity of Bradyrhizobium strains isolated from the host plant, Arachis hypogaea along the coastline of Tamil Nadu. Different populations collected from varying environmental conditions were analysed for salt and pH tolerance. Genetic diversity among the strains was studied using RAPD markers and PCR-RFLP of 16S rDNA and nifD genes. The approaches used in this study yielded consistent results, which revealed a high degree of heterogeneity among strains and detection of two distinct genetic groups. DA - 2001/2/1/ PY - 2001/2/1/ DO - 10.1139/w00-139 UR - http://dx.doi.org/10.1139/w00-139 ER - TY - JOUR TI - Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea T2 - Biology and Fertility of Soils DA - 2001/9/1/ PY - 2001/9/1/ DO - 10.1007/s003740100391 UR - http://dx.doi.org/10.1007/s003740100391 KW - diversity KW - nodulation KW - Bradyrhizobium KW - nifD KW - 16S rDNA ER - TY - MGZN TI - Highway runoff effects on Freshwater Mussel Health AU - Levine, J.F. T2 - Centerline: Environmental Quarterly Newsletter DA - 2001/10// PY - 2001/10// SP - 7 PB - NC Department of Transportation ER - TY - CONF TI - Hemolymph Collection in Elliptio complanata AU - Gustafson, L. AU - Levine, J.F. AU - Bogan, A. AU - Showers, W. AU - Hanlon, S. AU - Stoskopf, M. T2 - NCSU College of Veterinary Medicine Research Forum C2 - 2001/3// CY - Raleigh, NC DA - 2001/3// PY - 2001/3// ER - TY - CONF TI - Population Dynamics in Neutered and Intact Feral Cat Colonies AU - Nutter, F. AU - Levine, J.F. AU - Stoskopf, M. T2 - NCSU College of Veterinary Medicine Research Forum C2 - 2001/3// CY - Raleigh, NC DA - 2001/3// PY - 2001/3// ER - TY - CONF TI - Determination of Host Fish Species for the Propagation of Endangered Freshwater Mussels AU - Tuttle, A. AU - Hanlon, S. AU - Levine, J.F. T2 - NCSU College of Veterinary Medicine Research Forum C2 - 2001/3// CY - Raleigh, NC DA - 2001/3// PY - 2001/3// ER - TY - CONF TI - Prevalence of Bacterial Food-borne Pathogens in Shellfish AU - Tlamka, B. AU - Pitts, T. AU - Levine, J.F. AU - French, J.B. AU - Mare, CI AU - Joens, L.A. T2 - Eighty-Second Conference of Research Workers in Animal Diseases C2 - 2001/// CY - St. Louis, Missouri DA - 2001/// PY - 2001/11// ER - TY - CONF TI - A Method For Measuring Growth In Living Freshwater Mussels AU - Molina, R. AU - Levine, J.F. AU - Hanlon, S. AU - Savidge, T. AU - Bogan, A. AU - Johnson, J. T2 - Freshwater Mollusk Conservation Society Symposium C2 - 2001/3// DA - 2001/3// PY - 2001/3// ER - TY - RPRT TI - Freshwater mussels of North Carolina AU - Levine, J.F. AU - Hanlon, S. DA - 2001/// PY - 2001/// M3 - poster ER - TY - CONF TI - Affects Of Flow On Juveniles Of Lampsilis Radiata Radiata Reared In An Indoor Recirculating Culture System AU - Hanlon, S. AU - Levine, J.F. AU - Savidge, T. T2 - Freshwater Mollusk Conservation Society Symposium C2 - 2001/3// CY - Pittsburgh, PA DA - 2001/3// PY - 2001/3// ER - TY - BOOK TI - Freshwater mussels: A learning resource and activity book AU - Levine, J.F. AU - Hanlon, S. DA - 2001/// PY - 2001/// PB - N.C. Freshwater Mussel Conservation Partnership ER - TY - CONF TI - Nonlethal Hemolymph Collection for Assessing Freshwater Mollusk Health AU - Gustafson, L. AU - Levine, J.F. AU - Bogan, A. AU - Showers, W. AU - Hanlon, S. AU - Stoskopf, M. T2 - Freshwater Mussel Conservation Society Symposium C2 - 2001/3// CY - Pittsburgh, PA DA - 2001/3// PY - 2001/3// ER - TY - RPRT TI - The Life Cycle of Freshwater Mussels AU - Levine, J.F. AU - Hanlon, S AU - Bogan, A DA - 2001/// PY - 2001/// M3 - poster ER - TY - CONF TI - Use of a Multitiered Approach to Assess Health Status of Coastal North Carolina Fish AU - Law, J.M. AU - Choi, K.J. AU - Johnson, A.K. AU - Lehmann, D.W. AU - Pettengill, M. AU - Levine, J. AU - Harms, C T2 - 22nd Annual Society of Environmental Toxicology and Chemistry (SETAC) Meeting C2 - 2001/11// CY - Baltimore, Maryland DA - 2001/11// PY - 2001/11// ER - TY - BOOK TI - Hyperthermophilic Enzymes, Part C A3 - Adams, M.W.W. A3 - Kelly, R.M. DA - 2001/// PY - 2001/// VL - 334 PB - Academic Press SE - 3-526 ER - TY - BOOK TI - Hyperthermophilic Enzymes, Part B A3 - Adams, M.W.W. A3 - Kelly, R.M. DA - 2001/// PY - 2001/// VL - 331 PB - Academic Press SE - 3-494 ER - TY - BOOK TI - Hyperthermophilic Enzymes, Part A A3 - Adams, M.W.W. A3 - Kelly, R.M. DA - 2001/// PY - 2001/// VL - 330 PB - Academic Press SE - 3–513 ER - TY - JOUR TI - β-Mannanases from Thermotoga species AU - Chhabra, S.R. AU - Parker, K.N. AU - Lam, D. AU - Callen, W. AU - Snead, M.A. AU - Mathur, E.J. AU - Short, J.M. AU - Kelly, R.M. T2 - Methods in Enzymology AB - Thermostable mannanases have been identified from Thermotoga neapolitana, Rhodothermus marinus, Bacillus stearotherrnophilus, Thermo- anaerobacterium polysaccharolyticum, Caldocellosiruptor saccharolyticus," and Dictyoglomus thermophilum. The β-mannanases from T. Neapolitana (family 5) and R. marinus (family 26) are the most thermostable of these. The mannanase from T. neapolitana is less stable at 85°, but more stable at 90°, than the corresponding enzyme from R. marinus. The former has a half-life of 34 hr at 85° and 13 hr at 90°, whereas the latter has a half life of 45.3 and 4.5 hr at the respective temperatures. Hyperthermophilic mannanases are useful in several industrial applications where thermostability and thermoactivity are desirable. These include coffee extraction, oil/gas well stimulation, and wood pulp treatment. This chapter describes the purification and characterization of a β-mannanase from T. neapolitana, as well as cloning and sequencing of this enzyme from T. neapolitana and T. maritima. DA - 2001/// PY - 2001/// DO - 10.1016/S0076-6879(01)30378-6 VL - 330 SP - 224–238 ER - TY - RPRT TI - Methods and compositions for fracturing subterranean formations AU - Kelly, R.M. AU - Khan, S.A. AU - Leduc, P. AU - Tayal, A. AU - Prud'homme, R. DA - 2001/3// PY - 2001/3// M1 - 6,197,730 M3 - U.S. Patent SN - 6,197,730 ER - TY - JOUR TI - Anion adsorption on an Au colloid monolayer based cysteamine-modified gold electrode AU - Hu, X.Y AU - Xiao, Y. AU - Chen, H.Y T2 - Chemical Research in Chinese Universities DA - 2001/5// PY - 2001/5// VL - 17 IS - 2 SP - 159 – 167 ER - TY - JOUR TI - Primer for non-immunologists on immune-deficient mice and their applications in research AU - Croy, B. AU - Linder, K. AU - Yager, J. T2 - Comparative Medicine DA - 2001/// PY - 2001/// VL - 45 IS - 4 SP - 300–313 ER - TY - CHAP TI - Homomultimeric protease and putative bacteriocin homolog from Thermotoga maritima AU - Hicks, Paula M AU - Chang, Lara S AU - Kelly, Robert M T2 - Methods in Enzymology AB - Publisher Summary Thermotoga maritima is an anaerobic heterotroph belonging to the bacterial order Thermotogales that grows optimally at 80° by fermentation of carbohydrates and proteins, including starch, glucose, galactose, glycogen, and yeast extract. The bacterium is also able to reduce thiosulfate to sulfide, with an improved growth rate. Although the microorganism is a facultative sulfur reducer, the reduction of sulfur does not provide an energetic boost as seen by the lack of effect on growth yields and fermentation balances. T. maritima appears to be motile, migrating at a speed proportional to the temperature. This chapter describes the purification protocols used to isolate maritimacin, as well as the biochemical assays used to measure its activity. PY - 2001/// DO - 10.1016/s0076-6879(01)30397-x SP - 455-460 OP - PB - Elsevier SN - 9780121822316 UR - http://dx.doi.org/10.1016/s0076-6879(01)30397-x DB - Crossref ER - TY - CHAP TI - Protease I from Pyrococcus furiosus AU - Chang, Lara S AU - Hicks, Paula M AU - Kelly, Robert M T2 - Methods in Enzymology AB - Pyrococcus furiosus is a hyperthermophilic archaeon from the order Thermococcales capable of growth on a variety of proteinaceous and carbohydrate-containing substrates. Analysis of gelatin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) gels indicates that at least 11 endoproteinases are active in the cell extracts of this organism 2'3 and the following proteases have been characterized: protease I (PfpI), pyrolysin, proteasome, prolyl oligopeptidase, and proline dipeptidase. The gene encoding the 19-kDa subunit of PfpI has homologs in nearly every organism and cell examined to date, ranging from Escherichia coli to Homo sapiens; this ubiquity and evolutionary conservation indicates that it may play a fundamental physiological role. Efforts to study this issue have been exacerbated by difficulties encountered in obtaining significant amounts of a particular assembly of PfpI in either a native or a recombinant form. Native PfpI undergoes autoproteolysis and/or disassembly during direct purification from P. furiosus biomass and exists in multiple (singleto multisubunit) forms in vitro. The production of a recombinant form of PfpI is also problematic due to its toxicity toward mesophilic hosts. This chapter describes several methods that have been used to purify PfpI directly from P. Furiosus cell extracts are described here, together with an assay to detect proteolytic activity, a procedure to determine its molecular mass, and approaches to minimize PfpI-catalyzed proteolysis of other P. furious proteins. PY - 2001/// DO - 10.1016/s0076-6879(01)30392-0 SP - 403-413 OP - PB - Elsevier SN - 9780121822316 UR - http://dx.doi.org/10.1016/s0076-6879(01)30392-0 DB - Crossref ER - TY - CHAP TI - β-Endoglucanase from Pyrococcus furiosus AU - Cady, Susan G AU - Bauer, Michael W AU - Callen, Walter AU - Snead, Marjory A AU - Mathur, Eric J AU - Short, J.M AU - Kelly, Robert M T2 - Methods in Enzymology AB - Glycosylhydrolases have been isolated from a variety of heterotrophic hyperthermophiles and include glucanases, hemicellulases, and cellulases. Pyrococcus furiosus, a hyperthermophilic heterotroph isolated by Fiala and Stetter from geothermal regions of Vulcano Island, Italy, grows on a wide range of α- and β-1inked carbohydrates, a property supported by the enzyme inventory revealed in its genome sequence. This chapter describes the approaches used for the cloning and expression in Escherichia coli of the eglA gene, which encodes the P. furiosus endoglucanase, and protocols used to study the biochemical properties of the recombinant enzyme. It is also interesting to know that P. furiosus and other heterotrophic hyperthermophilic archaea do not seem to produce enzymes for the hydrolysis of nonglucan polysaccharides, such as mannan or xylan, even though Thermotoga maritima, a hyperthermophilic bacterium, does. Whether this is a distinguishing feature of archaeal growth physiology remains to be seen. PY - 2001/// DO - 10.1016/s0076-6879(01)30387-7 SP - 346-354 OP - PB - Elsevier SN - 9780121822316 UR - http://dx.doi.org/10.1016/s0076-6879(01)30387-7 DB - Crossref ER - TY - CHAP TI - αa-D-Galactosidases from Thermotoga species AU - Miller, E.S, Jr. AU - Parker, Kimberley N AU - Liebl, Wolfgang AU - Lam, David AU - Callen, Walter AU - Snead, Mabjory A AU - Mathur, Eric J AU - Short, J.M AU - Kelly, Robert M T2 - Methods in Enzymology AB - Publisher Summary Based on similarities in primary structure and hydrophobic cluster analyses, αGals have been grouped into three well-conserved families in the general classification of glycosylhydrolases Those from bacteria have been grouped into families 4 and 36 and those of eukaryotic origin into family 27. To date, only α Gals of the hyperthermophilic bacteria Thermotoga maritima ( Tm GalA) and T. neapolitana ( Tn GalA) have demonstrated activity and prolonged stability above 75°. These two enzymes are therefore of considerable interest from a biotechnological standpoint. Potential applications include the high temperature hydrolysis of galactomannans used for well stimulation in the oil and gas industry and oligosaccharide synthesis via glycosyltransferase reactions. Genes encoding both Tm GalA and Tn GalA have been cloned and expressed in Escherichia coli . Although these enzymes are structurally related, they exhibit different biochemical properties in terms of pH optima, activity, and thermostability. This chapter discusses the purification, cloning, and expression of recombinant α Gal from T. neapolitana and T. maritima , in addition to some of their biochemical properties. PY - 2001/// DO - 10.1016/s0076-6879(01)30380-4 SP - 246-260 OP - PB - Elsevier SN - 9780121822316 UR - http://dx.doi.org/10.1016/s0076-6879(01)30380-4 DB - Crossref ER - TY - CHAP TI - Xylose isomerases from Thermotoga AU - Vieille, Claire AU - Sriprapundh, Dinlaka AU - Kelly, Robert M AU - Zeikus, J.Gregory T2 - Methods in Enzymology AB - Typically present in microorganisms that grow on xylose, xylose isomerise (XI) converts xylose to xylulose, which is then phosphorylated and enters the pentose-phosphate pathway. Because it also accepts glucose as substrate, XI is used extensively to isomerize glucose to fructose in the manufacture of high fructose corn syrup (HFCS). Performed at temperatures around 55-60°, this isomerization process requires thermostable enzymes. XIs have been characterized from a number of eubacterial sources and from rice and barley, but they have not been reported in fungi or archaea. XIs from the hyperthermophilic eubacteria Thermotoga maritima and Thermotoga neapolitana are the most thermostable yet characterized XIs. PY - 2001/// DO - 10.1016/s0076-6879(01)30377-4 SP - 215-224 OP - PB - Elsevier SN - 9780121822316 UR - http://dx.doi.org/10.1016/s0076-6879(01)30377-4 DB - Crossref ER - TY - CHAP TI - Carboxylesterase from Sulfolobus solfataricus P1 AU - Sehgal, A.C AU - Callen, Walter AU - Mathur, Eric J AU - Short, J.M AU - Kelly, Robert M T2 - Methods in Enzymology AB - To date, relatively few investigations regarding the purification and characterization of thermostable (Topt > 60 °) esterases have been conducted. Thus far, esterases from Bacillus acidocaldarius, Pyrococcus furiosus, Bacillus stearothermophilus, Sulfolobus shibatae, Thermoanaerobacterium sp, Pyrococcus abyssi, and Sulfolobus acidocaldarius have been studied to varying extents. Those thermostable esterases that have been evaluated vary significantly with respect to molecular mass, although their biochemical properties, such as pH optimum and substrate specificity, are quite similar. This chapter describes the cloning, expression, purification, and characterization of a recombinant carboxylesterase (Sso P1 carboxylesterase) from the extreme thermoacidophile Sulfolobus solfataricus P1. S. solfataricus, which is a member of the Crenarchaeota, can be found in sulfurous caldrons and volcanic muds. PY - 2001/// DO - 10.1016/s0076-6879(01)30398-1 SP - 461-471 OP - PB - Elsevier SN - 9780121822316 UR - http://dx.doi.org/10.1016/s0076-6879(01)30398-1 DB - Crossref ER - TY - CHAP TI - α-Glucosidase from Pyrococcus furiosus AU - Chang, Stephen T AU - Parker, Kimberley N AU - Bauer, Michael W AU - Kelly, Robert M T2 - Methods in Enzymology AB - Hyperthermophilic α-glucosidases could also provide valuable insights into protein function, structure, and stability at high temperatures. Indeed, it is the intrinsic high temperature activity and stability of these proteins that have fueled considerable effort into the development of these and other glycosylhydrolases for use in starch conversion technology. Currently employed mesophilic enzymes exhibit limited tolerance to the high temperatures and pH variations encountered during starch solubilization and degradation. These mesophilic enzymes often have metal ion requirements for activity, whereas their counterpart hyperthermophilic versions often do not. Although pullulanases and glucoamylases (also known as amyloglucosidases) are typically used for saccharification of intermediate starch degradation products to glucose, heat-stable α-glucosidases, together with pullulanases, could theoretically fill that role more efficiently. However, despite the potential impact of hyperthermophilic enzymes on industrial processes, including starch conversion, their application is still largely unrealized. One readily apparent obstacle is developing a costefficient method for producing sufficient quantities of enzyme either directly from the source organism or through recombinant means. PY - 2001/// DO - 10.1016/s0076-6879(01)30381-6 SP - 260-269 OP - PB - Elsevier SN - 9780121822316 UR - http://dx.doi.org/10.1016/s0076-6879(01)30381-6 DB - Crossref ER - TY - CHAP TI - β-Mannosidase from Thermotoga species AU - Parker, Kimberley N AU - Chhabra, Swapnil AU - Lam, David AU - Snead, Marjorie A AU - Mathur, Eric J AU - Kelly, Robert M T2 - Methods in Enzymology AB - Publisher Summary β -Mannosidase is an exo-acting glycosylhydrolase whose function is to cleave mannose residues from the nonreducing termini of mannan oligosaccharides. In microorganisms, β -mannosidases often act in concert with endo-acting β -mannanases to completely hydrolyze mannan-based carbohydrates to be subsequently used for nutritional purposes. In mammals, the deficiency of mannosidase can lead to β -mannosidosis, a genetic disorder resulting from the storage and excretion of undegraded substrates. Hyperthermophilic β -mannosidases have been identified in archaea such as Pyrococcus furiosus and in the bacteria Thermotoga maritima and T. neapolitana . This chapter presents the protocols used to purify the β -mannosidase from T. Neapolitana together with the results of cloning and expression of genes encoding the analogous enzyme of T. maritima in Escherichia coli. PY - 2001/// DO - 10.1016/s0076-6879(01)30379-8 SP - 238-246 OP - PB - Elsevier SN - 9780121822316 UR - http://dx.doi.org/10.1016/s0076-6879(01)30379-8 DB - Crossref ER - TY - SOUND TI - Nonstationary Spatial Modeling for Multiple Point Sources AU - Ghosh, S. DA - 2001/1/25/ PY - 2001/1/25/ ER - TY - CONF TI - Bayesian Capture-Recapture Analysis Allowing for Heterogeneity Between Animals AU - Ghosh, S. T2 - Joint Statistical Meetings C2 - 2001/8/5/ CY - Atlanta, Georgia DA - 2001/8/5/ PY - 2001/8/5/ ER - TY - SOUND TI - Discussion on Bayesian Models for Enhancing Cross-population Comparability of Survey Results AU - Ghosh, S. DA - 2001/// PY - 2001/// ER - TY - CONF TI - Bayesian Imputation Methods to Measure Quality of Life with Missing Data AU - Umbach, A.T. AU - Ghosh, S.K. T2 - American Statistical Association C2 - 2001/// C3 - 2001 proceedings : papers presented at the Annual Meeting of the American Statistical Association, Joint Statistical Meetings, Atlanta, Georgia, August 5-9, 2001, and other ASA-sponsored conferences CY - Atlanta, Georgia DA - 2001/// PY - 2001/8/5/ PB - American Statistical Association ER - TY - SOUND TI - Bayesian Unit Root Tests in Stochastic Volatility Models (SVM) AU - Ghosh, S. DA - 2001/11/29/ PY - 2001/11/29/ ER - TY - CONF TI - A Conditional Nonparametric Approach to Estimating Survival Functions AU - Huang, Y. AU - Ghosh, S.K. T2 - American Statistical Association C2 - 2001/// C3 - 2001 proceedings : papers presented at the Annual Meeting of the American Statistical Association, Joint Statistical Meetings, Atlanta, Georgia, August 5-9, 2001, and other ASA-sponsored conferences CY - Atlanta, Georgia DA - 2001/// PY - 2001/8/5/ PB - American Statistical Association ER - TY - CONF TI - Bayesian Analysis of Circular Data Using Wrapped Distributions AU - Ravindran, P. AU - Ghosh, S.K. T2 - American Statistical Association C2 - 2001/// C3 - 2001 proceedings : papers presented at the Annual Meeting of the American Statistical Association, Joint Statistical Meetings, Atlanta, Georgia, August 5-9, 2001, and other ASA-sponsored conferences CY - Atlanta, Georgia DA - 2001/// PY - 2001/8/5/ PB - American Statistical Association ER - TY - JOUR TI - Bayesian Analysis of Interval-censored Survival Data using Penalized Likelihood AU - Ghosh, S.K. AU - Sinha, D. T2 - Sankhyā: The Indian Journal of Statistics, Series A DA - 2001/2// PY - 2001/2// VL - 63 IS - 1 SP - 1–14 ER - TY - CONF TI - Heart or lung: The culprit in cough AU - Hawkins, E.C. AU - Atkins, C. C2 - 2001/// C3 - Iams-Pfizer Senior Care Proceedings DA - 2001/// PB - The Gloyd Group ER - TY - CHAP TI - Heart or Lung: The culprit in cough? AU - Hawkins, E.C. AU - Atkins, C.E. T2 - Senior care 2001 : the impact of aging on everyday cases : proceedings from a symposium held at the North American Veterinary Conference and the Western Veterinary Conference PY - 2001/// SP - 85–97 PB - The Gloyd Group ER - TY - CONF TI - Nasal discharge in cats: cases to learn by AU - Hawkins, E.C. T2 - North American Veterinary Conference C2 - 2001/// C3 - Proceedings of the North American Veterinary Conference CY - Orlando, FL DA - 2001/// PY - 2001/1/13/ VL - 5 SP - 562-564 M1 - 15 PB - Eastern States Veterinary Association ER - TY - JOUR TI - Magnetic resonance imaging of spongy degeneration of the central nervous system in a Labrador retriever AU - Mariani, Christopher L. AU - Clemmons, Roger M. AU - Graham, John P. AU - Phillips, Lynette A. AU - Chrisman, Cheryl L. T2 - Veterinary Radiology & Ultrasound AB - A 7-month-old, neutered female Labrador Retriever was evaluated for tetraparesis and subtle cerebellar dysfunction. Clinical signs progressed over a period of 6 weeks to severe ataxia, hypermetria, intention tremors, and finally non-ambulatory tetraparesis. On magnetic resonance imaging of the brain there were large, bilaterally symmetrical, ovoid lesions in the region of the deep cerebellar nuclei that were hyperintense on T2-weighted and proton density images and hypointense on T1-weighted images. There were similar but smaller bilaterally symmetrical lesions present within the thalamus. Euthanasia was performed and lesions consistent with the previously described spongy degeneration of Labrador Retrievers were identified. This disease and its relation to similar human heritable leukodystrophies are discussed. DA - 2001/7// PY - 2001/7// DO - 10.1111/j.1740-8261.2001.tb00941.x VL - 42 IS - 4 SP - 285-290 SN - 1058-8183 1740-8261 UR - http://dx.doi.org/10.1111/j.1740-8261.2001.tb00941.x KW - dog KW - canine KW - spongy degeneration KW - Labrador Retrievers KW - Canavan's disease KW - cerebellum KW - MRI ER - TY - JOUR TI - Perspectives on the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for short tandem repeat genotyping in the post‐genome era AU - Null, A.P. AU - Muddiman, D.C. T2 - Journal of Mass Spectrometry (Special Feature) AB - Abstract The recent completion of the first rough draft of the human genome has provided fundamental information regarding our genetic make‐up; however, the post‐genome era will certainly require a host of new technologies to address complex biological questions. In particular, a rapid and accurate approach to characterize genetic markers, including short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) is demanded. STRs are the most informative of the two polymorphisms owing to their remarkable variability and even dispersity throughout eukaryotic genomes. Mass spectrometry is rapidly becoming a significant method in DNA analysis and has high probability of revolutionizing the way in which scientists probe the human genome. It is our responsibility as biomolecular mass spectrometrists to understand the issues in genetic analysis and the capabilities of mass spectrometry so that we may fulfill our role in developing a rapid, reliable technology to answer specific biological questions. This perspective is intended to familiarize the mass spectrometry community with modern genomics and to report on the current state of mass spectrometry, specifically electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, for characterization of STRs. Copyright © 2001 John Wiley & Sons, Ltd. DA - 2001/6/21/ PY - 2001/6/21/ DO - 10.1002/jms.172 VL - 36 IS - 6 SP - 589–606 KW - electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry KW - short tandem repeats KW - genotyping KW - DNA KW - human genome ER - TY - JOUR TI - Companion animals, too, can get pemphigus AU - Olivry, T. T2 - Quarterly - The Journal of the National Pemphigus Foundation DA - 2001/// PY - 2001/// VL - 26 SP - 11, ER - TY - JOUR TI - Oral manifestations of autoimmune blistering diseases AU - Chan, L.S. AU - Olivry, T. AU - Lozada-Nur, F. T2 - eMedicine Journal DA - 2001/// PY - 2001/// VL - 2 IS - 9 UR - http://www.emedicine.com/derm/topic661.htm ER - TY - JOUR TI - Homogeneous Preparations of 3′-Phosphoglycolate-Terminated Oligodeoxynucleotides from Bleomycin-Treated DNA as Verified by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry AU - Chen, Shuang AU - Hannis, James C. AU - Flora, Jason W. AU - Muddiman, David C. AU - Charles, Kwabena AU - Yu, Yin AU - Povirk, Lawrence F. T2 - Analytical Biochemistry AB - Single- and double-strand breaks bearing 3′-phosphoglycolate termini are among the most frequent lesions formed in DNA by ionizing radiation and other oxidative mutagens. In order to obtain homogeneous preparations of defined 3′-phosphoglycolate substrates for repair studies, 5′-32P-end-labeled partial duplex DNAs were treated with bleomycin, and individual cleavage products were isolated from polyacrylamide gels. The fragments were then treated with alkaline phosphatase and further purified by reverse-phase HPLC. Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry of the purified oligomers produced molecular ions of the expected masses, with no detectable contaminants. Gas-phase sequencing by tandem mass spectrometry of these single species yielded the expected sequence ions and confirmed the presence of phosphoglycolate on the 3′-terminal fragments only. The fragments could be relabeled with polynucleotide kinase to yield highly purified, high-specific-activity substrates for repair studies. DA - 2001/2// PY - 2001/2// DO - 10.1006/abio.2000.4936 VL - 289 IS - 2 SP - 274-280 J2 - Analytical Biochemistry LA - en OP - SN - 0003-2697 UR - http://dx.doi.org/10.1006/abio.2000.4936 DB - Crossref ER - TY - JOUR TI - Realistic Distributions of Infectious Periods in Epidemic Models: Changing Patterns of Persistence and Dynamics AU - Lloyd, Alun L. T2 - Theoretical Population Biology AB - Most mathematical models used to study the epidemiology of childhood viral diseases, such as measles, describe the period of infectiousness by an exponential distribution. The effects of including more realistic descriptions of the infectious period within SIR (susceptible/infectious/recovered) models are studied. Less dispersed distributions are seen to have two important epidemiological consequences. First, less stable behaviour is seen within the model: incidence patterns become more complex. Second, disease persistence is diminished: in models with a finite population, the minimum population size needed to allow disease persistence increases. The assumption made concerning the infectious period distribution is of a kind routinely made in the formulation of mathematical models in population biology. Since it has a major effect on the central issues of population persistence and dynamics, the results of this study have broad implications for mathematical modellers of a wide range of biological systems. DA - 2001/// PY - 2001/// DO - http://dx.doi.org/10.1006/tpbi.2001.1525 VL - 60 IS - 1 SP - 59–71 ER - TY - JOUR TI - Infection dynamics on scale-free networks AU - May, Robert AU - Lloyd, Alun T2 - Physical Review E AB - We discuss properties of infection processes on scale-free networks, relating them to the node-connectivity distribution that characterizes the network. Considering the epidemiologically important case of a disease that confers permanent immunity upon recovery, we derive analytic expressions for the final size of an epidemic in an infinite closed population and for the dependence of infection probability on an individual's degree of connectivity within the population. As in an earlier study [R. Pastor-Satorras and A. Vesipignani, Phys. Rev. Lett. 86, 3200 (2001); Phys. Rev. E. 63, 006117 (2001)] for an infection that did not confer immunity upon recovery, the epidemic process--in contrast with many traditional epidemiological models--does not exhibit threshold behavior, and we demonstrate that this is a consequence of the extreme heterogeneity in the connectivity distribution of a scale-free network. Finally, we discuss effects that arise from finite population sizes, showing that networks of finite size do exhibit threshold effects: infections cannot spread for arbitrarily low transmission probabilities. DA - 2001/// PY - 2001/// DO - http://dx.doi.org/10.1103/physreve.64.066112 VL - 64 IS - 6 ER - TY - CHAP TI - Electrochemical or Separations-Based Sensing of Neurotransmitter Dynamics and Storage at Cells and Varicosities in Culture AU - Ewing, A.G. AU - Sombers, L.A. AU - Woods, L.A. AU - Colliver, T.L. AU - Achalabun, M. AU - Moran, K. AU - Lapos, J. AU - Paxon, T. AU - Cans, A.S T2 - Monitoring molecules in neuroscience : proceedings of the 9th International Conference on In Vivo Methods, June 16-19, 2001, University College Dublin, Ireland A2 - O'Connor, W. T. A2 - Lowry, J. P. A2 - O'Connor, J. J. A2 - O'Neill, R. D. PY - 2001/// SP - 57–58 PB - University College Dublin ER - TY - JOUR TI - Genotyping of Simple and Compound Short Tandem Repeat Loci Using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry AU - Null, Allison P. AU - Hannis, James C. AU - Muddiman, David C. T2 - Analytical Chemistry AB - The utility of electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry as a new approach for genotyping short tandem repeats (STRs) is demonstrated. STRs are currently valued as a powerful source of genetic information with repeats that range in structure from simple to hypervariable. Two tetranucleotide STR loci were chosen to evaluate ESI-FTICR mass spectrometry as a tool for genotyping: HUMTH01, a simple STR with nonconsensus alleles, and vWA, a compound STR with nonconsensus alleles. For HUMTH01, the genotype (i.e., repeat number of each allele) was determined for each of 30 individuals using mass measurements of double-stranded amplicons. Low-intensity peaks observed in the spectra of amplicons derived from heterozygous individuals were identified by mass as heteroduplexes that had formed between nonhomologous strands. Mass measurement of the double-stranded vWA amplicon was not sufficient for determining whether the individual was homozygous for allele subtype 18 or 18‘ since the amplicons differ by only 0.99 Da. Therefore, single-stranded amplicons were generated by incorporating a phosphorylated primer, prepared using T4 polynucleotide kinase, into the PCR phase and subsequently digesting the bottom strand using λ-exonuclease. Accurate mass measurements were obtained for the single-stranded amplicons using internal calibration and the addition of a correction factor to adjust for the natural variation of isotopic abundances, confirming that the individual is homozygous for allele 18. Our results clearly demonstrate that ESI-FTICR mass spectrometry is a powerful approach to characterize both simple and compound STRs beyond the capabilities of electrophoretic technologies. DA - 2001/9// PY - 2001/9// DO - 10.1021/ac0103928 VL - 73 IS - 18 SP - 4514-4521 J2 - Anal. Chem. LA - en OP - SN - 0003-2700 1520-6882 UR - http://dx.doi.org/10.1021/ac0103928 DB - Crossref ER - TY - JOUR TI - Selective, Sensitive, and Rapid Phosphopeptide Identification in Enzymatic Digests Using ESI-FTICR-MS with Infrared Multiphoton Dissociation AU - Flora, Jason W. AU - Muddiman, David C. T2 - Analytical Chemistry AB - Rapid screening for phosphopeptides within complex proteolytic digests involving electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the negative ion mode with infrared multiphoton dissociation (IRMPD) accompanied by improved phosphopeptide sensitivity and selectivity is demonstrated with the tryptic digests of the naturally phosphorylated proteins bovine alpha- and beta-casein. All peptides in a complex proteolytic digest can be examined simultaneously for phosphorylation with a 4-s IR laser pulse at 7-11 W where phosphopeptide signature ions form upon irradiation, as the low energy of activation phosphate moiety cleavage transpires without the dissociation of the unphsophorylated peptide population. The tyrosine phosphorylated peptide HGLDN-pY-R, its nonphosphorylated analogue HGLDNYR, the kinase domain of insulin receptor unphosphorylated TRDIYETDYYRK, monophosphorylated TRDIYED-pY-YRK, and triphosphorylated TRDI-pY-ETD-pY-pY-RK were also used as model peptides in this research. The sensitivity and selectivity of phosphopeptides is shown to greatly improve when the volatile base piperidine is used to adjust the pH of th DA - 2001/7// PY - 2001/7// DO - 10.1021/ac010333u VL - 73 IS - 14 SP - 3305-3311 J2 - Anal. Chem. LA - en OP - SN - 0003-2700 1520-6882 UR - http://dx.doi.org/10.1021/ac010333u DB - Crossref ER - TY - JOUR TI - Complete sequencing of mono—deprotonated peptide nucleic acids by sustained off-resonance irradiation collision—induced dissociation AU - Flora, Jason W. AU - Muddiman, David C. T2 - Journal of the American Society for Mass Spectrometry AB - Complete peptide nucleic acids (PNAs) sequence information is obtained from the unimolecular decomposition of singly-charged PNA oligomers in the negative-ion mode using electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) and sustained off-resonance irradiation collision induced dissociation. The 4-mers, n-CATT-c, n-AGCT-c, n-AACT-c, and n-acetylated-AACT-c and two 6-mers, n-AAAAAA-c and n-CCCCCC-c, were investigated to explore the unimolecular decomposition of mixed-nucleobase and homopolymer PNAs representing purine and pyrimidine oligomers, respectively. PNA decomposition is explored using a product-ion appearance curve and double resonance experiments. A decomposition mechanism for sequence ion formation (PNA amide bond cleavage) is proposed. DA - 2001/7// PY - 2001/7// DO - 10.1016/s1044-0305(01)00259-8 VL - 12 IS - 7 SP - 805-809 J2 - J Am Soc Mass Spectrom LA - en OP - SN - 1044-0305 1879-1123 UR - http://dx.doi.org/10.1016/s1044-0305(01)00259-8 DB - Crossref ER - TY - JOUR TI - High-Mass Accuracy of Product Ions Produced by SORI-CID Using a Dual Electrospray Ionization Source Coupled with FTICR Mass Spectrometry AU - Flora, Jason W. AU - Hannis, James C. AU - Muddiman, David C. T2 - Analytical Chemistry AB - High-mass accuracy is demonstrated using internal calibration for product ions produced by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) of a 15-mer oligonucleotide, 5'-(CTG)5-3'. Internal calibration for this tandem MS experiment was accomplished using a dual electrospray ionization (ESI) source coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) utilizing hexapole accumulation and gated trapping. The pulse sequence entails injection, trapping, and gas-phase isolation of the precursor ion of interest followed by the SORI-CID of this ion and, subsequently, injection and trapping of the internal mass calibrant (i.e., poly(ethylene glycol) with a 1000 Da average mass). The product ions and the poly(ethylene glycol) ions are then simultaneously excited by a broadband frequency chirp excitation waveform and detected. This technique corrects for space-charge effects on the measurement of an ion's cyclotron frequency experienced when externally calibrated data are used. While external calibration for FTICR-MS can result in mass errors of greater than 100 ppm, this internal standardization method demonstrated significantly more consistent accurate mass measurements with average mass errors ranging from -1.2 to -3.2 ppm for the 15-mer oligonucleotide used in this study. This method requires limited modifications to ESI-FTICR mass spectrometers and is applicable for both positive and negative modes of ionization as well as other sample types (e.g., pharmaceuticals, proteins, etc.). DA - 2001/3// PY - 2001/3// DO - 10.1021/ac0011282 VL - 73 IS - 6 SP - 1247-1251 J2 - Anal. Chem. LA - en OP - SN - 0003-2700 1520-6882 UR - http://dx.doi.org/10.1021/ac0011282 DB - Crossref ER - TY - JOUR TI - Genotyping short tandem repeats using flow injection and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry AU - Hannis, James C. AU - Muddiman, David C. T2 - Rapid Communications in Mass Spectrometry AB - Characterizing polymerase chain reaction (PCR) amplicons has been accomplished for the first time using flow injection analysis coupled to electrospray ionization mass spectrometry (ESI-MS). The PCR amplicons were amplified at the human tyrosine hydroxylase short tandem repeat locus from an individual homozygotic for the 9.3 allele. One product was amplified using Pfu polymerase and yielded a blunt-ended amplicon of 82 base-pairs (bp) in length. The second PCR product was amplified using Taq polymerase that resulted in an amplicon with cohesive termini of 82 bp plus either mono- or diadenylation. The two PCR amplicons were alternatively injected using a 0.5-microL loop at 2 microM for the Pfu amplicon and 1 microM for the Taq amplicon with a flow rate of 200 nL/min during data acquisition. Both PCR amplicons were accurately identified using mass measurements illustrating the compatibility of ESI-MS for genotyping short tandem repeat sequences and the potential for high-throughput genotyping of large PCR amplicons. DA - 2001/// PY - 2001/// DO - 10.1002/rcm.234 VL - 15 IS - 5 SP - 348-350 J2 - Rapid Commun. Mass Spectrom. LA - en OP - SN - 0951-4198 1097-0231 UR - http://dx.doi.org/10.1002/rcm.234 DB - Crossref ER - TY - JOUR TI - Detection of double-stranded PCR amplicons at the attomole level electrosprayed from low nanomolar solutions using FT-ICR mass spectrometry AU - Hannis, James C. AU - Muddiman, David C. T2 - Fresenius' Journal of Analytical Chemistry DA - 2001/2/14/ PY - 2001/2/14/ DO - 10.1007/s002160000612 VL - 369 IS - 3-4 SP - 246-251 J2 - Fresenius' Journal of Analytical Chemistry OP - SN - 0937-0633 1432-1130 UR - http://dx.doi.org/10.1007/s002160000612 DB - Crossref ER - TY - JOUR TI - Impact of ion cloud densities on the measurement of relative ion abundances in Fourier transform ion cyclotron resonance mass spectrometry: experimental observations of coulombically induced cyclotron radius perturbations and ion cloud dephasing rates AU - Gordon, Eric F. AU - Muddiman, David C. T2 - Journal of Mass Spectrometry AB - Fundamental research into the quantitative properties of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has yielded interesting observations, especially in terms of factors affecting the accuracy of relative ion abundances. However, most of the previous discussions have focused on theoretical systems, or systems of limited scope. In this paper, we document ion motion attributes of a 30 spectra (six samples, five replicates each) system previously established as linear over two orders of magnitude. Observed behaviors include the perturbation of one charged species (cyclosporin A, CsA) of low ion density to a cyclotron orbit of greater radius than that of an almost identical, but slightly mass-separated species (CsG) with a higher ion density. This radial perturbation is attributed to the coulombic repulsion between the two ion clouds as they interact during the excitation process, as previously proposed by Uechi and Dunbar. Magnitudes of the perturbation were confirmed by making cyclotron radii determinations utilizing the ratio of the third-to-first harmonics for the charged species of interest. It was found that these radial differences can account for as much as a 55% signal bias in favor of CsA for a single sample and a >20% positive bias in the slope of the regressed data set. A second behavior noted that also contributes to the potential inaccuracy of relative ion abundance measurements is the difference in signal decay rates for CsA and CsG. Damping constants and initial time domain signal amplitudes were evaluated using segmented Fourier transforms. Discrepancies in decay rates were not expected from two species that have essentially identical collisional cross-sections. However, it has been observed that the faster decay rates are observed by the species of lower ion cloud density. We have attributed this differential signal decay phenomenon to the rates of loss of phase coherence for the two ion clouds. Previously, others have reported that less dense ion clouds are more susceptible to shearing and other disruptive forces during the course of their excited cyclotron motion. Our experimental evidence supports that it is the loss of cloud coherence that accounts for the signal loss over time, with the less dense cloud de-phasing more quickly. As the ion populations of the two investigated species near equivalence, so do their time constants. DA - 2001/// PY - 2001/// DO - 10.1002/jms.121 VL - 36 IS - 2 SP - 195-203 J2 - J. Mass Spectrom. LA - en OP - SN - 1076-5174 1096-9888 UR - http://dx.doi.org/10.1002/jms.121 DB - Crossref KW - space-charge KW - ion cloud density KW - signal decay KW - proteomics KW - quantification ER - TY - JOUR TI - Essential Wavelets for Statistical Applications and Data Analysis AU - Ghosh, Sujit K T2 - Technometrics AB - "Essential Wavelets for Statistical Applications and Data Analysis." Technometrics, 43(1), p. 105 DA - 2001/2// PY - 2001/2// DO - 10.1198/tech.2001.s557 VL - 43 IS - 1 SP - 105-105 J2 - Technometrics LA - en OP - SN - 0040-1706 1537-2723 UR - http://dx.doi.org/10.1198/tech.2001.s557 DB - Crossref ER - TY - BOOK TI - Ch. 31. Bayesian and frequentist methods in change-point problems AU - Ebrahimi, N. AU - Ghosh, S.K. DA - 2001/// PY - 2001/// DO - 10.1016/S0169-7161(01)20033-9 VL - 20 SE - 777-787 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-33845427195&partnerID=MN8TOARS ER - TY - JOUR TI - Magnetic resonance imaging of cerebral cortical necrosis (polioencephalomalacia) in a dog. AU - Mariani, Christopher L. AU - Platt, Simon R. AU - Newell, Susan M. AU - Terrell, Scott P. AU - Chrisman, Cheryl L. AU - Clemmons, Roger M. T2 - Veterinary Radiology Ultrasound AB - A 3-year-old neutered female mixed breed dog was examined because of severe, generalized seizure activity, tetraparesis, and encephalopathic signs. Cerebrospinal fluid (CSF) evaluation was unremarkable except for a mild increase in protein. Serum and CSF titers for infectious diseases were negative. Magnetic resonance (MR) imaging examination of the brain was performed and lesions were found within the cerebral gray matter of the temporal and parietal lobes. The lesions had increased signal intensity on T1, T2, and proton density-weighted images. There was mild inhomogeneous enhancement following intravenous contrast medium administration. Neurologic status improved and the seizures were well controlled, but the dog never regained normal mentation and euthanasia was performed 10 weeks after initial evaluation. At necropsy, severe cerebral cortical necrosis was found in the regions corresponding to the lesions seen on MR imaging examination. Large numbers of fat-containing macrophages (gitter cells) were found within these areas, and are thought to be responsible for the characteristic hyperintensity seen on the MR images. DA - 2001/11// PY - 2001/11// DO - 10.1111/j.1740-8261.2001.tb00981.x VL - 42 IS - 6 SP - 524-531 SN - 1058-8183 1740-8261 UR - http://dx.doi.org/10.1111/j.1740-8261.2001.tb00981.x KW - dog KW - canine KW - cerebral cortical necrosis KW - polioencephalomalacia KW - seizures KW - magnetic resonance imaging ER - TY - CONF TI - Complete Phase Diagrams for Binary Mixtures Via Gibbs Duhem Integration AU - Hitchcock, M.R. AU - Hall, C.K. T2 - First International Conference on Molecular Modeling and Simulation A2 - Cummings, P.T. A2 - Westmoreland, P.R. T3 - AlChE Symposium Series C2 - 2001/// C3 - Foundations of Molecular Modeling and Simulation: proceedings of the First International Conference on Molecular Modeling and Simulation, Keystone, Colorado, July 23-28, 2000 CY - Keystone, Colorado DA - 2001/// PY - 2000/7/23/ SP - 195 PB - American Institute of Chemical Engineers SN - 9780816908394 ER - TY - CHAP TI - Cherry eye AU - Gilger, B.C. T2 - The Five Minute Veterinary Consult A2 - Tilley, P. A2 - Smith, T. PY - 2001/// ET - 2nd SP - 969 PB - Williams & Wilkins ER - TY - CHAP TI - Third eyelid, protruding AU - Gilger, B.C. T2 - The Five Minute Veterinary Consult A2 - Tilley, P. A2 - Smith, T. PY - 2001/// ET - 2nd SP - 166–167 PB - Williams & Wilkins ER - TY - CHAP TI - Epiphora AU - Gilger, B.C. T2 - The Five Minute Veterinary Consult A2 - Tilley, P. A2 - Smith, T. PY - 2001/// ET - 2nd SP - 62–63 PB - Williams & Wilkins ER - TY - JOUR TI - (F8TPP)FeII/O2 Reactivity Studies {F8TPP = Tetrakis(2,6-difluorophenyl)porphyrinate(2−)}:  Spectroscopic (UV−Visible and NMR) and Kinetic Study of Solvent-Dependent (Fe/O2= 1:1 or 2:1) Reversible O2-Reduction and Ferryl Formation AU - Ghiladi, Reza A. AU - Kretzer, Ryan M. AU - Guzei, Ilia AU - Rheingold, Arnold L. AU - Neuhold, Yorck-Michael AU - Hatwell, Karen R. AU - Zuberbühler, Andreas D. AU - Karlin, Kenneth D. T2 - Inorganic Chemistry AB - In this report, we describe in detail the O(2)-binding chemistry of the metalloporphyrin (F(8)TPP)Fe(II) (1). This complex was synthesized from aqueous dithionite reduction of (F(8)TPP)Fe(III)-Cl (X-ray structure reported: C(55)H(36)ClF(8)FeN(4)O; a = 13.6517(2) A, b = 13.6475(2) A, c = 26.3896(4), alpha = 90 degrees, beta = 89.9776(4) degrees, gamma = 90 degrees; monoclinic, P2(1)/c, Z = 4). Complex 1 crystallizes from toluene/heptane solvent system as a bis(toluene) solvate, (F(8)TPP)Fe(II).(C(7)H(8))(2), with ferrous ion in the porphyrin plane (C(58)H(36)F(8)FeN(4); a = 20.9177(2) A, b = 11.7738(2) A, c = 19.3875(2), alpha = 90 degrees, beta = 108.6999(6) degrees, gamma = 90 degrees; monoclinic, C2/c, Z = 4; Fe-N(4)(av) = 2.002 A; N-Fe-N (all) = 90.0 degrees ). Close metal-arene contacts are also observed at 3.11-3.15 A. Upon oxygenation of 1 at 193 K in coordinating solvents, UV-visible and (2)H and (19)F NMR spectroscopies revealed the presence of a reversibly formed dioxygen adduct, formulated as the heme-superoxo complex (S)(F(8)TPP)Fe(III)-(O(2)(-)) (2) (S = solvent) [(i) tetrahydrofuran (THF) solvent: UV-visible, 416 (Soret), 536 nm; (2)H NMR: delta(pyrrole) 8.9 ppm; (ii) EtCN solvent: UV-visible, 414 (Soret), 536 nm; (iii) acetone solvent: UV-visible, 416 (Soret), 537 nm; (2)H NMR: delta(pyrrole) 8.9 ppm]. Dioxygen-uptake manometry (THF, 193 K) revealed an O(2):1 oxygenation stoichiometry of 1.02:1, consistent with the heme-superoxo formulation of 2. Stopped-flow UV-visible spectrophotometry studies of the (F(8)TPP)Fe(II) (1)/O(2) reaction in EtCN and THF solvents were able to provide kinetic and thermodynamic insight into the reversible formation of 2 [(i) EtCN: Delta H degrees = -40 +/- 5 kJ/mol; Delta S degrees = -105 +/- 23 J/(K mol); k(1) = (5.57 +/- 0.04) x 10(3) M(-)(1) s(-)(1) (183 K); Delta H(++) = 38.6 +/- 0.2 kJ/mol; Delta S(++) = 42 +/- 1 J/(K mol); (ii) THF: Delta H* = -37.5 +/- 0.4 kJ/mol; Delta S* = -109 +/- 2 J/(K mol)]. The (F(8)TPP)Fe(II) (1)/O(2) reaction was also examined at reduced temperatures in noncoordinating solvents (toluene, CH(2)Cl(2)), where UV-visible and (2)H and (19)F NMR spectroscopies also revealed the presence of a reversibly formed adduct, formulated as the peroxo-bridged dinuclear complex [(F(8)TPP)Fe(III)](2)-(O(2)(2)(-)) (3) [CH(2)Cl(2): UV-visible, 414 (Soret), 535 nm; (2)H NMR, delta(pyrrole) 17.5 ppm]. Dioxygen-uptake spectrophotometric titrations revealed a stoichiometry of 2 (F(8)TPP)Fe(II) (1) per O(2) upon full formation of 3. Addition of a nitrogenous base, 4-(dimethylamino)pyridine, to a cold solution of 3 in dichloromethane gave rapid formation of the iron(IV)-oxo ferryl species (DMAP)(F(8)TPP)Fe(IV)==O (4), based upon UV-visible [417 (Soret), 541 nm] and (2)H NMR (delta(pyrrole) = 3.5 ppm) spectroscopic characterization. These detailed investigations into the O(2)-adducts and "ferryl" species formed from (F(8)TPP)Fe(II) (1) may be potentially important for a full understanding of our ongoing heme-copper oxidase model studies, which employ 1 or similar "tethered" (i.e., covalently attached Cu-chelate) porphyrin analogues in heme/Cu heterobinuclear systems. DA - 2001/11// PY - 2001/11// DO - 10.1021/ic0105866 VL - 40 IS - 23 SP - 5754–5767 SN - 0020-1669 1520-510X UR - http://dx.doi.org/10.1021/ic0105866 ER - TY - JOUR TI - Multifrequency electron paramagnetic resonance of ultramarine blue AU - Eaton, G.R. AU - Eaton, S.S. AU - Stoner, J.W. AU - Quine, R.W. AU - Rinard, G.A. AU - Smirnov, A.I. AU - Weber, R.T. AU - Krzystek, J. AU - Hassan, A.K. AU - Brunel, L.-C. AU - Demortier, A. T2 - Applied Magnetic Resonance DA - 2001/// PY - 2001/// DO - 10.1007/BF03162429 VL - 21 IS - 3-4 SP - 563-570 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035528071&partnerID=MN8TOARS ER - TY - JOUR TI - Studies of Cetylpyridinium Chloride and Cetylpyridinium Salicylate in Solution and Adsorbed on Silica Surfaces Using X- and W-Band Electron Paramagnetic Resonance Spectroscopy AU - Bakker, Martin G. AU - Granger, Edward L. AU - Smirnov, Alex I. T2 - Langmuir AB - The electron paramagnetic resonance (EPR) spin probe 4-[N,N-dimethyl-N-(n-hexadecyl)ammonium]-2,2,6,6-tetramethylpiperindinyl-N‘-oxyl (HTAB*) has been used to study the adsorption and aggregation of cetylpyridinium chloride (CPC) and cetylpyridinium salicylate (CPSa) on silica particles. In CPC micelles, the local viscosity, as determined from the rotational correlation times of HTAB*, was found to be approximately constant, as was the local polarity determined from Aiso, giso, and HTAB* equilibrium constants. In CPSa micelles at concentrations above the sphere-to-rod transition, significant decreases in polarity and local viscosity were observed. The affinity of CPC for the strong binding site on the silica surface was found to be higher than the affinities of CPSa and HTAB. The polarities and local viscosities of the surfactant aggregates and the corresponding micelles were found to be similar. From analysis of the line widths of the EPR spectra, it was concluded that CPSa on silica surfaces forms two coexisting aggregate phases, one which excludes HTAB* and one in which HTAB* is concentrated. The HTAB* is believed to be excluded from the former phase because of ordering of the salicylate counterion and pyridinium headgroups as proposed by Favoriti and Treiner.1 DA - 2001/4// PY - 2001/4// DO - 10.1021/la001517r VL - 17 IS - 8 SP - 2346-2356 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035901799&partnerID=MN8TOARS ER - TY - JOUR TI - Redox Properties of C 6 S 8 n - and C 3 S 5 n - ( n = 0, 1, 2):  Stable Radicals and Unusual Structural Properties for C−S−S−C Bonds AU - Breitzer, Jonathan G. AU - Smirnov, Alex I. AU - Szczepura, Lisa F. AU - Wilson, Scott R. AU - Rauchfuss, Thomas B. T2 - Inorg. Chem. AB - The new anionic carbon sulfides C6S10(2-) and C12S16(2-) are described and crystallographically characterized. The C12S16(2-) anion consists of two C6S8 units connected by an exceptionally long (2.157(12) A) S-S bond. In solution, C12S16(2-) exists in equilibrium with the radical C6S8(-*). The equilibrium constant for radical formation (293 K, THF) is 1.2 x 10(-4) M, as determined by optical spectroscopy at varying concentrations. Radical formation occurs through scission of the S-S bond. On the basis of variable temperature EPR spectra, the thermodynamic parameters of this process are DeltaH = +51.5 +/- 0.5 kJ x mol(-1) and DeltaS = +110 +/- 3 J x mol(-1) x K(-1). C6S10(2-) is an oxidation product of C3S5(2-) and consists of two C3S5 units connected by an S-S bond. The S-S bond length (2.135(4) A) is long, and the CS-SC torsion angle is unusually acute (52.1 degrees ), which is attributed to an attractive interaction between C3S2 rings. The oxidation of (Me4N)2C3S5 occurs at -0.90 V vs Fc+/Fc in MeCN, being further oxidized at -0.22 V. The similarity of the cyclic voltammogram of (Me4N)2C6S10 to that of (Me4N)2C3S5 indicates that C6S10(2-) is the initial oxidation product of C3S5(2-). DA - 2001/3// PY - 2001/3// DO - 10.1021/ic000999r VL - 40 IS - 7 SP - 1421-1429 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035952488&partnerID=MN8TOARS ER - TY - JOUR TI - Information on parasitic gastrointestinal tract infections in cats (Letter to the Editor) AU - Levy, M.G. AU - Gookin, J.L. AU - Poore, M.F. AU - Litaker, R.W. AU - Dykstra, M. T2 - Journal of the American Veterinary Medical Association DA - 2001/// PY - 2001/// VL - 218 IS - 2 SP - 194–195 ER - TY - JOUR TI - Adverse reactions to sulfonamide administration AU - Hopwood, R. AU - Papich, M. AU - Gookin, J.L. T2 - Standards of Care in Emergency and Critical Care Medicine DA - 2001/// PY - 2001/// VL - 3 SP - 5–12 ER - TY - CHAP TI - Surgical Disorders of the Large Intestine AU - Blikslager, A.T. T2 - Large Animal Internal Medicine A2 - Smith, B.P. PY - 2001/// SP - 662–665 PB - Mosby ER - TY - CHAP TI - Surgical Disorders of the Small Intestine AU - Blikslager, A.T. T2 - Large Animal Internal Medicine A2 - Smith, B.P. PY - 2001/// SP - 649–653 PB - Mosby ER - TY - JOUR TI - Use of ambulatory electrocardiography for detection of ventricular premature complexes in healthy dogs AU - Meurs, K.M. AU - Spier, A.W. AU - Wright, N.A. AU - Hamlin, R.A. T2 - Journal of the American Veterinary Medical Association AB - To evaluate the use of 24-hour ambulatory electrocardiography (AECG) for the detection of ventricular premature complexes (VPC) in healthy dogs.Case series.50 healthy mature dogs.A 24-hour AECG was performed on each dog and evaluated for the presence of VPC.Fifty dogs weighing between 18.2 to 40.9 kg (40 and 90 lb) representing 13 breeds were evaluated; there were 4 sexually intact females, 21 spayed females, 4 sexually intact males, and 21 castrated males. Ages ranged from 1 to 12 years. Thirty-four dogs had no VPC; 16 dogs had between 1 and 24 VPC. The grade of arrhythmia ranged from 1 to 4, with 4 dogs having an arrhythmia with a grade > 1. Significant differences were not detected between the group of dogs with VPC and those without VPC with regard to sex, age, and minimum, maximum, or mean heart rate.We conclude that healthy mature dogs have infrequent VPC, as detected by use of 24-hour AECG. The presence of numerous or sequential VPC may be suggestive of cardiac or systemic disease and may indicate the need for thorough clinical evaluation. DA - 2001/4// PY - 2001/4// DO - 10.2460/javma.2001.218.1291 VL - 218 IS - 8 SP - 1291–1295 ER - TY - CHAP TI - Hypertrophic Cardiomyopathy AU - Meurs, K.M. AU - Spier, A.W. T2 - Consultations in Feline Internal Medicine PY - 2001/// VL - IV SP - 56–78 PB - WB Saunders ER - TY - CHAP TI - Inflammatory Disorders of the Central Nervous System AU - Muñana, K.R. T2 - Consultations in Feline Internal Medicine A2 - August, J.R. PY - 2001/// ET - 4th SP - 425–433 PB - WB Saunders ER - TY - JOUR TI - Selection of hairy root clones of Artemisia annua L. for artemisinin production AU - Xie, Deyu AU - Ye, Hechun AU - Li, Guofeng AU - Guo, Zhongchen AU - Zou, Zhuorong AU - Guo, Zhongchen T2 - Israel Journal of Plant Sciences AB - Hairy roots were induced from two kinds of explants and selection of hairy root clones was studied for artemisin in production. Leaf blade pieces and petiole segments of Artemisia annua plantlets were infected with Agrobacterium rhizogenesstrain 1601. The efficiency of leaf blade pieces forming hairy roots was higher than that of petiole segments. Light promoted hairy root induction and branching. Two hundred and twenty hairy root clones showed considerable variations in capacity of growth and branching. Six hairy root clones were established in suspension culture and showed obvious differences in their biomass and artemisin in content. Among six clones, clone 1601-L-3 produced the highest biomass, more than 70 times the inoculum, while clone 1601-L-1 gave the lowest biomass. The artemisin in content of clone 1601-L-1 was the highest, 1.195 mg/g DW, and this line yielded the highest artemisinin level of 9.08 mg/L. DA - 2001/1/1/ PY - 2001/1/1/ DO - 10.1560/n11n-6blg-er7c-xkct VL - 49 IS - 2 SP - 129-134 J2 - Israel Journal of Plant Sciences OP - SN - 0792-9978 UR - http://dx.doi.org/10.1560/n11n-6blg-er7c-xkct DB - Crossref ER - TY - JOUR TI - Regeneration of Acacia mangium through somatic embryogenesis AU - Xie, D. Y. AU - Hong, Y. T2 - Plant Cell Reports DA - 2001/1/15/ PY - 2001/1/15/ DO - 10.1007/s002990000288 VL - 20 IS - 1 SP - 34-40 J2 - Plant Cell Reports OP - SN - 0721-7714 1432-203X UR - http://dx.doi.org/10.1007/s002990000288 DB - Crossref ER - TY - JOUR TI - AU - Xie, Deyu AU - Hong, Yan T2 - Plant Cell, Tissue and Organ Culture DA - 2001/// PY - 2001/// DO - 10.1023/a:1010632619342 VL - 66 IS - 3 SP - 167-173 OP - SN - 0167-6857 UR - http://dx.doi.org/10.1023/a:1010632619342 DB - Crossref KW - micropropagation KW - regeneration KW - thidiazuron ER - TY - JOUR TI - Dioxygen Reactivity of Mononuclear Heme and Copper Components Yielding A High-Spin Heme−Peroxo−Cu Complex AU - Ghiladi, Reza A. AU - Hatwell, Karen R. AU - Karlin, Kenneth D. AU - Huang, Hong-wei AU - Moënne-Loccoz, Pierre AU - Krebs, Carsten AU - Huynh, Boi Hanh AU - Marzilli, Lisa A. AU - Cotter, Robert J. AU - Kaderli, Susan AU - Zuberbühler, Andreas D. T2 - Journal of the American Chemical Society AB - ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTDioxygen Reactivity of Mononuclear Heme and Copper Components Yielding A High-Spin Heme−Peroxo−Cu ComplexReza A. Ghiladi, Karen R. Hatwell, Kenneth D. Karlin, Hong-wei Huang, Pierre Moënne-Loccoz, Carsten Krebs, Boi Hanh Huynh, Lisa A. Marzilli, Robert J. Cotter, Susan Kaderli, and Andreas D. ZuberbühlerView Author Information Department of Chemistry, The Johns Hopkins University Charles and 34th Streets, Baltimore, Maryland 21218 Department of Biochemistry and Molecular Biology Oregon Graduate Institute, Beaverton, Oregon 97006 Department of Physics, Emory University Atlanta, Georgia 30322 Department of Pharmacology and Molecular Sciences The Johns Hopkins School of Medicine Baltimore, Maryland 21205 Institute für Anorganische Chemie, University of Basel CH-4056 Basel, Switzerland Cite this: J. Am. Chem. Soc. 2001, 123, 25, 6183–6184Publication Date (Web):June 1, 2001Publication History Received6 March 2001Published online1 June 2001Published inissue 1 June 2001https://doi.org/10.1021/ja010602yCopyright © 2001 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views749Altmetric-Citations76LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (52 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Oxides,Pyrroles,Quantum mechanics,Resonance structures,Saturation Get e-Alerts DA - 2001/6// PY - 2001/6// DO - 10.1021/ja010602y VL - 123 IS - 25 SP - 6183-6184 J2 - J. Am. Chem. Soc. LA - en OP - SN - 0002-7863 1520-5126 UR - http://dx.doi.org/10.1021/ja010602y DB - Crossref ER - TY - JOUR TI - Epidemiology: How Viruses Spread Among Computers and People AU - Lloyd, A.L. AU - May, R.M. T2 - Science AB - The spread of diseases in human and other populations can be described in terms of networks, where individuals are represented by nodes and modes of contact by edges. Similar models can be applied to the spread of viruses on the Internet. In their Perspective, Lloyd and May discuss the similarities and differences between the dynamics of computer viruses and infections of human and other populations. DA - 2001/5/18/ PY - 2001/5/18/ DO - 10.1126/SCIENCE.1061076 VL - 292 IS - 5520 SP - 1316-1317 SN - 0036-8075 1095-9203 UR - http://dx.doi.org/10.1126/science.1061076 ER - TY - JOUR TI - Correlation of QT dispersion with indices used to evaluate the severity of familial ventricular arrhythmias in Boxers AU - Spier, Alan W. AU - Meurs, Kathryn M. AU - Muir, William W. AU - Lehmkuhl, Linda B. AU - Hamlin, Robert L. T2 - American Journal of Veterinary Research AB - To measure QT interval duration and QT dispersion in Boxers and to determine whether QT variables correlate with indices of disease severity in Boxers with familial ventricular arrhythmias, including the number of ventricular premature complexes per day, arrhythmia grade, and fractional shortening.25 Boxers were evaluated by ECG and echocardiography.The QT interval duration was measured from 12-lead ECG and corrected for heart rate (QTc), using Fridericia's formula. The QT and QTc were calculated for each lead, from which QT and QTc dispersion were determined. Echocardiography and 24-hour ambulatory ECG were performed to evaluate for familial ventricular arrhythmias. Total number of ventricular premature complexes, arrhythmia grade, and fractional shortening were determined and used as indices of disease severity.There was no correlation between any QT variable and total number of ventricular premature complexes, arrhythmia grade, or fractional shortening. No difference between QT dispersion and QTc dispersion was identified, and correction for heart rate did not affect the results.QT interval duration and dispersion did not correlate with indices of disease severity for familial ventricular arrhythmias. Heart rate correction of the QT interval did not appear to be necessary for QT dispersion calculation in this group of dogs. QT dispersion does not appear to be a useful noninvasive diagnostic tool in the evaluation of familial ventricular arrhythmias of Boxers. Identification of affected individuals at risk for sudden death remains a challenge in the management of this disease. DA - 2001/9// PY - 2001/9// DO - 10.2460/ajvr.2001.62.1481 VL - 62 IS - 9 SP - 1481-1485 J2 - American Journal of Veterinary Research LA - en OP - SN - 0002-9645 UR - http://dx.doi.org/10.2460/ajvr.2001.62.1481 DB - Crossref ER - TY - JOUR TI - Comparison of in-hospital versus 24-hour ambulatory electrocardiography for detection of ventricular premature complexes in mature Boxers AU - Meurs, Kathryn M. AU - Spier, Alan W. AU - Wright, Nicola A. AU - Hamlin, Robert L. T2 - Journal of the American Veterinary Medical Association AB - To evaluate the use of in-hospital electrocardiography (ECG) for detection of ventricular premature complexes (VPC), compared with 24-hour ambulatory ECG.Original study.188 Boxers > 9 months old; 31 had a history of syncope, and 157 were healthy (no history of syncope).In-hospital ECG was performed on all Boxers for at least 2 minutes. Within 7 days after the in-hospital ECG was completed, 24-hour ambulatory ECG was performed.The specificity of in-hospital ECG was 100% for the detection of at least 50 VPC in a 24-hour period in dogs with syncope and 93% in healthy dogs. In-hospital ECG had poor sensitivity, although sensitivity increased as the number of VPC per 24 hours increased.Use of in-hospital ECG is highly specific for detection of at least 50 VPC during a 24-hour period. However, in-hospital ECG is insensitive, and a lack of VPC does not suggest that the dog does not have a substantial number of VPC during that same period. The use of in-hospital ECG appears to be inadequate for screening purposes and therapeutic evaluations in mature Boxers with ventricular arrhythmic disease. DA - 2001/1// PY - 2001/1// DO - 10.2460/javma.2001.218.222 VL - 218 IS - 2 SP - 222-224 J2 - Journal of the American Veterinary Medical Association LA - en OP - SN - 0003-1488 UR - http://dx.doi.org/10.2460/javma.2001.218.222 DB - Crossref ER - TY - JOUR TI - Use of western immunoblot for evaluation of myocardial dystrophin, -sarcoglycan, and -dystroglycan in dogs with idiopathic dilated cardiomyopathy AU - Spier, Alan W AU - Meurs, Kathryn M. AU - Coovert, Daniel D AU - Lehmkuhl, Linda B. AU - O'Grady, Michael R. AU - Freeman, Lisa M. AU - Burghes, Arthur H. AU - Towbin, Jeffrey A. T2 - American Journal of Veterinary Research AB - To evaluate the potential importance of dystrophin, alpha-sarcoglycan (adhalin), and beta-dystroglycan, by use of western blot analysis, in several breeds of dogs with dilated cardiomyopathy.Myocardial samples obtained from 12 dogs were evaluated, including tissues from 7 dogs affected with dilated cardiomyopathy, 4 control dogs with no identifiable heart disease (positive control), and 1 dog affected with Duchenne muscular dystrophy (negative control for dystrophin). Of the affected dogs, 4 breeds were represented (Doberman Pinscher, Dalmatian, Bullmastiff, and Irish Wolfhound).Western blot analysis was used for evaluation of myocardial samples obtained from dogs with and without dilated cardiomyopathy for the presence of dystrophin and 2 of its associated glycoproteins, alpha-sarcoglycan and beta-dystroglycan.Detectable differences were not identified between dogs with and without myocardial disease in any of the proteins evaluated.Abnormalities in dystrophin, alpha-sarcoglycan, and beta-dystroglycan proteins were not associated with the development of dilated cardiomyopathy in the dogs evaluated in this study. In humans, the development of molecular biological techniques has allowed for the identification of specific causes of dilated cardiomyopathy that were once considered to be idiopathic. The use of similar techniques in veterinary medicine may aid in the identification of the cause of idiopathic dilated cardiomyopathy in dogs, and may offer new avenues for therapeutic intervention. DA - 2001/1// PY - 2001/1// DO - 10.2460/ajvr.2001.62.67 VL - 62 IS - 1 SP - 67-71 J2 - American Journal of Veterinary Research LA - en OP - SN - 0002-9645 UR - http://dx.doi.org/10.2460/ajvr.2001.62.67 DB - Crossref ER - TY - JOUR TI - Clinical features of dilated cardiomyopathy in Great Danes and results of a pedigree analysis: 17 cases (1990-2000) AU - Meurs, Kathryn M. AU - Miller, Matthew W. AU - Wright, Nicola A. T2 - Journal of the American Veterinary Medical Association AB - To determine clinical features of dilated cardiomyopathy (DCM) in Great Danes and to determine whether DCM is familial in this breed.Retrospective study.17 Great Danes with DCM.Medical records of Great Danes in which DCM was diagnosed on the basis of results of echocardiography (fractional shortening < 25%, end-systolic volume index > 30 ml/m2 of body surface area) were reviewed. Pedigrees were obtained for affected animals, as well as for other Great Danes in which DCM had been diagnosed.Dilated cardiomyopathy appeared to be familial and was characterized by ventricular dilatation, congestive heart failure (left-sided or biventricular), and atrial fibrillation. Pedigree analysis suggested that DCM was inherited as an X-linked recessive trait, but the mode of inheritance could not be definitively identified.Results suggest that DCM may be an X-linked recessive trait in Great Danes. Thus, dogs with DCM probably should not be used for breeding, and female offspring of affected dogs should be used cautiously. Male offspring of affected females are at an increased risk of developing DCM and should be evaluated periodically for early signs of disease. Results of pedigree analysis were preliminary and should be used only as a guide for counseling breeders, rather than as a basis for making breeding decisions. DA - 2001/3// PY - 2001/3// DO - 10.2460/javma.2001.218.729 VL - 218 IS - 5 SP - 729-732 J2 - Journal of the American Veterinary Medical Association LA - en OP - SN - 0003-1488 UR - http://dx.doi.org/10.2460/javma.2001.218.729 DB - Crossref ER - TY - JOUR TI - Evaluation of the cardiac actin gene in Doberman Pinschers with dilated cardiomyopathy AU - Meurs, Kathryn M. AU - Magnon, Alex L. AU - Spier, Alan W. AU - Miller, Mathew W. AU - Lehmkuhl, Linda B. AU - Towbin, Jeffrey A. T2 - American Journal of Veterinary Research AB - To evaluate the coding region of the cardiac actin gene in Doberman Pinschers with dilated cardiomyopathy (DCM) for mutations that could be responsible for the development of the condition28 dogs (16 Doberman Pinschers with DCM and 12 mixed-breed control dogs).Ten milliliters of blood was collected from each dog for DNA extraction. Polymerase chain reaction (PCR) primers were designed to amplify canine exonic regions, using the sequences of exons 2 to 6 of the cardiac actin gene. Single-stranded conformational polymorphism analysis was performed for each exon with all samples. Autoradiographs were analyzed for banding patterns specific to affected dogs. The DNA sequencing was performed on a selected group of affected and control dogs.Molecular analysis of exons 2 to 6 of the cardiac actin gene did not reveal any differences in base pairs between affected dogs and control dogs selected for DNA evaluation.Mutations in exons 5 and 6 of the cardiac actin gene that have been reported in humans with familial DCM do not appear to be the cause of familial DCM in Doberman Pinschers. Additionally, evaluation of exons 2 to 6 for causative mutations did not reveal a cause for inherited DCM in these Doberman Pinschers. Although there is evidence that DCM in Doberman Pinschers is an inherited problem, a molecular basis for this condition remains unresolved. Evaluation of other genes coding for cytoskeletal proteins is warranted. DA - 2001/1// PY - 2001/1// DO - 10.2460/ajvr.2001.62.33 VL - 62 IS - 1 SP - 33-36 J2 - American Journal of Veterinary Research LA - en OP - SN - 0002-9645 UR - http://dx.doi.org/10.2460/ajvr.2001.62.33 DB - Crossref ER - TY - JOUR TI - Interplay of intrinsic and environmental effects on the magnetic properties of free radicals issuing from H-atom addition to cytosine. AU - Adamo, C AU - Heitzmann, M AU - Meilleur, F AU - Rega, N AU - Scalmani, G AU - Grand, A AU - Cadet, J AU - Barone, V T2 - Journal of the American Chemical Society AB - Possible radical reaction products issuing from H-atom addition to cytosine have been characterized and analyzed by means of a comprehensive quantum mechanical approach including density functional computations (B3LYP), together with simulation of the solvent by the polarizable continuum model (PCM), and averaging of spectroscopic properties over the most important vibrational motions. The hyperfine couplings of the semirigid 5,6-dihydrocytos-6yl radical computed at the optimized geometry are in good agreement with their experimental counterparts. On the other hand, vibrational averaging is mandatory for obtaining an effectively planar structure for the 5,6-dihydrocytos-5yl radical with the consequent equivalence of β-hydrogens. Finally, only proper consideration of environmental effects restores the agreement between computed and experimental couplings for the base anion protonated at N3. DA - 2001/7// PY - 2001/7// DO - 10.1021/ja004284z VL - 123 IS - 29 SP - 7113–7117, UR - http://europepmc.org/abstract/med/11459491 ER - TY - JOUR TI - The 14th annual international mammalian genome society conference: A glimpse into the future of murine functional genomics AU - Strunk, Karen E. AU - Roberts, Reade Bruce T2 - genesis AB - genesisVolume 29, Issue 4 p. 153-155 Graduate Student Commentary The 14th annual international mammalian genome society conference: A glimpse into the future of murine functional genomics † Karen E. Strunk, Karen E. Strunk Department of Genetics, University of North Carolina-Chapel HillSearch for more papers by this authorReade Bruce Roberts, Corresponding Author Reade Bruce Roberts reader@med.unc.edu Department of Genetics, University of North Carolina-Chapel HillDepartment of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7264Search for more papers by this author Karen E. Strunk, Karen E. Strunk Department of Genetics, University of North Carolina-Chapel HillSearch for more papers by this authorReade Bruce Roberts, Corresponding Author Reade Bruce Roberts reader@med.unc.edu Department of Genetics, University of North Carolina-Chapel HillDepartment of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7264Search for more papers by this author First published: 10 April 2001 https://doi.org/10.1002/gene.1018Citations: 1 † Editor's note: We encourage graduate students to submit commentaries of interest to Terry Magnuson. The commentaries will be reviewed by the editors and outside referees consisting of two graduate students. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume29, Issue4April 2001Pages 153-155 RelatedInformation DA - 2001/// PY - 2001/// DO - 10.1002/gene.1018 VL - 29 IS - 4 SP - 153-155 J2 - genesis LA - en OP - SN - 1526-954X 1526-968X UR - http://dx.doi.org/10.1002/gene.1018 DB - Crossref ER - TY - JOUR TI - Epidemiological and ecological characteristics of past dengue virus infection in Santa Clara, Peru AU - Reiskind, Michael H. AU - Baisley, Kathy J. AU - Calampa, Carlos AU - Sharp, Trueman W. AU - Watts, Douglas M. AU - Wilson, Mark L. T2 - Tropical Medicine and International Health AB - To determine risk factors associated with dengue (DEN) virus infection among residents of Santa Clara, Peru, a rural Amazonian village near Iquitos, a cross‐sectional serological, epidemiological and environmental survey was conducted. Demographic, social and behavioural information was obtained by standardized questionnaire from 1225 Santa Clara residents (61.3%) aged 5 years or older. Additional data were obtained on the environmental variables and immature mosquito species and abundance surrounding each household ( n =248). Sera that had been collected previously by the Peruvian Ministry of Health from residents were tested by an enzyme‐linked immunosorbent assay (ELISA) for DEN virus IgG antibody. Antibody identity was verified as DEN by plaque reduction neutralization test. Data on individuals were analysed by univariate and multivariable methods, and independent sample t ‐tests. Spatial clustering was evaluated by comparing distances among DEN positive households. Overall, antibody prevalence was 29.4% and more than doubled from the youngest to the oldest age groups, but did not differ by sex. Curiously, length of residence in Santa Clara was negatively associated with DEN virus antibodies. More frequent travel to Iquitos was positively associated with seroprevalence. Residents who obtained water from a river source rather than a local well also had significantly higher antibody prevalence. None of the environmental variables measured at each household corresponded to the patterns of antibody distribution. Of the larval mosquitoes found around residences, all were determined to be species other than Aedes . No evidence of spatial autocorrelation among antibody‐positive households was detected. These results strongly suggested that recent DEN virus transmission did not occur in the village and that most infections of residents of this rural village were acquired while visiting the city of Iquitos. DA - 2001/3// PY - 2001/3// DO - 10.1046/j.1365-3156.2001.00703.x VL - 6 IS - 3 SP - 212-218 J2 - Trop Med Int Health LA - en OP - SN - 1360-2276 1365-3156 UR - http://dx.doi.org/10.1046/j.1365-3156.2001.00703.x DB - Crossref KW - dengue fever KW - virus antibody serology KW - Aedes aegypti KW - spatial pattern KW - vector ecology KW - Peru ER - TY - JOUR TI - Liquid diets accelerate the growth of early-weaned pigs and the effects are maintained to market weight AU - Kim, J.H. AU - Heo, K.N. AU - Odle, J. AU - Han, I.K. AU - Harrell, R.J. T2 - Journal of Animal Science DA - 2001/// PY - 2001/// VL - 79 IS - 2 SP - 427-434 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035256719&partnerID=MN8TOARS ER - TY - JOUR TI - Differential induction of peroxisomal β-oxidation enzymes by clofibric acid and aspirin in piglet tissues AU - Yu, Xing Xian AU - Odle, J. AU - Drackley, J.K. T2 - American Journal of Physiology - Regulatory Integrative and Comparative Physiology DA - 2001/// PY - 2001/// VL - 281 IS - 5 50-5 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035195482&partnerID=MN8TOARS ER - TY - JOUR TI - Determination of Carnitine Renal Threshold and Effect of Medium-Chain Triglycerides on Carnitine Profiles in Newborn Pigs AU - Heo, K.N. AU - Odle, J. AU - Lin, X. AU - Van Kempen, T.A.T.G. AU - Han, I.K. T2 - Asian-Australasian Journal of Animal Sciences DA - 2001/// PY - 2001/// VL - 14 IS - 2 SP - 237-242 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0347670167&partnerID=MN8TOARS ER - TY - JOUR TI - A mechanism for combinatorial regulation of electrical activity: Potassium channel subunits capable of functioning as Src homology 3-dependent adaptors AU - Nitabach, M. N. AU - Llamas, D. A. AU - Araneda, R. C. AU - Intile, J. L. AU - Thompson, I. J. AU - Zhou, Y. I. AU - Holmes, T. C. T2 - Proceedings of the National Academy of Sciences DA - 2001/1/16/ PY - 2001/1/16/ DO - 10.1073/pnas.031446198 VL - 98 IS - 2 SP - 705-710 J2 - Proceedings of the National Academy of Sciences LA - en OP - SN - 0027-8424 1091-6490 UR - http://dx.doi.org/10.1073/pnas.98.2.705 DB - Crossref ER - TY - JOUR TI - Genome Sequence of the Plant Pathogen and Biotechnology Agent Agrobacterium tumefaciens C58 AU - Goodner, B. AU - Hinkle, G. AU - Gattung, S. AU - Miller, N. AU - Blanchard, M. AU - Qurollo, B. AU - Goldman, B.S. AU - Cao, Y. AU - Askenazi, M. AU - Halling, C. AU - Mullin, L. AU - Houmiel, K. AU - Gordon, J. AU - Vaudin, M. AU - Iartchouk, O. AU - Epp, A. AU - Liu, F. AU - Wollam, C. AU - Allinger, M. AU - Doughty, D. AU - Scott, C. AU - Lappas, C. AU - Markelz, B. AU - Flanagan, C. AU - Crowell, C. AU - Gurson, J. AU - Lomo, C. AU - Sear, C. AU - Strub, G. AU - Cielo, C. AU - Slater, S. T2 - Science AB - Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants. DA - 2001/12/14/ PY - 2001/12/14/ DO - 10.1126/science.1066803 VL - 294 IS - 5550 SP - 2323-2328 SN - 0036-8075 1095-9203 UR - http://dx.doi.org/10.1126/science.1066803 ER - TY - CONF TI - Preliminary report: Comparison of 980-nm, 808-nm diode laser enhanced with Indocyanine green to the Nd: YAG laser applied to equine respiratory tissue AU - L.P., Tate, Jr. AU - Blikslager, A.T. AU - Campbell, N.B. AB - The Neodynium: Yttrium Aluminum Garnet (Nd:YAG) laser has been the mainstay of performing upper respiratory laser surgery in the equine since 1984. The 808-nm diode laser has also been applied transendoscopically as well as the 980-nm diode laser over recent years. It has been shown that Indocyanine Green (ICG) enhances the performance of the 808- nm laser because it is absorbed at 810 nm of light. The 808- nm laser's tissue interaction combined with ICG is equivalent to or greater than the Nd:YAG laser's cutting ability. The 980-nm diode laser performance was similar to that of the Nd:YAG as determined by the parameters of this study. This study compared the depths and widths of penetration achieved with the 808-nm diode laser after intravenous injection of ICG on equine respiratory tissue. It also compared depths and widths of penetration achieved by the non-contact application of the 980-nm diode laser delivering the same energy of 200 joules. The depths and widths of penetration of both diode lasers were compared to themselves and to the Nd:YAG laser with all factors remaining constant. C2 - 2001/// C3 - Proceedings of SPIE - The International Society for Optical Engineering DA - 2001/// DO - 10.1117/12.427772 VL - 4244 SP - 583-590 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0034935283&partnerID=MN8TOARS KW - chromophore KW - indocyanine green KW - diode laser KW - equine KW - Nd : YAG laser ER - TY - JOUR TI - Neodymium:Yttrium-aluminum-garnet laser ablation of a urethral web to relieve urinary outflow obstruction in a horse T2 - Journal of the American Veterinary Medical Association DA - 2001/// PY - 2001/// VL - 218 IS - 12 SP - 1970-1972 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035877844&partnerID=MN8TOARS ER - TY - JOUR TI - The future of antiinflammatory therapy. AU - Jones, SL AU - Blikslager, A T2 - The Veterinary clinics of North America. Equine practice DA - 2001/// PY - 2001/// VL - 17 IS - 2 SP - 245–62, UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-1942520108&partnerID=MN8TOARS ER - TY - JOUR TI - Deep digital flexor tenotomy for treatment of severe laminitis in a cow AU - Gayle, J.M. AU - Burrell, G.A. AU - Anderson, K.L. AU - Rich Redding, W. AU - Blikslager, A.T. T2 - Journal of the American Veterinary Medical Association DA - 2001/// PY - 2001/// VL - 219 IS - 5 SP - 644-646+613 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0040621851&partnerID=MN8TOARS ER - TY - JOUR TI - The ACVD task force on canine atopic dermatitis: Forewords and lexicon AU - Olivry, T. AU - DeBoer, D.J. AU - Griffin, C.E. AU - Halliwell, R.E.W. AU - Hill, P.B. AU - Hillier, A. AU - Marsella, R. AU - Sousa, C.A. T2 - Veterinary Immunology and Immunopathology DA - 2001/// PY - 2001/// DO - 10.1016/S0165-2427(01)00343-9 VL - 81 IS - 3-4 SP - 143-146 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035922087&partnerID=MN8TOARS ER - TY - JOUR TI - Erratum: A spontaneous canine model of mucous membrane (Cicatricial) pemphigold, an autoimmune blistering disease affecting mucosae and mucocutaneous junctions' (Journal of Autoimmunity 16 (411-421)) AU - Olivry, T. T2 - Journal of Autoimmunity DA - 2001/// PY - 2001/// DO - 10.1006/jaut.2001.0536 VL - 17 IS - 2 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035445848&partnerID=MN8TOARS ER - TY - JOUR TI - Nouvelles dermatoses auto-immunes du chien et du chat 1re partie : Maladies ayant pour cible le follicule pileux et les kératinocytes basaux AU - Rivierre, Ch. AU - Olivry, Th. T2 - Pratique Medicale et Chirurgicale de l'Animal de Compagnie DA - 2001/// PY - 2001/// VL - 36 IS - 6 SP - 635-645 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035667903&partnerID=MN8TOARS ER - TY - JOUR TI - Cross-species and interassay comparisons of phytoestrogen action. AU - Whitten, P L AU - Patisaul, H B T2 - Environmental Health Perspectives AB - This paper compiles animal and human data on the biologic effects and exposure levels of phytoestrogens in order to identify areas of research in which direct species comparisons can be made. In vitro and in vivo assays of phytoestrogen action and potency are reviewed and compared to actions, dose-response relationships, and estimates of exposure in human subjects. Binding studies show that the isoflavonoid phytoestrogens are high-affinity ligands for estrogen receptors (ERs), especially ER beta, but have lower potency in whole-cell assays, perhaps because of interactions with binding proteins. Many other enzymatic actions require concentrations higher than those normally seen in plasma. In vivo data show that phytoestrogens have a wide range of biologic effects at doses and plasma concentrations seen with normal human diets. Significant in vivoresponses have been observed in animal and human tests for bone, breast, ovary, pituitary, vasculature, prostate, and serum lipids. The doses reported to be biologically active in humans (0.4--10 mg/kg body weight/day) are lower than the doses generally reported to be active in rodents (10--100 mg/kg body weight/day), although some studies have reported rodent responses at lower doses. However, available estimates of bioavailability and peak plasma levels in rodents and humans are more similar. Steroidogenesis and the hypothalamic-pituitary-gonadal axis appear to be important loci of phytoestrogen actions, but these inferences must be tentative because good dose-response data are not available for many end points. The similarity of reported proliferative and antiproliferative doses illustrates the need for fuller examination of dose-response relationships and multiple end points in assessing phytoestrogen actions. DA - 2001/3// PY - 2001/3// DO - 10.1289/ehp.01109s15 VL - 109 IS - suppl 1 SP - 5-20 J2 - Environmental Health Perspectives LA - en OP - SN - 0091-6765 1552-9924 UR - http://dx.doi.org/10.1289/ehp.01109s15 DB - Crossref ER - TY - JOUR TI - Coumestrol Antagonizes Neuroendocrine Actions of Estrogen via the Estrogen Receptor α AU - Jacob, Dena A. AU - Temple, Jennifer L. AU - Patisaul, Heather B. AU - Young, Larry J. AU - Rissman, Emilie F. T2 - Experimental Biology and Medicine AB - The phytoestrogen coumestrol has estrogenic actions on peripheral reproductive tissues. Yet in the brain this compound has both estrogenic and anti-estrogenic effects. We used estrogen receptor alpha knockout mice (ERalphaKO) to determine whether coumestrol has estrogenic actions in mice and also if these effects are mediated by the classic ERalpha. Female wild-type (WT) and ERalphaKO mice were ovariectomized and treated with estradiol (E2), dietary coumestrol, both, or neither compound. Ten days later the animals were sacrificed, blood was collected, and brain tissues were perfused. Fixed brains were sectioned and immunocytochemistry was employed to quantify progesterone receptors (PR) in the medial preoptic (POA) and ventromedial nucleus of the hypothalamus (VMN). Plasma was assayed for luteinizing hormone (LH). Estrogen treatment induced PR immunoreactivity in both regions in brains of WT females. In ERalphaKO mice, lower levels of PR were induced. The stimulatory effects of E2 on PR were attenuated in the POA by cotreatment with coumestrol, and the same trend was noted in the VMN. WT ovariectomized females treated with E2 had low levels of LH, while LH was high in untreated females and even higher in ovariectomized females treated with coumestrol. ERalphaKO females in all treatment groups had high levels of LH. Taken together, the results show that coumestrol has anti-estrogenic actions in the brain and pituitary and that ERalpha mediates these effects. DA - 2001/4// PY - 2001/4// DO - 10.1177/153537020122600406 VL - 226 IS - 4 SP - 301-306 J2 - Exp Biol Med (Maywood) LA - en OP - SN - 1535-3702 1535-3699 UR - http://dx.doi.org/10.1177/153537020122600406 DB - Crossref KW - phytoestrogen KW - progesterone receptor KW - luteinizing hormone KW - nutrition KW - estrogen receptor beta ER - TY - JOUR TI - Soy Isoflavone Supplements Antagonize Reproductive Behavior and Estrogen Receptor α- and β-Dependent Gene Expression in the Brain* AU - Patisaul, Heather B. AU - Dindo, Marietta AU - Whitten, Patricia L. AU - Young, Larry J. T2 - Endocrinology AB - Epidemiological evidence suggests that isoflavone phytoestrogens may reduce the risk of cancer, osteoporosis, and heart disease, effects at least partially mediated by estrogen receptors alpha and beta (ERalpha and ERbeta). Because isoflavone dietary supplements are becoming increasingly popular and are frequently advertised as natural alternatives to estrogen replacement therapy, we have examined the effects of one of these supplements on estrogen-dependent behavior and ERalpha- and ERbeta-dependent gene expression in the brain. In the adult female rat brain, 17beta-estradiol treatment decreased ERbeta messenger RNA signal in the paraventricular nucleus by 41%, but supplement treatment resulted in a 27% increase. The regulation of ERbeta in the paraventricular nucleus is probably via an ERbeta-dependent mechanism. Similarly, in the ventromedial nucleus of the hypothalamus, supplement treatment diminished the estrogen-dependent up-regulation of oxytocin receptor by 10.5%. The regulation of oxytocin receptor expression in the ventromedial nucleus of the hypothalamus is via an ERalpha-dependent mechanism. Supplement treatment also resulted in a significant decrease in receptive behavior in estrogen- and progesterone-primed females. The observed disruption of sexual receptivity by the isoflavone supplement is probably due to antiestrogenic effects observed in the brain. These results suggest that isoflavone phytoestrogens are antiestrogenic on both ERalpha- and ERbeta-dependent gene expression in the brain and estrogen-dependent behavior. DA - 2001/7/1/ PY - 2001/7/1/ DO - 10.1210/endo.142.7.8241 VL - 142 IS - 7 SP - 2946-2952 LA - en OP - SN - 0013-7227 1945-7170 UR - http://dx.doi.org/10.1210/endo.142.7.8241 DB - Crossref ER - TY - JOUR TI - Atypical Reaction to Acetaminophen Intoxication in a Dog AU - Mariani, Christopher L. AU - Fulton, Robert B. T2 - Journal of Veterinary Emergency and Critical Care AB - Abstract Objective: To report an unusual manifestation of acetaminophen intoxication in a dog. Case Summary: A Miniature Pinscher was presented for evaluation of lethargy and facial swelling after ingestion of 500–750 mg/kg of acetaminophen. Laboratory testing revealed eccentrocytes, Heinz bodies, hemoglobinemia, and a declining packed red cell volume consistent with oxidative damage to the erythrocytes. Hepatocellular damage, as monitored by serum alanine aminotransferase, was not documented. The dog responded well to therapeutic intervention, and was discharged from the hospital. The dog returned to the hospital 2 days later with clinical signs and diagnostic tests consistent with acute keratoconjunctivitis sicca, which was assumed to be due to a toxic etiology. The dog responded well to therapy, regaining normal levels of tear production within six weeks. New or Unique Information Provided: Acetaminophen intoxication may be associated with severe erythrocytic oxidative damage in the absence of detectable hepatic injury in dogs. Acetaminophen intoxication may have the potential to cause acute keratoconjunctivitis sicca in dogs. ( J Vet Emerg Crit Care 2001; 11(2): 123–126 ) DA - 2001/6// PY - 2001/6// DO - 10.1111/j.1476-4431.2001.tb00078.x VL - 11 IS - 2 SP - 123-126 J2 - J Veter Emer Crit LA - en OP - SN - 1479-3261 1476-4431 UR - http://dx.doi.org/10.1111/j.1476-4431.2001.tb00078.x DB - Crossref ER - TY - JOUR TI - The dependence of viral parameter estimates on the assumed viral life cycle: limitations of studies of viral load data AU - Lloyd, A. L. T2 - Proceedings of the Royal Society of London. Series B: Biological Sciences AB - Estimation of viral parameters, such as the basic reproductive number (R0) and infected cell life span, is central to the quantitative study of the within-host dynamics of viral diseases such as human immunodeficiency virus, hepatitis B or hepatitis C. As these parameters can rarely be determined directly, they are usually estimated indirectly by fitting mathematical models to viral load data. This paper investigates how parameter estimates obtained by such procedures depend on the assumptions made concerning the viral life cycle. It finds that estimates of the basic reproductive number obtained using viral load data collected during the initial stages of infection can depend quite sensitively on these assumptions. The use of models which neglect the intracellular delay before virion production can lead to severe underestimates of R0 and, hence, to overly optimistic predictions of how efficacious treatment must be in order to prevent or eradicate the disease. These results are also of importance for attempts at estimating R0 from similar epidemiological data as there is a correspondence between within-host and between-host models. Estimates of the life span of infected cells obtained from viral load data collected during drug treatment studies also depend on the assumptions made in modelling the virus life cycle. The use of more realistic descriptions of the life cycle is seen to increase estimates of infected cell life span, in addition to providing a new explanation for the shoulder phase seen during drug treatment. This study highlights the limitations of what can be learnt by fitting mathematical models to infectious disease data without detailed independent knowledge of the life cycle of the infectious agent. DA - 2001/4/22/ PY - 2001/4/22/ DO - 10.1098/rspb.2000.1572 VL - 268 IS - 1469 SP - 847-854 J2 - Proceedings of the Royal Society of London. Series B: Biological Sciences LA - en OP - SN - 1471-2954 UR - http://dx.doi.org/10.1098/rspb.2000.1572 DB - Crossref KW - virus dynamics KW - parameter estimation KW - basic reproductive number KW - cell life span KW - non-exponential distribution ER - TY - JOUR TI - Destabilization of epidemic models with the inclusion of realistic distributions of infectious periods AU - Lloyd, A. L. T2 - Proceedings of the Royal Society of London. Series B: Biological Sciences AB - Most mathematical models used to understand the dynamical patterns seen in the incidence of childhood viral diseases, such as measles, employ a simple, but epidemiologically unrealistic, description of the infection and recovery process. The inclusion of more realistic descriptions of the recovery process is shown to cause a significant destabilization of the model. When there is seasonal variation in disease transmission this destabilization leads to the appearance of complex dynamical patterns with much lower levels of seasonality than previously predicted. More generally, this study illustrates how detailed dynamical properties of a model may depend in an important way on the assumptions made in the formulation of the model. DA - 2001/5/7/ PY - 2001/5/7/ DO - 10.1098/rspb.2001.1599 VL - 268 IS - 1470 SP - 985-993 J2 - Proceedings of the Royal Society of London. Series B: Biological Sciences LA - en OP - SN - 1471-2954 UR - http://dx.doi.org/10.1098/rspb.2001.1599 DB - Crossref KW - epidemic model KW - infectious period distribution KW - seasonally forced dynamics ER - TY - JOUR TI - Realistic Distributions of Infectious Periods in Epidemic Models: Changing Patterns of Persistence and Dynamics AU - Lloyd, Alun L. T2 - Theoretical Population Biology AB - Most mathematical models used to study the epidemiology of childhood viral diseases, such as measles, describe the period of infectiousness by an exponential distribution. The effects of including more realistic descriptions of the infectious period within SIR (susceptible/infectious/recovered) models are studied. Less dispersed distributions are seen to have two important epidemiological consequences. First, less stable behaviour is seen within the model: incidence patterns become more complex. Second, disease persistence is diminished: in models with a finite population, the minimum population size needed to allow disease persistence increases. The assumption made concerning the infectious period distribution is of a kind routinely made in the formulation of mathematical models in population biology. Since it has a major effect on the central issues of population persistence and dynamics, the results of this study have broad implications for mathematical modellers of a wide range of biological systems. DA - 2001/8// PY - 2001/8// DO - 10.1006/tpbi.2001.1525 VL - 60 IS - 1 SP - 59-71 J2 - Theoretical Population Biology LA - en OP - SN - 0040-5809 UR - http://dx.doi.org/10.1006/tpbi.2001.1525 DB - Crossref ER - TY - JOUR TI - Infection dynamics on scale-free networks AU - May, Robert M. AU - Lloyd, Alun L. T2 - Physical Review E AB - We discuss properties of infection processes on scale-free networks, relating them to the node-connectivity distribution that characterizes the network. Considering the epidemiologically important case of a disease that confers permanent immunity upon recovery, we derive analytic expressions for the final size of an epidemic in an infinite closed population and for the dependence of infection probability on an individual's degree of connectivity within the population. As in an earlier study [R. Pastor-Satorras and A. Vesipignani, Phys. Rev. Lett. 86, 3200 (2001); Phys. Rev. E. 63, 006117 (2001)] for an infection that did not confer immunity upon recovery, the epidemic process--in contrast with many traditional epidemiological models--does not exhibit threshold behavior, and we demonstrate that this is a consequence of the extreme heterogeneity in the connectivity distribution of a scale-free network. Finally, we discuss effects that arise from finite population sizes, showing that networks of finite size do exhibit threshold effects: infections cannot spread for arbitrarily low transmission probabilities. DA - 2001/11/19/ PY - 2001/11/19/ DO - 10.1103/physreve.64.066112 VL - 64 IS - 6 SP - 066112 J2 - Phys. Rev. E LA - en OP - SN - 1063-651X 1095-3787 UR - http://dx.doi.org/10.1103/physreve.64.066112 DB - Crossref ER - TY - JOUR TI - Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability AU - Qurollo, Barbara A. AU - Bishop, Paul E. AU - Hassan, Hosni M. T2 - Canadian Journal of Microbiology DA - 2001/// PY - 2001/// DO - 10.1139/cjm-47-1-63 VL - 47 IS - 1 SP - 63-71 J2 - Rev. can. microbiol. OP - SN - 1480-3275 0008-4166 UR - http://dx.doi.org/10.1139/cjm-47-1-63 DB - Crossref ER - TY - JOUR TI - Sodium and Chloride Requirements of Growing Broiler Chickens (Twenty-one to Forty-two Days of Age) Fed Corn-Soybean Diets AU - Murakami, A. E. AU - Oviedo-Rondon, E. O. AU - Martins, E. N. AU - Pereira, M. S. AU - Scapinello, C. T2 - Poultry Science AB - Two trials were conducted to determine Na+ and Cl- nutritional requirements and dietary electrolyte balance (DEB) and its effects on acid-base balance, litter moisture, and incidence of tibial dyschondroplasia (TD) in broiler chickens during the growing period. Cobb broilers were distributed in a completely randomized design (30 pens) with six treatments, five replicates, and 50 birds per experimental unit at 21 d of age. Treatments used in both trials were a basal diet with 0.10% Na+ (Trial 1) or Cl- (Trial 2) supplemented to result in diets with Na+ or Cl- levels of 0.10, 0.15, 0.20, 0.25, 0.30, and 0.35%. In the first trial, the results indicated an optimum Na+ requirement of 0.15%. The Na+ levels, obtained with supplemental NaHCO3, did not affect blood gas parameters and TD incidence. Litter moisture increased linearly with Na+ levels. In the second trial, the Cl- requirement was estimated at 0.23%. Increasing Cl- levels, provided by NaCl with NaHCO3 to balance Na+, caused a linear effect (P < or = 0.01) on blood gas parameters, with an estimated equilibrium at 0.19% dietary Cl-. No effect (P > or = 0.05) of Cl- levels on litter moisture was observed. The hypertrophic area of growth plate in the proximal tibiotarsus increased with Cl- levels (P < or = 0.001). A nonlinear model describes this response. The best dietary electrolyte balance (DEB) was between 250 to 261 mEq/kg in Trial 1 and 249 to 257 mEq/kg in Trial 2. We concluded that the Na+ requirement was 0.15%, and the Cl- requirement was 0.23% for maximum performance of growing chickens between 21 and 42 d of age, and the best DEB was between 249 and 261 mEq/kg. DA - 2001/3/1/ PY - 2001/3/1/ DO - 10.1093/ps/80.3.289 VL - 80 IS - 3 SP - 289-294 J2 - Poultry Science LA - en OP - SN - 0032-5791 1525-3171 UR - http://dx.doi.org/10.1093/ps/80.3.289 DB - Crossref KW - acid-base balance KW - chloride requirements KW - sodium KW - tibial dyschondroplasia ER - TY - JOUR TI - Sodium and Chloride Requirements of Young Broiler Chickens Fed Corn-Soybean Diets (One to Twenty-One Days of Age) AU - Oviedo-Rondon, E. O. AU - Murakami, A. E. AU - Furlan, A. C. AU - Moreira, I. AU - Macari, M. T2 - Poultry Science AB - Sodium (Na+) and chloride (Cl-) nutritional requirements, dietary electrolyte balance (DEB), and their effects on acid-base balance, litter moisture, and tibial dyschondroplasia (TD) incidence for young broiler chickens were evaluated in two trials. One-day-old Cobb broilers were distributed in a completely randomized design with six treatments, five replicates, and 50 birds per experimental unit. Treatments used in both experiments were a basal diet with 0.10% Na+ (Experiment 1) or Cl- (Experiment 2) supplemented to result in diets with Na+ or Cl- levels of 0.10, 0.15, 0.20, 0.25, 0.30, or 0.35%, respectively. In Experiment 1, results indicated an optimum Na+ requirement of 0.26%. Sodium levels caused a linear increase in arterial blood gas parameters, indicating an alkalogenic effect of Na+. The hypertrophic area of growth plate in the proximal tibiotarsi decreased with Na+ levels. The TD incidence decreased with increases in dietary Na+. Litter moisture increased linearly with sodium levels. In Experiment 2, the Cl- requirement was estimated as 0.25%. Chloride levels caused a quadratic effect (P < or = 0.01) on blood gas parameters, with an estimated equilibrium [blood base excess (BE) = 0] at 0.30% of dietary Cl-. No Cl- treatment effects (P > or = 0.05) were observed on litter moisture or TD incidence. The best DEB for maximum performance was 298 to 315 mEq/kg in Experiment 1 and 246 to 264 mEq/kg in Experiment 2. We concluded that the Na+ and Cl- requirements for optimum performance of young broiler chickens were 0.28 and 0.25%, respectively. DA - 2001/5/1/ PY - 2001/5/1/ DO - 10.1093/ps/80.5.592 VL - 80 IS - 5 SP - 592-598 J2 - Poultry Science LA - en OP - SN - 0032-5791 1525-3171 UR - http://dx.doi.org/10.1093/ps/80.5.592 DB - Crossref KW - acid-base balance KW - chloride KW - sodium KW - tibial dyschondroplasia ER - TY - JOUR TI - Corrigendum to: “The zebrafish fth1, slc3a2, men1, pc, fgf3 and cycd1 genes define two regions of conserved synteny between linkage group 7 and human chromosome 11q13” [Gene 261 (2000) 235–242] AU - Yoder, Jeffrey A AU - Litman, Gary W T2 - Gene DA - 2001/5// PY - 2001/5// DO - 10.1016/s0378-1119(01)00443-7 VL - 269 IS - 1-2 SP - 227 ER - TY - JOUR TI - Preemptive analgesia: managing pain before it begins AU - Shafford, H.L. AU - Lascelles, B.D.X. AU - Hellyer, P.W. T2 - Veterinary Medicine DA - 2001/6// PY - 2001/6// SP - 478-492 ER - TY - CHAP TI - Drugs used in the treatment of the musculoskeletal system and joints AU - Lascelles, B.D.X. T2 - The Veterinary Formulary A2 - Bishop, Y. PY - 2001/// ET - 5th SP - 469-488 PB - Pharmaceutical Press ER - TY - JOUR TI - Surgical reconstruction of ectrodactyly deformity in four dogs AU - Innes, JF AU - McKee, WM AU - Mitchell, RAS AU - Lascelles, BDX AU - Johnson, KA T2 - Veterinary and Comparative Orthopaedics and Traumatology DA - 2001/// PY - 2001/// VL - 14 IS - 04 SP - 201-209 ER - TY - JOUR TI - Morphine, pethidine and buprenorphine disposition in the cat AU - Taylor, PM AU - Robertson, SA AU - Dixon, MJ AU - Ruprah, M AU - Sear, JW AU - Lascelles, BDX AU - Waters, C AU - Bloomfield, M T2 - Journal of veterinary pharmacology and therapeutics DA - 2001/// PY - 2001/// VL - 24 IS - 6 SP - 391-398 ER - TY - JOUR TI - Evaluation of the clinical efficacy of meloxicam in cats with painful locomotor disorders AU - Lascelles, BDX AU - Henderson, AJ AU - Hackett, IJ T2 - Journal of Small Animal Practice DA - 2001/// PY - 2001/// VL - 42 IS - 12 SP - 587-593 ER - TY - JOUR TI - Combined omental pedicle grafts and thoracodorsal axial pattern flaps for the reconstruction of chronic, nonhealing axillary wounds in cats AU - Lascelles, B. Duncan X. AU - White, Richard A. S. T2 - Veterinary Surgery AB - To assess the results of an omental pedicle graft in combination with a thoracodorsal axial pattern flap for the reconstruction of chronic nonhealing axillary wounds in 10 cats caused by forelimb entrapment within a collar.A prospective, clinical trial. ANIMALS USED: Ten client-owned domestic shorthair cats.Routine biochemical and hematologic evaluation was performed on each cat, and all were tested for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Microbial culture was performed on samples from the wounds. After surgical debridement, omentalization using a vascular pedicle of greater omentum, and closure of the chronic axillary wounds, using a thoracodorsal axial pattern flap, was performed. All excised tissue was examined histologically.The sex distribution was 7 males and 3 females, with a mean age of 3.5 years. The cats had undergone a median number of 3 previous repair attempts over a 1.5- to 25-month period before referral (mean, 10.2 months). No hematologic or biochemical abnormalities were noted apart from moderately elevated creatine kinase and aspartate transaminase concentrations in some cats. All cats were negative for FIV and FeLV. Histologic examination of resected tissue revealed hair (foreign body) in 2 cats and an unidentified foreign-body reaction in 3 other cats. Complete healing occurred in all cats (mean follow-up period of 21.7 months), with 2 cats requiring further surgery: 1 for flap dehiscence at 4 days after surgery, and 1 for donor-site dehiscence at 4 days after surgery. One other cat developed a large seroma in the axilla that resolved by 10 days following surgery.The use of an omental pedicle graft in combination with a thoracodorsal axial pattern flap is the first consistently successful 1-step technique for the management of chronic nonhealing axillary wounds in cats. DA - 2001/8// PY - 2001/8// DO - 10.1053/jvet.2001.24396 VL - 30 IS - 4 SP - 380-385 UR - https://dx.doi.org/10.1053/jvet.2001.24396 ER - TY - JOUR TI - Inactivation of an Astrovirus Associated with Poult Enteritis Mortality Syndrome AU - Schultz-Cherry, Stacey AU - King, Daniel J. AU - Koci, Matthew D. T2 - Avian Diseases AB - Outbreaks of poult enteritis mortality syndrome (PEMS) continue to cause financial losses to the turkey industry. Clinically, PEMS is defined by mortality profiles, diarrhea, flock unevenness, and immunosuppression. PEMS is a very difficult disease to control and prevent. Depopulation of PEMS-affected flocks and thorough cleaning of the contaminated housing have failed to prevent infection (disease) in subsequent flock placements. The relationship of PEMS to other enteric disease complexes of young turkeys is unknown, partly because the causative agent of PEMS remains unknown. Recently, we isolated a unique astrovirus strain from the thymus and intestines of PEMS-infected poults. This strain is molecularly and serologically distinct from the astrovirus that circulated in turkeys in the 1980s. Mammalian astroviruses are very resistant to inactivation. In these studies, we examined the stability of partially purified PEMS-associated astrovirus to inactivation with heat, laboratory disinfectants, and commercial disinfectants used in commercial turkey houses in an embryonated egg model system. Similar to mammalian astroviruses, the PEMS-associated astrovirus is resistant to inactivation by heat, acidification, detergent treatment, and treatment with phenolic, quaternary ammonium, or benzalkonium chloride-based products. Only treatment with formaldehyde, beta-propriolactone, or the peroxymonosulfate-based product Virkon S completely inactivated the astrovirus in the embryo model. These studies provide an alternate means to potentially control at least one virus associated with PEMS through the use of specific disinfectants. DA - 2001/1// PY - 2001/1// DO - 10.2307/1593014 VL - 45 IS - 1 SP - 76 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035066975&partnerID=MN8TOARS KW - astrovirus KW - poult enteritis mortality syndrome KW - PEMS KW - disinfectants ER - TY - JOUR TI - Landscape-scale patterns of shrub-species abundance in California chaparral - The role of topographically mediated resource gradients AU - Meentemeyer, RK AU - Moody, A AU - Franklin, J T2 - PLANT ECOLOGY DA - 2001/9// PY - 2001/9// DO - 10.1023/a:1011944805738 VL - 156 IS - 1 SP - 19-41 SN - 1385-0237 KW - digital terrain model KW - environmental gradient KW - predictive mapping KW - resource availability KW - RHESSys KW - vegetation modeling ER - TY - JOUR TI - Environmental factors influencing spatial patterns of shrub diversity in chaparral, Santa Ynez Mountains, California AU - Moody, A. AU - Meentemeyer, Ross K. T2 - Journal of Vegetation Science AB - We examined patterns of shrub species diversity relative to landscape‐scale variability in environmental factors within two watersheds on the coastal flank of the Santa Ynez Mountains, California. Shrub species richness and dominance was sampled at a hierarchy of spatial units using a high‐powered telescope from remote vantage points. Explanatory variables included field estimates of total canopy cover and percentage rock cover, and modeled distributions of slope, elevation, photosynthetically active radiation, topographic moisture index, and local topographic variability. Correlation, multiple regression, and regression tree analyses showed consistent relationships between field‐based measurements of species richness and dominance, and topographically‐mediated environmental variables. In general, higher richness and lower dominance occurred where environmental conditions indicated greater levels of resource limitation with respect to soil moisture and substrate availability. Maximum richness in shrub species occurred on high elevation sites with low topographic moisture index, rocky substrate, and steep slopes. Maximum dominance occurred at low elevation sites with low topographic variability, high potential solar insolation, and high total shrub canopy cover. The observed patterns are evaluated with respect to studies on species‐environment relations, resource use, and regeneration of shrubs in chaparral and coastal sage scrub. The results are discussed in the context of existing species‐diversity hypotheses that hinge on reduced competitive dominance and increased resource heterogeneity under conditions of resource limitation. DA - 2001/// PY - 2001/// DO - 10.1111/j.1654-1103.2001.tb02615.x VL - 12 IS - 1 SP - 41–52 ER - TY - JOUR TI - Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA AU - Dong, A. AU - Yoder, J.A. AU - Zhang, X. AU - Zhou, L. AU - Bestor, T.H. AU - Cheng, X. T2 - Nucleic Acids Research AB - DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m(5)C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m(5)C MTases, including the consensus S:-adenosyl-L-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-L-homocysteine (AdoHcy) has been determined at 1.8 A resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HHAI, a confirmed bacterial m(5)C MTase, and the smaller target recognition domains of DNMT2 and M.HHAI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HHAI. DNMT2 binds AdoHcy in the same conformation as confirmed m(5)C MTases and, while DNMT2 shares all sequence and structural features with m(5)C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif. C2 - 29660 DA - 2001/1/15/ PY - 2001/1/15/ DO - 10.1093/nar/29.2.439 VL - 29 IS - 2 SP - 439-448 SN - 1362-4962 UR - http://dx.doi.org/10.1093/nar/29.2.439 ER - TY - JOUR TI - Novel immune-type receptor genes AU - Litman, GW AU - Hawke, NA AU - Yoder, JA T2 - IMMUNOLOGICAL REVIEWS AB - Novel immune-type receptor (NITR) genes, which initially were identified in the Southern pufferfish (Spheroides nephelus), encode products which consist of an extracellular variable (V) and V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail, which typically possesses an immunoreceptor tyrosine-based inhibition motif (ITIM). Multiple NITR genes have been identified in close, contiguous chromosomal linkage. The V regions of NITRs resemble prototypic forms defined for immunoglobulin (Ig) and T-cell antigen receptor (TCR), are present in multiple families and exhibit regionalized variation in sequence, which also occurs in Ig and TCR. Comparisons of exons encoding transmembrane and cytoplasmic regions of multiple NITRs suggest that exon shuffling has factored in the diversification of the NITR gene complex. Zebrafish (Danio rerio) NITRs exhibit many of these characteristics. NITRs that have been identified in additional species of bony fish demonstrate additional variation in the number of extracellular domains as well as in the presence of intramembranous charged residues, cytoplasmic tails and ITIMs. The presence in NITRs of V regions that are related closely to those found in Ig and TCR, as well as regulatory motifs and other structural features that are characteristic of immune inhibitory receptors encoded at the leukocyte receptor cluster, suggests that the NITRs are representative of an integral stage in the evolution of innate and adaptive immune function. DA - 2001/6// PY - 2001/6// DO - 10.1034/j.1600-065X.2001.1810121.x VL - 181 IS - 1 SP - 250-259 SN - 0105-2896 ER - TY - JOUR TI - Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster AU - Yoder, J. A. AU - Mueller, M. G. AU - Wei, S. AU - Corliss, B. C. AU - Prather, D. M. AU - Willis, T. AU - Litman, R. T. AU - Djeu, J. Y. AU - Litman, G. W. T2 - Proceedings of the National Academy of Sciences AB - An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of approximately 40 nitr genes are contiguous in the genome and span approximately 0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster. DA - 2001/5/29/ PY - 2001/5/29/ DO - 10.1073/pnas.121101598 VL - 98 IS - 12 SP - 6771-6776 J2 - Proceedings of the National Academy of Sciences LA - en OP - SN - 0027-8424 1091-6490 UR - http://dx.doi.org/10.1073/pnas.121101598 DB - Crossref ER - TY - JOUR TI - Extraordinary variation in a diversified family of immune-type receptor genes AU - Hawke, N.A. AU - Yoder, Jeffrey AU - Haire, R.N. AU - Mueller, M.G. AU - Litman, R.T. AU - Miracle, A.L. AU - Stuge, T. AU - Shen, L. AU - Miller, N. AU - Litman, G.W. AU - al. T2 - Proceedings of the National Academy of Sciences AB - Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified in Ictalurus punctatus (channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners. DA - 2001/11/6/ PY - 2001/11/6/ DO - 10.1073/pnas.231418598 VL - 98 IS - 24 SP - 13832-13837 J2 - Proceedings of the National Academy of Sciences LA - en OP - SN - 0027-8424 1091-6490 UR - http://dx.doi.org/10.1073/pnas.231418598 DB - Crossref ER - TY - JOUR TI - Cloning and sequence analysis of a zebrafish cDNA encoding DNA (cytosine-5)-methyltransferase-1 AU - Mhanni, AA AU - Yoder, JA AU - Dubesky, C AU - McGowan, RA T2 - GENESIS AB - Abstract Summary: The zebrafish has become a well‐established animal model for the analysis of development and of several disease phenotypes. Several of the favorable traits that make it a popular model organism would also be beneficial for the study of normal and abnormal vertebrate development in which DNA methylation may play a role. We report the determination of the full‐length cDNA sequence corresponding to the zebrafish DNA (cytosine‐5‐) methyltransferase gene, Dnmt1 . It is 4,907 bases long and has an open reading frame predicted to encode a 1,499 amino acid protein that is similar in size and sequence to a number of other methyltransferases identified in other organisms. genesis 30:213–219, 2001. © 2001 Wiley‐Liss, Inc. DA - 2001/8// PY - 2001/8// DO - 10.1002/gene.1067 VL - 30 IS - 4 SP - 213-219 SN - 1526-968X UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0034861153&partnerID=MN8TOARS KW - DNA cytosine-5-methyltransferase KW - DNA mtase KW - Dnmt1 KW - zebrafish KW - methylation KW - development ER - TY - JOUR TI - Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: effect on Brucella abortus survival AU - Barthel, R. AU - Feng, J. AU - Piedrahita, J. A. AU - McMurray, D. N. AU - Templeton, J. W. AU - Adams, L. G. T2 - Infection and Immunity AB - Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. Bovine NRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovine NRAMP1 as stable transgenes under the regulatory control of the bovine NRAMP1 promoter in the murine RAW264.7 macrophage cell line (Bcg(s)) to analyze the regulation of the NRAMP1 gene and its role in macrophage function. We demonstrated that the 5'-flanking region of bovine NRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expression of a transgene in murine macrophages. A polymorphism within a microsatellite in the 3' untranslated region critically affects the expression of bovine NRAMP1 and the control of in vitro replication of Brucella abortus but not Salmonella enterica serovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-gamma)- and IFN-gamma-lipopolysaccharide (LPS)-treated transfected cell lines, yet the resistant transfected cell lines produced significantly less NO than other cell lines, following stimulation with LPS at 24 and 48 h. DA - 2001/// PY - 2001/// DO - 10.1128/IAI.69.5.3110-3119.2001 VL - 69 IS - 5 SP - 3110-3119 ER - TY - JOUR TI - Inactivation of the whey acidic protein (WAP) gene by site-specific recombination in mouse embryonic stem cells AU - Lee, C. K. AU - Piedrahita, J. A. T2 - Journal of Animal Science DA - 2001/// PY - 2001/// VL - 42 IS - 6 SP - 941-956 ER - TY - CHAP TI - Surface characteristics of mineral filled polypropylene filaments AU - McCord, M.G. AU - George, B.R. AU - Hudson, S.M. T2 - Surface characteristics of fibers and textiles A2 - C. M. Pastore, A2 - Kiekens, P. PY - 2001/// PB - New York: M. Dekker SN - 9780824700027 ER - TY - CHAP TI - Penicillins and related beta-Lactam antibiotics AU - Vaden, S. AU - Riviere, J. E. T2 - Veterinary pharmacology and therapeutics (8th ed.) PY - 2001/// SP - 818-827 PB - Ames, IA: Iowa State University Press SN - 0813817439 ER - TY - CHAP TI - Pesticide disposition: dermal absorption AU - Baynes, R. AU - Riviere, J. E. T2 - Handbook of Pesticide Toxicology (2nd ed.) AB - Percutaneous absorption is reported as the possible route of entry in 65-85% of all cases of occupational exposure with pesticides. This chapter focuses on mechanisms and pathways of dermal absorption and experimental models used to assess dermal absorption by in vitro, ex vivo, and in vivo methods. The effects of biological variability, pesticide chemistry and formulations, and environmental variables that influence dermal absorption are discussed. The skin is relatively impermeable to aqueous solutions and ions, but it may be permeable in varying degrees to a large number of drugs or xenobiotics. Drug or xenobiotic delivery pathways can hypothetically involve intercellular and intracellular passive diffusion across the epidermis and dermis and/or transappendageal routes via hair follicles and sweat pores. Regional variation in skin permeability in different body sites may be related to skin thickness, number of cell layers, cell size of the epidermis and stratum corneum, and distribution of hair follicles and sweat pores. PY - 2001/// DO - 10.1016/b978-012426260-7.50025-2 SP - 515-530 PB - San Diego: Academic Press SN - 9780124262614 ER - TY - CHAP TI - MARCKS protein: a potential modulator of airway mucin secretion. AU - Li, Y. AU - Martin, L. D. AU - Adler, K. B. T2 - Cilia and mucus: from development to respiratory disease. PY - 2001/// SP - 179-193 ER - TY - CHAP TI - Interleukin-13 induced mucous cell hyperplasia in airway epithelium. AU - Martin, L. D. AU - Macchione, M. AU - Bonner, J. C. AU - Booth, B. W. AU - Akley, N. J. AU - Adler, K. B. T2 - Cilia and mucus: from development to respiratory disease. PY - 2001/// SP - 253-263 ER - TY - JOUR TI - Chelonian shell-fracture repair techniques AU - Kishimori, J. AU - Lewbart, G. A. AU - Marcellin-Little, D. J. AU - Roe, S. AU - Trogdon, M. AU - Henson, H. AU - Stoskopf, M. K. T2 - Exotic DVM DA - 2001/// PY - 2001/// VL - 3 IS - 5 SP - 35-41 ER - TY - JOUR TI - MARCKS protein interaction with the cellular contractile machinery may regulate mucin secretion by human airway epithelium. AU - Li, Y. AU - Pettersen, C. A. AU - Martin, L. D. AU - Adler, K. B. T2 - American Journal of Respiratory and Critical Care Medicine DA - 2001/// PY - 2001/// VL - 163 SP - A225 ER - TY - JOUR TI - Differentiation of murine tracheal epithelial cells in vitro. AU - Macchione, M. AU - Akley, N. J. AU - Adler, K. B. AU - Martin, L. D. T2 - American Journal of Respiratory and Critical Care Medicine DA - 2001/// PY - 2001/// VL - 163 SP - A225 ER - TY - JOUR TI - Creation of polymer films with novel structures and properties by processing with inclusion compounds AU - Huang, L. AU - Gerber, M. AU - Taylor, H. AU - Lu, J. AU - Tapaszi, E. AU - Wutkowski, M. AU - Hill, M. AU - Funahlee, F. N. AU - Harvey, A. AU - Rusa, C. C. AU - Porbeni, F. E. AU - Edeki, E. T2 - ACS Symposium Series AB - We have begun to fabricate polymer films whose compositions, structures, and properties may be developed and controlled during their formation with inclusion compounds (ICs). ICs formed with either urea(U) or cyclodextrin(CD) hosts and containing guest polymers or small-molecule additives are embedded into carrier polymer films either by solution casting or melt pressing methods. Once embedded, the IC crystals are left undisturbed or are disrupted by solvent treatment, which removes the host (U or CD), but not the carrier polymer nor the coalesced IC-guest. In this manner polymer-polymer composite and additive-filled films have been fabricated. Employment of polymer-U or CD-ICs produces composite films containing two different polymers or two populations of the same polymer. In the latter case, the morphologies of the carrier and IC-coalesced chains may differ, because of chain-folded and chain-extended crystallization, respectively. We may, for example, control film permeabilities by either controlling the compositions or the morphologies of DA - 2001/// PY - 2001/// DO - 10.1021/bk-2001-0790.ch014 VL - 790 ER - TY - JOUR TI - Autocrine production of TGFa mediates interleukin 13-induced proliferation of human airway epithelial cells during development of a mucous phenotype in vitro. AU - Booth, B. AU - Bonner, J. C. AU - Adler, K. B. AU - Martin, L. D. T2 - American Journal of Respiratory and Critical Care Medicine DA - 2001/// PY - 2001/// VL - 163 SP - A738 ER - TY - JOUR TI - A hybrid bioabsorbable wound dressing AU - London-Brown, A. AU - Hudson, S. M. AU - Tonelli, A. AU - Vigo, T. AU - Edwards, R. AU - Gupta, B. S. T2 - ACS Symposium Series DA - 2001/// PY - 2001/// VL - 792 ER - TY - JOUR TI - Evaluation of potential health risks to Eastern Elliptio (Elliptio complanata) (Mollusca: Bivalvia: Unionida: Unionidae) and implications for sympatric endangered freshwater mussel species AU - Chittick, B. AU - Stoskopf, M. AU - Law, M. AU - Overstreet, R. AU - Levine, J. T2 - Journal of Aquatic Ecosystem Stress and Recovery DA - 2001/// PY - 2001/// VL - 9 IS - 1 SP - 35 ER - TY - PAT TI - Tightly coupled porphyrin macrocycles for molecular memory storage AU - Gryko, D. T. AU - Clausen, P. C. AU - Bocian, D. F. AU - Kuhr, W. G. AU - Lindsey, J. S. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - The latest in antimicrobials for pet fish AU - Lewbart, G. A. T2 - Exotic DVM DA - 2001/// PY - 2001/// VL - 3 IS - 3 SP - 80 ER - TY - JOUR TI - Surgical techniques in the koi patient AU - Lewbart, G. A. T2 - Exotic DVM DA - 2001/// PY - 2001/// VL - 3 IS - 3 SP - 43 ER - TY - JOUR TI - Identification of avian mycoplasma strains by random amplification of polymorphic DNA (RAPD) AU - Ley, D. H. T2 - Zootecnica International DA - 2001/// PY - 2001/// IS - 6 SP - 46 ER - TY - PAT TI - High density non-volatile memory device incorporating thiol-derivatized porphyrins AU - Gryko, D. T. AU - Clausen, P. C. AU - Roth, K. M. AU - Bocian, D. F. AU - Kuhr, W. G. AU - Lindsey, J. S. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - Current approaches to anesthesia and analgesia in fish AU - Lewbart, G. A. T2 - Exotic DVM DA - 2001/// PY - 2001/// VL - 3 IS - 3 SP - 19 ER - TY - JOUR TI - Chemical analysis of six commercial adult iguana [Iguana iguana], diets AU - Hurty, C. A. AU - Diaz, D. E. AU - Campbell, J. L. AU - Lewbart, Gregory T2 - Journal of Herpetological Medicine and Surgery AB - Reptile keeping is one of the most rapidly expanding areas in the pet industry. In response to this trend, several pet food companies have formulated specialized diets and supplements that cater to the unique nutritional needs of different reptile species. Since nutrition is a key variable of captive reptile husbandry, we investigated the nutrient composition of six commercially available adult iguana, Iguana iguana, feeds that are intended for use as principle diets. We compared the results of our analyses to the nutrient information provided on packaging labels and to available information on the suggested nutrient recommendations for iguanas. Crude protein contents ranged from 13.21% dry matter (DM) to 27.15% DM with two of the six diets containing a lower protein than indicated on the label. Crude fat content ranged from 1.46 ± 0.44% DM to 10.25 ± 0.05% DM with three of the six diets having fat contents below the amount stated on labels. The acid detergent fiber (ADF) content, a measure of insoluble fiber, ranged from 5.42 ± 0.54% DM to 13.95 ± 0.27% DM. Great variety in concentrations of iron (Fe), copper (Cu), calcium (Ca), phosphorus (P), zinc (Zn), magnesium (Mg), and manganese (Mn) was demonstrated. DA - 2001/// PY - 2001/// DO - 10.5818/1529-9651.11.3.23 VL - 11 IS - 3 SP - 23 ER - TY - JOUR TI - Degenerative mucinotic mural folliculitis in cats AU - Gross, Thelma Lee AU - Olivry, Thierry AU - Vitale, Carlo B. AU - Power, Helen T. T2 - Veterinary Dermatology AB - A novel form of mural folliculitis is described in seven cats. Clinically, all cats exhibited generalized alopecia with scaling or crusting that was more pronounced over the head, neck, and shoulders. The face and muzzle of all cats was unusually thickened. Six of seven cats were progressively lethargic but did not demonstrate any other consistent systemic abnormalities. Histologically, there was severe mixed inflammation of the wall of the follicular isthmus in all cats, accompanied by some follicular destruction in five cats. Sebaceous glands were not affected. All cats had variable, but often striking, follicular mucin deposition, as well as epidermal hyperkeratosis and crusting. The cause of the severe mural folliculitis was not identified, and all cats responded poorly to immunomodulating therapy. Follicular mucinosis may be a nonspecific finding, likely reflective of the follicular lymphocytic milieu, and does not always herald follicular lymphoma. DA - 2001/10// PY - 2001/10// DO - 10.1046/j.0959-4493.2001.00229.x VL - 12 IS - 5 SP - 279-283 J2 - Vet Dermatol LA - en OP - SN - 0959-4493 1365-3164 UR - http://dx.doi.org/10.1046/j.0959-4493.2001.00229.x DB - Crossref KW - alopecia KW - cats KW - follicle KW - mucinosis KW - mural folliculitis ER - TY - JOUR TI - A photolysis-triggered heme ligand switch in H93G myoglobin AU - Franzen, S AU - Bailey, J AU - Dyer, RB AU - Woodruff, WH AU - Hu, RB AU - Thomas, MR AU - Boxer, SG T2 - BIOCHEMISTRY AB - Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR) spectroscopy have been used to identify the ligation state of ferrous heme iron for the H93G proximal cavity mutant of myoglobin in the absence of exogenous ligand on the proximal side. Preparation of the H93G mutant of myoglobin has been previously reported for a variety of axial ligands to the heme iron (e.g., substituted pyridines and imidazoles) [DePillis, G., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. The present study examines the ligation states of heme in preparations of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93G, resonance Raman spectroscopic evidence shows water to be the axial (fifth) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe-C stretching frequencies for the CO adduct indicates that it is six-coordinate with a histidine trans ligand. Following photolysis of CO, a time-dependent change in ligation is evident in both step-scan FTIR and saturation resonance Raman spectra, leading to the conclusion that a conformationally driven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine ligation, while conformational strain favors the dissociation of histidine following photolysis of CO. The replacement of histidine by water in the five-coordinate complex is estimated to occur in < 5 micros. The results demonstrate that the H93G myoglobin cavity mutant has potential utility as a model system for studying the conformational energetics of ligand switching in heme proteins such as those observed in nitrite reductase, guanylyl cyclase, and possibly cytochrome c oxidase. DA - 2001/5/1/ PY - 2001/5/1/ DO - 10.1021/bi0023403 VL - 40 IS - 17 SP - 5299-5305 SN - 0006-2960 ER - TY - BOOK TI - Polymers from the inside out: An introduction to macromolecules AU - Tonelli, A. AU - Srinivasarao, M. AB - Preface. Acknowledgments. Chapter Summary. Introduction. Step-Growth Polymerization. Chain-Growth Polymerization. The Microstructures of Polymers. The Conformational Characteristics of Polymers. Solution Properties of Polymers. Bulk Properties of Polymers. Naturally Occurring Biopolymers. Index. CN - QD381 .T65 2001 DA - 2001/// PY - 2001/// DO - 10.5860/choice.39-1583 PB - New York: Wiley-Interscience SN - 0471381381 ER - TY - JOUR TI - Blood chemistry values of juvenile red pacu (Piaractus brachypolmus) AU - Sakamoto, K AU - Lewbart, GA AU - Smith, TM T2 - VETERINARY CLINICAL PATHOLOGY AB - Blood samples were collected from 29 juvenile red pacu (Piaractus brachypomus), ornamental freshwater fish, to establish baseline blood chemistry values. Mean (minimum-maximum) values, obtained by automated bichromatic analysis and ion selective electrode analysis, were as follows: sodium, 150.4 (146-159) mmol/L; potassium, 3.93 (2.7-5.0) mmol/L; chloride, 138.7 (128-150) mmol/L; total CO2, 7.5 (6-10) mmol/L; albumin, 0.86 (0.5-1.0) g/dL; lactate dehydrogenase, 237.8 (65-692) IU/L; aspartate aminotransferase, 49.1 (0-125) IU/L; creatinine, 0.31 (0.2-0.4) mg/dL; calcium, 10.80 (9.5-12.5) mg/dL; anion gap, 6.89 (1.2-12.5) mmol/L; and phosphorus, 7.29 (4.1-8.9) mg/dL. DA - 2001/// PY - 2001/// DO - 10.1111/j.1939-165X.2001.tb00257.x VL - 30 IS - 2 SP - 50-52 SN - 0275-6382 KW - blood chemistry KW - freshwater fish KW - Piaractus brachypomus KW - red pacu ER - TY - JOUR TI - Mortality patterns associated with poult enteritis mortality syndrome (PEMS) and coronaviral enteritis in turkey flocks raised in PEMS-affected regions AU - Carver, DK AU - Vaillancourt, JP AU - Stringham, M AU - Guy, JS AU - Barnes, HJ T2 - AVIAN DISEASES AB - Poult enteritis mortality syndrome (PEMS) is an economically devastating disease. To date, many questions about the syndrome remain unanswered, including its cause, transmission of causative agent(s), and control methods. Turkey coronavirus (TCV) infection has been associated with some outbreaks of PEMS, with areas having a higher prevalence of TCV infection also experiencing an increased incidence of PEMS. This study was designed to establish mortality patterns for flocks experiencing excess mortality and TCV infection in PEMS-affected regions and to delineate the possible role of TCV in PEMS-affected flocks. Fifty-four commercial turkey flocks on farms in areas with and without a history of TCV infection were monitored for weekly mortality and for antibodies to TCV. Flocks were chosen on the basis of placement dates and were monitored from day of placement until processing. All flocks were tested for TCV by an indirect fluorescent antibody assay. PEMS status was determined with the use of the clinical definition of mortality greater than 2% during any 3-wk period from 2 wk of age through the end of brooding due to unknown cause. Of the 54 flocks, 24 remained healthy, 23 experienced PEMS, and 7 tested positive for TCV but did not experience PEMS. Ten flocks experienced PEMS and tested positive for TCV, whereas 13 flocks experienced PEMS and did not test positive for TCV. Four health status groups were evident: healthy, PEMS positive, TCV positive, and PEMS + TCV positive. Distinct mortality patterns were seen for each of the four health status groups. Whereas TCV was associated with PEMS in 43% of PEMS cases, 13 cases (57%) of PEMS did not involve TCV. Additionally, 7 out of 17 cases of TCV (41%) did not experience excess mortality (PEMS) at any time during brooding of the flock. The results of this study indicate that TCV can be associated with PEMS but is neither necessary nor sufficient to cause PEMS. DA - 2001/// PY - 2001/// DO - 10.2307/1592878 VL - 45 IS - 4 SP - 985-991 SN - 0005-2086 KW - mortality KW - turkey KW - poult enteritis mortality syndrome KW - coronavirus KW - avian KW - disease KW - enteric ER - TY - JOUR TI - Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-alpha AU - Booth, BW AU - Adler, KB AU - Bonner, JC AU - Tournier, F AU - Martin, LD T2 - AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY AB - Remodeling of the airways, as occurs in asthmatic patients, is associated with the continual presence of inflammatory mediators and Th2 cytokines, especially interleukin (IL)-13, during cycles of epithelial injury and repair. In this study, we examined the effect of IL-13 on well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture. IL-13 induced proliferation of NHBE cells after 24 h exposure, as reflected by [(3)H]thymidine uptake and cell counts. The effects of IL-13 were mediated through the epidermal growth factor receptor (EGFR), as proliferation was attenuated by AG1478, an EGFR tyrosine kinase inhibitor. Proliferation appeared to be mediated by transforming growth factor (TGF)-alpha, a potent ligand for EGFR, which was released rapidly from NHBE cells in response to IL-13. Neutralizing antibody to TGF-alpha, but not antibodies against other potentially important growth factors (EGF, heparin binding epidermal growth factor-like growth factor [HB-EGF], platelet-derived growth factor [PDGF]), inhibited the mitogenic response to IL-13. This study provides the first experimental evidence that IL-13 can initiate a proliferative response of human airway epithelium in the absence of inflammatory cells or other cell types. The results are consistent with a mechanism whereby IL-13 induces release of TGF-alpha from the epithelial cells, which in turn binds via an autocrine/paracrine-type action to the EGFR, initiating proliferation. IL-13-induced airway remodeling in vivo may involve this epithelium-driven response. DA - 2001/12// PY - 2001/12// DO - 10.1165/ajrcmb.25.6.4659 VL - 25 IS - 6 SP - 739-743 SN - 1535-4989 ER - TY - JOUR TI - Genetic analysis of bipartite geminivirus tissue tropism AU - Qin, Y AU - Petty, ITD T2 - VIROLOGY AB - The bipartite geminiviruses bean golden mosaic virus (BGMV), cabbage leaf curl virus (CabLCV), and tomato golden mosaic virus (TGMV) exhibit differential tissue tropism in Nicotiana benthamiana. In systemically infected leaves, BGMV remains largely confined to vascular-associated cells (phloem-limited), whereas CabLCV and TGMV can escape into the surrounding mesophyll. Previous work established that TGMV BRi, the noncoding region upstream from the BR1 open reading frame (ORF), is required for mesophyll invasion, but the virus must also contain the TGMV AL23 or BL1/BR1 ORFs. Here we show that, in a BGMV-based hybrid virus, CabLCV AL23 also directed efficient mesophyll invasion in conjunction with TGMV BRi, which suggests that host-adaptation of AL23 is important for the phenotype. Cis-acting elements required for mesophyll invasion were delineated by analyzing BGMV-based hybrid viruses in which various parts of BRi were exchanged with those of TGMV. Interestingly, mesophyll invasion efficiency of hybrid viruses was not correlated with the extent of viral DNA accumulation. In conjunction with TGMV AL23, a 52-bp region of TGMV BRi with sequence homology to DNA A was sufficient for mesophyll invasion. This 52-bp sequence also directed mesophyll invasion in combination with the TGMV BL1/BR1 ORFs. Overall, these results are consistent with a model for mesophyll invasion in which AL2 protein, in association with host factors, acts through the 52-bp region in TGMV BRi to affect expression of the BR1 gene. DA - 2001/12/20/ PY - 2001/12/20/ DO - 10.1006/viro.2001.1205 VL - 291 IS - 2 SP - 311-323 SN - 0042-6822 KW - phloem-limited KW - mesophyll invasion KW - noncoding region KW - tomato golden mosaic virus KW - bean golden mosaic virus KW - cabbage leaf curl virus ER - TY - JOUR TI - Comparative development of avian primordial germ cells and production of germ line chimeras AU - S D'Costa, AU - Pardue, SL AU - Petitte, JN T2 - AVIAN AND POULTRY BIOLOGY REVIEWS DA - 2001/// PY - 2001/// DO - 10.3184/147020601783698477 VL - 12 IS - 4 SP - 151-168 SN - 1470-2061 KW - primordial germ cells KW - embryo KW - chimeras KW - chick KW - turkey KW - quail KW - migration KW - transgenic ER - TY - JOUR TI - Acute and chronic mineral oil pneumonitis in two horses AU - Davis, J. L. AU - Ramirez, S. AU - Campbell, N. AU - jones T2 - Equine Veterinary Education AB - Equine Veterinary EducationVolume 13, Issue 5 p. 230-234 Acute and chronic mineral oil pneumonitis in two horses J. L. Davis, J. L. Davis Departments of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.Search for more papers by this authorS. Ramirez, S. Ramirez Anatomy, Physiology and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.Search for more papers by this authorN. Campbell, N. Campbell Departments of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.Search for more papers by this authorS. L. Jones, Corresponding Author S. L. Jones Departments of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.†Departments of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.Search for more papers by this author J. L. Davis, J. L. Davis Departments of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.Search for more papers by this authorS. Ramirez, S. Ramirez Anatomy, Physiology and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.Search for more papers by this authorN. Campbell, N. Campbell Departments of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.Search for more papers by this authorS. L. Jones, Corresponding Author S. L. Jones Departments of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.†Departments of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.Search for more papers by this author First published: 05 January 2010 https://doi.org/10.1111/j.2042-3292.2001.tb00099.xCitations: 6AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume13, Issue5October 2001Pages 230-234 RelatedInformation DA - 2001/// PY - 2001/// DO - 10.1111/j.2042-3292.2001.tb00099.x VL - 13 IS - 5 SP - 230–234 ER - TY - JOUR TI - Rational syntheses of cyclic hexameric porphyrin arrays for studies of self-assembling light-harvesting systems AU - Yu, LH AU - Lindsey, JS T2 - JOURNAL OF ORGANIC CHEMISTRY AB - Two new cyclic hexameric arrays of porphyrins have been prepared in a rational, convergent manner. The porphyrins in each cyclic hexamer are joined by diphenylethyne linkers affording a wheel-like array with a diameter of approximately 35 A. One array is comprised of five zinc (Zn) porphyrins and one free base (Fb) porphyrin (cyclo-Zn(5)FbU) while the other is comprised of an alternating sequence of two Zn porphyrins and one Fb porphyrin (cyclo-Zn(2)FbZn(2)FbU). The prior synthesis employed a one-flask template-directed process and afforded alternating Zn and Fb porphyrins or all Zn porphyrins. More diverse metalation patterns are attractive for manipulating the flow of excited-state energy in the arrays. The rational synthesis of each array employed three Pd-mediated coupling reactions with four tetraarylporphyrin building blocks bearing diethynyl, diiodo, bromo/iodo, or iodo/ethynyl groups. The final ring closure yielding the cyclic hexamer was achieved by reaction of a porphyrin pentamer + porphyrin monomer or the joining of two porphyrin trimers. In the presence of a tripyridyl template, the yields of the 5 + 1 and 3 + 3 reactions ranged from 10 to 13%. The 5 + 1 reaction in the absence of the template proceeded in 3.5% yield, thereby establishing the structure-directed contribution to cyclic hexamer formation. The 3 + 3 route relied on successive ethyne + iodo/bromo coupling reactions. One template-directed route to cyclo-Zn(2)FbZn(2)FbU employed a magnesium porphyrin, affording cyclo-Zn(2)FbZn(2)MgU from which magnesium was selectively removed. The arrays exhibit absorption spectra that are nearly the sum of the spectra of the component parts, indicating weak electronic coupling. Fluorescence spectroscopy showed that the quantum yield of energy transfer in toluene at room temperature from the Zn porphyrins to the Fb porphyrin(s) was 60% in cyclo-Zn(5)FbU and 90% in cyclo-Zn(2)FbZn(2)FbU. Two dipyridyl-substituted porphyrins, a Zn tetraarylporphyrin and a Fb oxaporphyrin, have been synthesized for use as guests in the cyclic hexamers, affording self-assembled arrays for light-harvesting studies. DA - 2001/11/2/ PY - 2001/11/2/ DO - 10.1021/jo010742q VL - 66 IS - 22 SP - 7402-7419 SN - 0022-3263 ER - TY - JOUR TI - Natural history of hypertrophic cardiomyopathy and aortic thromboembolism in a family of domestic shorthair cats AU - Baty, CJ AU - Malarkey, DE AU - Atkins, CE AU - DeFrancesco, TC AU - Sidley, J AU - Keene, BW T2 - JOURNAL OF VETERINARY INTERNAL MEDICINE AB - A feline domestic shorthair queen and her 3 offspring were all diagnosed with asymptomatic hypertrophic cardiomyopathy (HCM). The family has been followed for 13 years, and 3 cats have died of aortic thromboembolism (ATE). This communication documents the long-term progression of HCM in these cats that presented with mild left ventricular hypertrophy and hyperdynamic systolic ventricular function, developed progressive left atrial enlargement, and eventually resulted in hypodynamic left ventricular systolic function with relative left ventricular chamber dilation at the time of ATE. DA - 2001/// PY - 2001/// DO - 10.1892/0891-6640(2001)015<0595:NHOHCA>2.3.CO;2 VL - 15 IS - 6 SP - 595-599 SN - 0891-6640 KW - cardiac myocyte disarray KW - end stage KW - spontaneous echo contrast KW - systolic function KW - ventricular remodeling ER - TY - JOUR TI - Mycoplasmosis in evening and pine grosbeaks with conjunctivitis in Quebec AU - Mikaelian, I AU - Ley, DH AU - Claveau, R AU - Lemieux, M AU - Berube, JP T2 - JOURNAL OF WILDLIFE DISEASES AB - An outbreak of conjunctivitis affected evening grosbeaks (Coccothraustes vespertinus) and pine grosbeaks (Pinicola enucleator) in Quebec (Canada) during the winter 1998-99. One to 30% of the individuals from these two species were sick at 13 feeding stations. Sick birds were thin and had unilateral or bilateral catarrhal and lymphoplasmacytic conjunctivitis and rhinitis, and mucopurulent infra-orbital sinusitis. Mycoplasmal organisms were isolated in cultures in an affected evening grosbeak and identified as Mycoplasma gallisepticum by direct immunofluorescence. Random amplified polymorphic DNA (RAPD) fingerprinting of this isolate resulted in a banding pattern that was identical to patterns of M. gallisepticum isolates made from similar lesions in house finches (Carpodacus mexicanus) and American gold finches (Carduelis tristis) throughout eastern North America. Mycoplasma gallisepticum was identified by polymerase chain reaction in another evening grosbeak and a pine grosbeak. These observations suggest that the same strain of M. gallisepticum is the likely etiology for the observed disease in evening and pine grosbeaks in Canada and represent an extension of the host-species range for the ongoing epidemic of M. gallisepticum conjunctivitis in eastern North America. DA - 2001/10// PY - 2001/10// DO - 10.7589/0090-3558-37.4.826 VL - 37 IS - 4 SP - 826-830 SN - 0090-3558 KW - case report KW - Coccothraustes vespertinus KW - conjunctivitis KW - evening grosbeak KW - mycoplasmosis KW - Mycoplasma gallisepticum KW - pine grosbeak KW - Pinicola enucleator ER - TY - JOUR TI - First isolation of La Crosse virus from naturally infected Aedes albopictus AU - Gerhardt, R. R. AU - Gottfried, K. L. AU - Apperson, C. S. AU - Davis, B. S. AU - Erwin, P. C. AU - Smith, A. B. AU - Panella, N. A. AU - Powell, E. E. AU - Nasci, R. S. T2 - Emerging Infectious Diseases AB - Abstract La Crosse (LAC) virus, a California serogroup bunyavirus, is the leading cause of pediatric arboviral encephalitis in the United States and an emerging disease in Tennessee, West Virginia, and North Carolina. Human cases of LAC encephalitis in Tennessee and North Carolina have increased above endemic levels during 1997 to 1999 and may represent an expansion of a new southeastern endemic focus. This report describes the isolation of LAC virus from the exotic mosquito Aedes albopictus. The discovery of LAC virus in wild populations of Ae. albopictus, coupled with its expanding distribution in the southeastern United States, suggests that this mosquito may become an important accessory vector, potentially increasing the number of human cases in endemic foci or expanding the range of the disease. DA - 2001/// PY - 2001/// DO - 10.3201/eid0705.010006 VL - 7 IS - 5 SP - 807-811 ER - TY - JOUR TI - Creation of novel polymer materials by processing with inclusion compounds AU - Huang, L AU - Gerber, M AU - Taylor, H AU - Lu, J AU - Tapaszi, E AU - Wutkowski, M AU - Hill, M AU - Lewis, C AU - Harvey, A AU - Herndon, A AU - Wei, M AU - Rusa, CC AU - Tonelli, AE T2 - MACROMOLECULAR SYMPOSIA AB - The processing of polymer materials from their inclusion compounds (ICs) formed with urea (U) and cyclodextrin (CD) hosts is described. Several examples are presented and serve to demonstrate the fabrication of unique polymer-polymer composites and blends, including intimate blends of normally incompatible polymers, and the delivery of additives to polymers by means of embedding polymer- or additive-U and CD- ICs into carrier polymer films and fibers, followed by coalescence of the IC guest, or by coalescence of two polymers or a polymer and an additive from their common CD-IC crystals. DA - 2001/11// PY - 2001/11// DO - 10.1002/1521-3900(200112)176:1<129::AID-MASY129>3.0.CO;2-M VL - 176 SP - 129-144 SN - 1022-1360 ER - TY - JOUR TI - Antimicrobials - Peptide antibiotics in mast cells of fish AU - Silphaduang, U AU - Noga, EJ T2 - NATURE DA - 2001/11/15/ PY - 2001/11/15/ DO - 10.1038/35104690 VL - 414 IS - 6861 SP - 268-269 SN - 0028-0836 ER - TY - JOUR TI - Synthesis of meso-substituted chlorins via tetrahydrobilene-a intermediates AU - Taniguchi, M AU - Ra, D AU - Mo, G AU - Balasubramanian, T AU - Lindsey, JS T2 - JOURNAL OF ORGANIC CHEMISTRY AB - Chlorin building blocks incorporating a geminal dimethyl group in the reduced ring and synthetic handles in specific patterns at the perimeter of the macrocycle are expected to have utility in biomimetic and materials chemistry. A prior route employed condensation of a dihydrodipyrrin (Western half) and a bromodipyrromethane-monocarbinol (Eastern half), followed by oxidative cyclization of the putative dihydrobilene-a to form the meso-substituted zinc chlorin in yields of ∼10%. The limited stability of the dihydrodipyrrin precluded study of the chlorin-forming process. We now have refined this methodology. A tetrahydrodipyrrin Western half (2,3,4,5-tetrahydro-1,3,3-trimethyldipyrrin) has been synthesized and found to be quite stable. The condensation of the Western half and an Eastern half (100 mM each) proceeded smoothly in CH3CN containing 100 mM TFA at room temperature for 30 min. The resulting linear tetrapyrrole, a 2,3,4,5-tetrahydrobilene-a, also is quite stable, enabling study of the conversion to chlorin. Refined conditions for the oxidative cyclization were found to include the following: the tetrahydrobilene-a (10 mM), AgTf (3−5 molar equiv), Zn(OAc)2 (15 molar equiv), and 2,2,6,6-tetramethylpiperidine (15 molar equiv) in CH3CN at reflux exposed to air for 4−6 h, affording the zinc chlorin. The chlorin-forming process could be implemented in either a two-flask process or a one-flask process. The two-flask process was applied to form six zinc chlorins bearing substituents such as pentafluorophenyl, 3,5-di-tert-butylphenyl, TMS-ethyl benzoate, iodophenyl, or ethynylphenyl (deprotection of the TMS-ethynyl group occurred during the oxidative cyclization process). The stepwise yields (isolated) for the condensation and oxidative cyclization processes forming the tetrahydrobilene and zinc chlorin were 32−72% and 27−62%, respectively, giving overall yields of zinc chlorin from the Eastern and Western halves of 12−45%. Taken together, the refinements introduced enable 100-mg quantities of chlorin building blocks to be prepared in a facile and rational manner. DA - 2001/11/2/ PY - 2001/11/2/ DO - 10.1021/jo0104835 VL - 66 IS - 22 SP - 7342-7354 SN - 0022-3263 ER - TY - JOUR TI - Investigation of two rational routes for preparing p-phenylene-linked porphyrin trimers AU - Yu, LH AU - Lindsey, JS T2 - TETRAHEDRON AB - Multiporphyrin arrays with p-phenylene linkers, aryl groups at the non-linking meso positions, and no β-substituents are attractive constructs for light-harvesting applications. Condensation of a free base porphyrin-benzaldehyde and 5-mesityldipyrromethane (10 mM each) in CH2Cl2 containing 100 mM TFA at room temperature for 30–40 min followed by oxidation with DDQ afforded a p-phenylene-linked porphyrin trimer in 36% yield. Suzuki coupling of an iodo-porphyrin and a bis(dioxaborolane)-porphyrin (20 and 10 mM, respectively) in toluene/DMF (2:1) containing K2CO3 (8 equiv.) at 90–95°C for ∼20 h afforded the same trimer in 66% yield. The former route was used to prepare a diethynyl substituted p-phenylene-linked porphyrin trimer. While the two routes are somewhat complementary in scope, both are convergent and proceed in a rational manner. DA - 2001/11/5/ PY - 2001/11/5/ DO - 10.1016/S0040-4020(01)00928-0 VL - 57 IS - 45 SP - 9285-9298 SN - 0040-4020 KW - porphyrins and analogues KW - pyrroles KW - Suzuki reactions KW - alkynes ER - TY - JOUR TI - A survey of acid catalysts in dipyrromethanecarbinol condensations leading to meso-substituted porphyrins AU - Geier, GR AU - Callinan, JB AU - Rao, PD AU - Lindsey, JS T2 - JOURNAL OF PORPHYRINS AND PHTHALOCYANINES AB - The successful use of dipyrromethanecarbinols in rational routes to porphyrinic macrocycles requires catalysis conditions that enable irreversible condensation, thereby avoiding substituent scrambling and formation of undesired porphyrin products. Previously, successful conditions of trifluoroacetic acid (TFA) (30 mM) in acetonitrile were identified following a lengthy survey of TFA and BF 3 -etherate catalysis in diverse solvents. In this study, focus was placed on the acid catalyst by examining 17 acids in CH 2 Cl 2 , the traditional solvent for two-step, one-flask porphyrin syntheses. In the self-condensation of the carbinol derived from 1-(4-methylbenzoyl)-5-phenyldipyrromethane, porphyrin yields of 9–55% were obtained from the various acids, compared to 20% under TFA catalysis in acetonitrile. A number of catalytic conditions that produce little to no porphyrin in reactions of pyrrole + benzaldehyde afforded good yields of porphyrin and the suppression of scrambling in reactions of dipyrromethanecarbinols. The four best acid catalysts ( InCl 3 , Sc ( OTf ) 3 , Yb ( OTf ) 3 , and Dy ( OTf ) 3 ) initially identified were then examined with dipyrromethanecarbinols bearing challenging substituents (alkyl, pyridyl, or no substituent). The greatest improvement was obtained with the pyridyl substrates. Selected reactions performed on a preparative scale (115 to 460 mg of isolated porphyrin) verified the results of the analytical-scale experiments and revealed the more facile isolation of the porphyrin from reactions performed in CH 2 Cl 2 rather than acetonitrile. This study provides alternatives to the use of TFA/acetonitrile that offer advantages in terms of yield and isolation of the porphyrin without sacrificing suppression of scrambling. Furthermore, the finding that poor catalysts for the benzaldehyde + pyrrole reaction can be excellent catalysts for dipyrromethanecarbinols provides guidance for the identification of other catalysts for use with reactive precursors in porphyrin-forming reactions. DA - 2001/12// PY - 2001/12// DO - 10.1002/jpp.387 VL - 5 IS - 12 SP - 810-823 SN - 1088-4246 KW - dipyrromethane KW - porphyrin KW - acid catalysis KW - pyridyl KW - lanthanide KW - laser desorption mass spectrometry (LD-MS) ER - TY - JOUR TI - Transforming growth factor-beta(1) modifies fibroblast growth factor-2 production in type II cells AU - Li, CM AU - Khosla, J AU - Hoyle, P AU - Sannes, PL T2 - CHEST AB - Transforming growth factor (TGF)-beta(1) is an inflammatory cytokine that plays multiple roles in pulmonary fibrosis. In vascular epithelium, it has been shown to regulate production and activity of fibroblast growth factor (FGF)-2, a potent type II cell mitogen in the lung. Such a relationship could have important consequences in prefibrotic change in the lung alveolus, where reepithelialization of alveolar surfaces is crucial. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-beta(1) or FGF-1, another type II cell mitogen. Isolated rat type II cells were exposed to 0 to 40 ng/mL of TGF-beta(1) or 0 to 500 ng/mL of FGF-1 in serum-free medium for 1 to 3 days. Using a specific immunoassay, significant increases in FGF-2 protein in type II cell lysates were achieved after 1 day of exposure to 100 ng/mL of FGF-1 and after 3 days of treatment with 8 ng/mL of TGF-beta(1). Similarly, transcripts for FGF-2 were dramatically increased with TGF-beta(1) or FGF-1, as were those for FGF receptor (FGFR)-1. These interactions were dramatically effected by the addition of heparin, a model sulfated extracellular matrix (ECM). Heparin as low as 0.01 mg/mL significantly downregulated expression of TGF-beta(1) and FGF-1-stimulated FGF-2 and FGFR-1. These results demonstrate important regulatory links between FGF-2, sulfated ECMs, and both TGF-beta(1) and FGF-1, which could contribute to the modulation of normal cell turnover, development, and repair processes attendant to fibrosis in the lung. DA - 2001/7// PY - 2001/7// DO - 10.1378/chest.120.1_suppl.S60 VL - 120 IS - 1 SP - 60S-61S SN - 0012-3692 KW - alveolar epithelium KW - extracellular matrix KW - growth factors ER - TY - JOUR TI - Role of receptor tyrosine kinases and mitogen-activated protein kinases in metal-induced pulmonary fibrosis AU - Bonner, JC AU - Wang, YZ AU - Zhang, P AU - Rice, A AU - Zhang, LM AU - Adler, K AU - Choe, N AU - Kagan, E T2 - CHEST AB - The proliferation of lung fibroblasts is a key component of pulmonary fibrosis. Several cell-surface receptor tyrosine kinases, including the platelet-derived growth factor receptor (PDGF-R) and epidermal growth factor receptor (EGF-R), mediate fibroblast mitogenesis via the activation of mitogen-activated protein (MAP) kinases. We have developed a model of metal-induced oxidative stress in rats using vanadium pentoxide (V2O5) that is characterized by interstitial and peribronchiolar fibrosis, airway smooth-muscle thickening, and mucous cell metaplasia. In vivo activation of the extracellular signal-regulated kinases (ERKs [ERK-1 and ERK-2]) was demonstrated by immunohistochemistry in fibrotic lesions caused by V2O5 exposure. Moreover, V2O5 injury upregulated platelet-derived growth factor α-receptor messenger RNA (mRNA) and protein in vivo. The mechanism of PDGF-Rα upregulation by V2O5 was elucidated in vitro and involved the release of interleukin-1β by alveolar macrophages, which then activated lung fibroblasts in a paracrine manner to activate p38 MAP kinase, which caused stabilization of PDGF-Rα mRNA. V2O5 also activated ERK-1 and ERK-2 in cultured lung fibroblasts in an oxidant-dependent manner that involved upstream activation of the EGF-R, Raf-1, MAP kinase kinase signaling cascade. In another study, V2O5 exposure of human bronchial epithelial cells in vitro caused the release of mitogenic activity for human lung fibroblasts that was abolished by a neutralizing antibody against heparin-binding epidermal growth factor-like growth factor. Induction of heparin-binding epidermal growth factor-like growth factor mRNA and protein by V2O5in vitro was reduced by the MAP kinase kinase inhibitor PD98059 and the p38 MAP kinase inhibitor SB203580. Finally, the intraperitoneal administration of tyrosine kinase inhibitors specific for either the PDGF-R or the EGF-R (tyrphostins AG1296 and AG1478, respectively) significantly reduced pulmonary fibrosis in rats exposed to V2O5. Collectively, these studies have identified signaling pathways and inducible genes activated by V2O5-stimulated oxidative stress that may offer potential targets for therapeutic intervention of pulmonary fibrosis. DA - 2001/7// PY - 2001/7// DO - 10.1378/chest.120.1_suppl.S55 VL - 120 IS - 1 SP - 55S-56S SN - 0012-3692 ER - TY - JOUR TI - PGE2 triggers recovery of transmucosal resistance via EP receptor cross talk in porcine ischemia-injured ileum AU - Blikslager, Anthony T. AU - Pell, Susan M. AU - Young, Karen M. T2 - American Journal of Physiology-Gastrointestinal and Liver Physiology AB - 16,16-Dimethyl-PGE2 (PGE2) may interact with one of four prostaglandin type E (EP) receptors, which signal via cAMP (via EP2 or EP4 receptors) or intracellular Ca(2+) (via EP1 receptors). Furthermore, EP3 receptors have several splice variants, which may signal via cAMP or intracellular Ca(2+). We sought to determine the PGE2 receptor interactions that mediate recovery of transmucosal resistance (R) in ischemia-injured porcine ileum. Porcine ileum was subjected to 45 min of ischemia, after which the mucosa was mounted in Ussing chambers. Tissues were pretreated with indomethacin (5 microM). Treatment with the EP1, EP2, EP3, and EP4 agonist PGE2 (1 microM) elevated R twofold and significantly increased tissue cAMP content, whereas the EP2 and EP4 agonist deoxy-PGE1 (1 microM) or the EP1 and EP3 agonist sulprostone (1 microM) had no effect. However, a combination of deoxy-PGE1 and sulprostone stimulated synergistic elevations in R and tissue cAMP content. Furthermore, treatment of tissues with deoxy-PGE1 and the Ca(2+) ionophore A-23187 stimulated synergistic increases in R and cAMP, indicating that PGE2 triggers recovery of R via EP receptor cross talk mechanisms involving cAMP and intracellular Ca(2+). DA - 2001/8// PY - 2001/8// DO - 10.1152/ajpgi.2001.281.2.g375 VL - 281 IS - 2 SP - G375-G381 J2 - American Journal of Physiology-Gastrointestinal and Liver Physiology LA - en OP - SN - 0193-1857 1522-1547 UR - http://dx.doi.org/10.1152/ajpgi.2001.281.2.G375 DB - Crossref KW - mucosa KW - barrier function KW - adenosine 3 ' 5 '-cyclic monophosphate KW - G protein KW - short-circuit current ER - TY - JOUR TI - Molecular simulation of complete phase diagrams for binary mixtures AU - Lamm, MH AU - Hall, CK T2 - AICHE JOURNAL AB - Abstract Vapor‐liquid, vapor‐solid and liquid‐solid coexistence lines are calculated for binary mixtures of Lennard‐Jones spheres using Monte Carlo simulation and the Gibbs‐Duhem integration technique. Complete phase diagrams showing equilibrium between vapor, liquid and solid phases are constructed for binary Lennard‐Jones mixtures with diameter ratios ranging from σ 11 /σ 22 = 0.85–1.0 and attractive well‐depth ratios ranging from ϵ 11 /ϵ 22 = 0.625−1.6, at a reduced pressure P*≡Pσ /ϵ 11 = 0.002. The Lorentz‐Berthelot combining rules are used to calculate the cross‐species interaction parameters σ 12 and ϵ 12 . The variation in the shape of the complete phase diagrams change as a function of the diameter ratio σ 11 /σ 22 and well‐depth ratio ϵ 11 /ϵ 22 is systematically explored. The phase diagrams found here resemble those found experimentally for argon‐methane, iodine‐sulfur, water‐sodium chloride, water‐silver nitrate, water‐potassium nitrate, and p‐dichlorobenzene‐p‐dibromobenzene. DA - 2001/7// PY - 2001/7// DO - 10.1002/aic.690470718 VL - 47 IS - 7 SP - 1664-1675 SN - 1547-5905 ER - TY - JOUR TI - Interleukin-13, a mediator of subepithelial fibrosis, enhances growth factor production and proliferation in human airway epithelial cells AU - Booth, B AU - Bonner, J AU - Akley, N AU - Macchione, M AU - Adler, K AU - Martin, LD T2 - CHEST AB - Subepithelial fibrosis is a prominent feature of the remodeled asthmatic airway. The cytokine interleukin (IL)-13, implicated as a mediator in the development of asthma, induces a significant degree of subepithelial fibrosis in the lungs of transgenic mice. Since IL-13 has been shown to exert effects on the airway epithelium, including the development of a mucous phenotype, we have begun to determine whether IL-13 provokes production of factors from the epithelium that could elicit the observed subepithelial fibrotic response. In the studies reported herein, injured airways with regions of regenerating/differentiating cells and regions of normal fully differentiated cells have been mimicked by examining the effects of IL-13 on normal human bronchial epithelial cells during mucociliary differentiation in air/liquid interface culture. Exposure of normal human bronchial epithelial cells to IL-13 resulted in increased production of soluble transforming growth factor (TGF)-α, with the growth factor interacting in an autocrine manner with the epidermal growth factor receptor. Production of soluble TGF-α was very rapid, with a threefold increase observed in response to IL-13 (10 ng/mL) by 1 h of exposure. Continuous exposure to IL-13 throughout the course of mucociliary differentiation (a total of 10 days) resulted in a twofold increase in cell number by day 7 when cells are differentiated. Exposure to IL-13 (10 ng/mL; 24 h) provoked a threefold increase in proliferation once the cells were differentiated, an effect that could be duplicated in differ- entiated, but not undifferentiated cells, by the direct addition of TGF-α (5 ng/mL or 25 ng/mL; 24 h). Proliferation of differentiated cells in response to continuous IL-13 treatment was followed 2 days later by a decrease in proliferation compared to control mice. Soluble TGF-α, however, continued to be produced from these nonproliferating cultures. Thus, an increase in soluble TGF-α in response to IL-13 may serve to promote proliferation of injured epithelial cells in an autocrine manner. Once this proliferative effect is no longer necessary, the soluble TGF-α may promote proliferation of other cells. These data suggest that any injury to the airway epithelium resulting in the production of IL-13 from infiltrating inflammatory cells may provoke the release of soluble TGF-α from the airway epithelium. The availability of this growth factor may contribute to the subepithelial fibrosis observed in chronic asthma. DA - 2001/7// PY - 2001/7// DO - 10.1378/chest.120.1_suppl.S15 VL - 120 IS - 1 SP - 15S-15S SN - 0012-3692 ER - TY - JOUR TI - Immunopathology of Bartonella vinsonii (berkhoffii) in experimentally infected dogs AU - Pappalardo, BL AU - Brown, TT AU - Tompkins, M AU - Breitschwerdt, EB T2 - VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY AB - Following natural infection with Bartonella, dogs and humans develop comparable disease manifestations including endocarditis, peliosis hepatis, and granulomatous disease. As the immunologic response to infection in these hosts has not been clearly established, data presented here was derived from the experimental infection of six specific pathogen free (SPF) beagles with a known pathogenic strain of Bartonella. Six dogs were inoculated intravenously with 10(9)cfu of B. vinsonii ssp. berkhoffii and six control dogs were injected intravenously with an equivalent volume of sterile saline. Despite production of substantial levels of specific antibody, blood culture and molecular analyses indicated that Bartonella established chronic infection in these dogs. Flow cytometric analysis of monocytes indicated impaired bacterial phagocytosis during chronic Bartonella infection. There was also a sustained decrease in the percentage of CD8+ lymphocytes in the peripheral blood. Moreover, modulation of adhesion molecule expression (downregulation of L-selectin, VLA-4, and LFA-1) on CD8+ lymphocytes suggested quantitative and qualitative impairment of this cell subset in Bartonella-infected dogs. When compared with control dogs, flow cytometric analysis of lymph node (LN) cells from B. vinsonii infected dogs revealed an expanded population of CD4+ T cells with an apparent naïve phenotype (CD45RA+/CD62L+/CD49D(dim)). However, fewer B cells from infected dogs expressed cell-surface MHC II, implicating impaired antigen presentation to helper T cells within LN. Taken together, results from this study indicate that B. vinsonii establishes chronic infection in dogs which may result in immune suppression characterized by defects in monocytic phagocytosis, an impaired subset of CD8+ T lymphocytes, and impaired antigen presentation within LN. DA - 2001/12// PY - 2001/12// DO - 10.1016/S0165-2427(01)00372-5 VL - 83 IS - 3-4 SP - 125-147 SN - 1873-2534 KW - dog KW - Bartonella KW - immunosuppression ER - TY - JOUR TI - Electron microscopic observations of stratum corneum intercellular lipids in normal and atopic dogs AU - Inman, AO AU - Olivry, T AU - Dunston, SM AU - Monteiro-Riviere, NA AU - Gatto, H T2 - VETERINARY PATHOLOGY AB - The barrier function of mammalian skin is maintained by intercellular stratum corneum lipids. In human patients with atopic dermatitis, an abnormal lipid barrier results in dry skin and increased transepidermal water loss. At this time, it is not known if a defective lipid barrier is present in atopic dogs. Normal and atopic canine skin were postfixed in ruthenium tetroxide and studied using transmission electron microscopy to determine structural differences within stratum corneum lipids. Intercellular lipid lamellae were graded on a semiquantitative scale. The deposition of stratum corneum lipid lamellae in atopic canine skin appeared markedly heterogeneous compared with that seen in normal canine skin. When present, the lamellae often exhibited an abnormal structure. The continuity and thickness of the intercellular lipid lamellae were significantly less in nonlesional atopic than in normal canine skin. These preliminary observations suggest that the epidermal lipid barrier is defective in atopic canine skin. Additional studies are needed to further characterize the biochemical defect and to possibly correct it with nutritional and/or pharmacologic intervention. DA - 2001/11// PY - 2001/11// DO - 10.1354/vp.38-6-720 VL - 38 IS - 6 SP - 720-723 SN - 1544-2217 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035197191&partnerID=MN8TOARS KW - barrier defect KW - canine atopic dermatitis KW - dogs KW - lipid lamellae KW - ruthenium tetroxide ER - TY - JOUR TI - Efficiency study of estimators for a treatment effect in a pretest-posttest trial AU - Yang, L AU - Tsiatis, AA T2 - AMERICAN STATISTICIAN AB - Several possible methods used to evaluate treatment effects in a randomized pretest–posttest trial with two treatment groups are the two-sample t test, the paired t test, analysis of covariance I (ANCOVA I), the analysis of covariance II (ANCOVA II), and generalized estimating equations (GEE). The ANCOVA I includes treatment and baseline response as covariates in a linear model and ANCOVA II additionally includes an interaction term between the baseline response and treatment indicator as a covariate. The parameters in the ANCOVAI and ANCOVAII models are generally estimated using ordinary least squares. In this article, a semiparametric model, which makes no assumptions about the response distributions, is used. The asymptotic properties of the estimators derived from these five methods and their relative efficiencies are discussed under this semiparametric model. We show that all these methods yield consistent estimators for the treatment effect which have asymptotically normal distributions under the semiparametric model. The GEE and the ANCOVA II estimators are asymptotically equivalent and the most efficient. The estimators from other three methods are less efficient except under some special conditions which are outlined in the article. DA - 2001/11// PY - 2001/11// DO - 10.1198/000313001753272466 VL - 55 IS - 4 SP - 314-321 SN - 0003-1305 KW - analysis of covariance KW - GEE model KW - paired t test KW - semiparametric model KW - two-sample t test ER - TY - JOUR TI - Effect of a charge relay on the vibrational frequencies of carbonmonoxy iron porphine adducts: The coupling of changes in axial ligand bond strength and porphine core size AU - Franzen, S T2 - JOURNAL OF THE AMERICAN CHEMICAL SOCIETY AB - The effect of a charge relay involving Asp-His-Fe in peroxidase enzymes is explored using density functional theory (DFT) calculations of vibrational spectra and potential energy surfaces of carbonmonoxy model systems. The series of models consists of a carbonmonoxy iron porphine molecule with a trans imidazole ligand hydrogen-bonded to six different partners at the Ndelta position. Calculations on the oxy system and on models of the Asp-His-Ser catalytic triad of serine proteases were also performed to obtain an understanding of how the redistribution of charge in these systems may contribute to enzymatic function. The goal of the study is to relate the experimental frequencies in resonance Raman and Fourier transform infrared studies to bonding that is important for the function of heme enzymes. Calculations of both axial and in-plane modes exhibit trends that agree with experimental data. Comparisons of the charge distribution on the different models show that polarization of iron carbonomonoxy bonds consistent with the mechanism for peroxidase function leads to a frequency reduction in the C-O stretching mode nuCO. The combination of axial trans sigma-bonding and pi-bonding effects that include expansion of the porphine core result in little change in the Fe-C stretching frequency nuFe-CO in the series of molecules studied with different Ndelta-H hydrogen bonding. A particular role for the core size is discussed that demonstrates the applicability of trends observed in vibrational spectroscopy of hemes to the charge relay mechanism and other axial ligation effects. The bonding interactions described account for the increase in electron density on bound diatomic ligands, which is required for peroxidase function. DA - 2001/12/19/ PY - 2001/12/19/ DO - 10.1021/ja0108988 VL - 123 IS - 50 SP - 12578-12589 SN - 1520-5126 ER - TY - JOUR TI - Bioavailability and disposition of sodium and procaine penicillin G (benzylpenicillin) administered orally with milk to calves AU - Musser, JMB AU - Anderson, KL T2 - JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS AB - Eighteen 1‐week‐old Holstein calves were randomly assigned to one of three groups: (a) sodium penicillin G administered intravenously, (b) sodium penicillin G administered orally, or (c) procaine penicillin G administered orally. All calves were dosed with penicillin G at 4.0 mg/kg BW. At 5 weeks of age, the calves were dosed again. Blood samples were taken serially for 24 h after both dosings. Plasma was assayed for penicillin G by high performance liquid chromatography (HPLC). For i.v. administration, the area under the concentration–time curve ( AUC ), 7456 and 5508 ng/mL h, and systemic clearance, 0.54 and 0.73 L/kg h, were significantly different ( P < 0.05) at 1 and 5 weeks of age, respectively. There were no significant differences between orally administered sodium and procaine penicillin G within the same age groups. Following oral (p.o.) administration, there were significant differences ( P < 0.01) at 1 and 5 weeks of age in the AUC , 760 and 409 ng/mL h, terminal half‐life, 2.1 and 1.6 h, time of maximum concentration ( T MAX ), 3.0 and 2.3 h, and maximum plasma concentration ( C MAX ), 85 and 58 ng/mL, respectively. Bioavailability was 10.2 and 7.4% at 1 and 5 weeks, respectively. DA - 2001/6// PY - 2001/6// DO - 10.1046/j.1365-2885.2001.00325.x VL - 24 IS - 3 SP - 161-169 SN - 0140-7783 ER - TY - JOUR TI - Autoimmune blistering dermatoses in domestic animals AU - Olivry, T AU - Chan, LS T2 - CLINICS IN DERMATOLOGY AB - Pemphigus foliaceus (PF) is the most common antibody-mediated autoimmune skin disease of dogs. Desmoglein-1 (DSG1), the major human PF antigen, represents only a minor autoantigen in canine PF (cPF). A recent immunomapping study proposed desmocollin-1 (DSC1) as a relevant candidate autoantigen for cPF. To investigate this hypothesis, 85 cPF sera were screened for the presence of anti-DSC1 IgG using indirect immunofluorescence (IIF) on live canine DSC1-transfected 293T cells. Seventy-five sera contained detectable antikeratinocyte IgG on IIF using footpad substrate (IIFpos cPF), while 10 did not (IIFneg cPF). Sera from 35 healthy dogs, eight from exfoliative superficial pyoderma (ESP)-affected dogs and 21 dogs with non-PF autoimmune blistering skin diseases served as controls. All sera were tested concurrently by IIF on canine DSG1-transfected as well as nontransfected cells. None of the healthy dog or ESP sera labelled any of the transfected or nontransfected cells. Fifty-seven of 75 IIFpos cPF (86%) and 7/10 of IIFneg cPF sera (70%) contained detectable anti-DSC1 IgG. None of these sera recognized nontransfected cells. Five cPF sera (6%) recognized DSG1 in addition to DSC1. Finally, 5/21 (24%) sera from dogs with non-PF autoimmune blistering diseases contained low anti-DSC1 IgG titers. In 7/10 dogs (70%), from whom serial serum samples were collected during treatment, anti-DSC1 IgG titers decreased in parallel with the reduction in disease clinical severity. Altogether, these findings suggest that DSC1 is a major autoantigen in cPF. DA - 2001/// PY - 2001/// DO - 10.1016/S0738-081X(00)00197-8 VL - 19 IS - 6 SP - 750-760 SN - 1879-1131 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0034756684&partnerID=MN8TOARS ER - TY - JOUR TI - Testing for tick-borne diseases in dogs - A roundtable discussion AU - Ford, R. B. AU - Lappin, M. AU - Levy, S. A. AU - Philipp, M. AU - Breitschwerdt, E. T2 - Veterinary Technician DA - 2001/// PY - 2001/// VL - 22 IS - 4 SP - 184-189 ER - TY - JOUR TI - T-helper cell response to woodchuck hepatitis virus antigens after therapeutic vaccination of chronically-infected animals treated with lamivudine AU - Hervas-Stubbs, S AU - Lasarte, JJ AU - Sarobe, P AU - Vivas, I AU - Condreay, L AU - Cullen, JM AU - Prieto, J AU - Borras-Cuesta, F T2 - JOURNAL OF HEPATOLOGY AB - Immunotherapy of patients chronically-infected with hepatitis B virus (HBV) may have the risk of fulminant hepatitis. This risk might be diminished if immunotherapy was carried out under conditions of low viremia.Five woodchucks chronically-infected with woodchuck hepatitis virus (WHV), a virus closely related to HBV, were treated with lamivudine for 23 weeks. At week 10, when viremia had decreased by 3-5 logs, three woodchucks were vaccinated with woodchuck hepatitis virus surface antigen (WHsAg) plus the T-helper determinant FISEAIIHVLHSR.It was found that the administration of lamivudine only, had no effect on the T-helper response against WHV antigens. By contrast, vaccination induced T-helper responses against WHV antigens, shifting the cytokine profile from Th2 to Th0/Th1, but was without effect on viremia, WHsAg levels, or anti-WHs antibodies. Analysis of liver biopsies showed that lamivudine administration may have reduced hepatic inflammation. By contrast, vaccination clearly enhanced hepatic inflammation. After lamivudine withdrawal, viremia returned to high levels.These results suggest that therapeutic vaccination of chronically-infected woodchucks under conditions of low viremia shifts the cytokine profile against viral antigens towards Th0/Th1. This shift may prevent the efficient induction of anti-WHs antibodies. DA - 2001/7// PY - 2001/7// DO - 10.1016/S0168-8278(01)00063-0 VL - 35 IS - 1 SP - 105-111 SN - 0168-8278 KW - woodchuck hepatitis virus KW - lamivudine KW - Th1/Th2 KW - therapeutic vaccination ER - TY - JOUR TI - Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer AU - Pop, C AU - Chen, YR AU - Smith, B AU - Bose, K AU - Bobay, B AU - Tripathy, A AU - Franzen, S AU - Clark, AC T2 - BIOCHEMISTRY AB - We have investigated the oligomeric properties of procaspase-3 and a mutant that lacks the pro-domain (called pro-less variant). In addition, we have examined the interactions of the 28 amino acid pro-peptide when added in trans to the pro-less variant. By sedimentation equilibrium studies, we have found that procapase-3 is a stable dimer in solution at 25 degrees C and pH 7.2, and we estimate an upper limit for the equilibrium dissociation constant of approximately 50 nM. Considering the expression levels of caspase-3 in Jurkat cells, we predict that procaspase-3 exists as a dimer in vivo. The pro-less variant is also a dimer, with little apparent change in the equilibrium dissociation constant. Thus, in contrast with the long pro-domain caspases, the pro-peptide of caspase-3 does not appear to be involved in dimerization. Results from circular dichroism, fluorescence anisotropy, and FTIR studies demonstrate that the pro-domain interacts weakly with the pro-less variant. The data suggest that the pro-peptide adopts a beta-structure when in contact with the protein, but it is a random coil when free in solution. In addition, when added in trans, the pro-peptide does not inhibit the activity of the mature caspase-3 heterotetramer. On the other hand, the active caspase-3 does not efficiently hydrolyze the pro-domain at the NSVD(9) sequence as occurs when the pro-peptide is in cis to the protease domain. Based on these results, we propose a model for maturation of the procaspase-3 dimer. DA - 2001/11/27/ PY - 2001/11/27/ DO - 10.1021/bi011037e VL - 40 IS - 47 SP - 14224-14235 SN - 0006-2960 ER - TY - JOUR TI - Recombinant Major Antigenic Protein 2 of Ehrlichia canis: a Potential Diagnostic Tool AU - Alleman, A. R. AU - McSherry, L. J. AU - Barbet, A. F. AU - Breitschwerdt, E. B. AU - Sorenson, H. L. AU - Bowie, M. V. AU - Belanger, M. T2 - Journal of Clinical Microbiology AB - The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum from an experimentally infected dog. The recombinant MAP2 (rMAP2) was tested in an ELISA format using 141 serum samples from E. canis immunofluorescent antibody (IFA)-positive and IFA-negative dogs. Fifty-five of the serum samples were from dogs experimentally or naturally infected with E. canis and were previously demonstrated to contain antibodies reactive with E. canis by indirect immunofluorescence assays. The remaining 86 samples, 33 of which were from dogs infected with microorganisms other than E. canis, were seronegative. All of the samples from experimentally infected animals and 36 of the 37 samples from naturally infected animals were found to contain antibodies against rMAP2 of E. canis in the ELISA. Only 3 of 53 IFA-negative samples tested positive on the rMAP2 ELISA. There was 100% agreement among IFA-positive samples from experimentally infected animals, 97.3% agreement among IFA-positive samples from naturally infected animals, and 94.3% agreement among IFA-negative samples, resulting in a 97.2% overall agreement between the two assays. These data suggest that rMAP2 of E. canis could be used as a recombinant test antigen for the serodiagnosis of canine monocytic ehrlichiosis. DA - 2001/7/1/ PY - 2001/7/1/ DO - 10.1128/JCM.39.7.2494-2499.2001 VL - 39 IS - 7 SP - 2494-2499 J2 - Journal of Clinical Microbiology LA - en OP - SN - 0095-1137 UR - http://dx.doi.org/10.1128/JCM.39.7.2494-2499.2001 DB - Crossref ER - TY - JOUR TI - Quenching of porphyrin excited states by adjacent or distant porphyrin cation radicals in molecular arrays AU - Lammi, RK AU - Ambroise, A AU - Wagner, RW AU - Diers, , JR AU - Bocian, DF AU - Holten, D AU - Lindsey, JS T2 - CHEMICAL PHYSICS LETTERS AB - The quenching of photoexcited porphyrins by porphyrin cation radicals at adjacent or distant sites in diphenylethyne-linked porphyrin dyads and triads has been studied using static and time-resolved optical spectroscopy. The excited-state quenching is highly efficient in all cases (>99%) and occurs in ⩽11 ps between adjacent sites (by through-bond energy/charge transfer) and in ⩽55 ps between distant sites (likely by superexchange-assisted energy transfer). The rates can be tuned using the porphyrin-linker connection motif. These results should prove useful in the design of arrays that use electro- or photo-chemically generated porphyrin cation radicals for molecular switching and other applications in molecular photonics. DA - 2001/6/15/ PY - 2001/6/15/ DO - 10.1016/S0009-2614(01)00452-3 VL - 341 IS - 1-2 SP - 35-44 SN - 0009-2614 ER - TY - JOUR TI - Purification of molecularly bridged metal nanoparticle arrays by centrifugation and size exclusion chromatography AU - Novak, JP AU - Nickerson, C AU - Franzen, S AU - Feldheim, DL T2 - ANALYTICAL CHEMISTRY AB - Size exclusion chromatography and centrifugation separation protocols were developed and compared for isolating enriched fractions of phenylethynyl-bridged metal nanoparticle dimers and trimers from the monomeric particle starting material. Both methods enabled the isolation of enriched fractions of a desired array without causing significant sample aggregation or replacement of the phenylethynyl bridge. Solutions containing ca. 70% bridged gold dimers were obtained using either method. The further development of methods for separating discrete arrays of covalently bridged nanoparticle homo and hetero structures is expected to help advance our understanding of collective metal particle electronic structure-function relationships. DA - 2001/12/1/ PY - 2001/12/1/ DO - 10.1021/ac010812t VL - 73 IS - 23 SP - 5758-5761 SN - 0003-2700 ER - TY - JOUR TI - Protein refolding versus aggregation: Computer simulations on an intermediate-resolution protein model AU - Smith, AV AU - Hall, CK T2 - JOURNAL OF MOLECULAR BIOLOGY AB - Computer simulations are performed on a system of eight model peptide chains to study how the competition between protein refolding and aggregation affects the optimal conditions for refolding of four-helix bundles. The discontinuous molecular dynamics algorithm is utilized along with an intermediate-resolution protein model that we developed for this work. Physically, the model is much more detailed than any model used to date for simulations of protein aggregation. Each model residue consists of a detailed, three-bead backbone and a simplified, single-bead side-chain. Excluded volume, hydrogen bond, and hydrophobic interactions are modeled with discontinuous (i.e. hard-sphere and square-well) potentials. Simulations efficiently sample conformational space, and complete folding trajectories from random initial configurations to two four-helix bundles are possible within two days on a single processor workstation. Folding of the bundles follows two main pathways, one through a trimeric intermediate and the other through an intermediate with two dimers. The proportion of trajectories that follow each route is significantly different for the eight-peptide system in this work than in a previously studied four-peptide system, which yields one four-helix bundle, suggesting, as our previous simulations have, that protein folding properties are strongly influenced by the presence of other proteins. Folding of the bundles is optimal within a fixed temperature range, with the high-temperature boundary a function of the complexity of the protein (or oligomer) to be folded and the low-temperature boundary a function of the complexity of the protein's environment. Above the optimal temperature range for folding, the model chains tend to unfold; below the optimal range, the model chains tend to aggregate. As has been seen previously, aggregates have substantial levels of native secondary structure, suggesting that aggregates are composed largely of partially folded intermediates, not denatured chains. DA - 2001/9/7/ PY - 2001/9/7/ DO - 10.1006/jmbi.2001.4845 VL - 312 IS - 1 SP - 187-202 SN - 1089-8638 KW - discontinuous molecular dynamics KW - protein folding KW - protein misfolding KW - aggregation KW - four-helix bundle ER - TY - JOUR TI - Phenotypic diversity of modern Chinese and North American soybean cultivars AU - Cui, ZL AU - Carter, TE AU - Burton, JW AU - Wells, R T2 - CROP SCIENCE AB - Chinese and North American (NA) soybean breeding programs have a 70‐yr history of genetic progress in relative isolation from each other. Because both programs rest upon a genetic base that is primarily Chinese in origin, the actual genetic distinctness of Chinese and NA breeding is not clear. The objectives of this study were to (i) develop a phenotypic similarity (PS) index for a large group of Chinese and NA cultivars, on the basis of biochemical, morphological, and agronomic traits, (ii) compare Chinese and NA cultivars for PS through cluster analysis, and (iii) use results to develop guidelines for management of the contrasting Chinese and NA breeding programs as reservoirs of diversity. Chinese (47) and NA (25) cultivars were evaluated for 25 traits in growth chambers. Traits pleiotropic to maturity were avoided. Significant ( P < 0.05) differences between Chinese and NA cultivars were noted for leaf and seed traits. Multivariate analysis captured 79% of the total genotypic variation among the 72 cultivars and was used to develop PS estimates. Cluster analysis of PS showed a much greater phenotypic diversity among Chinese than among NA cultivars and a striking distinctness between the two groups. The contrasting nature of Chinese and NA cultivars in this study is theorized to reflect that (i) the NA cultivars may trace to a subset of the Chinese cultivar genetic base, and/or (ii) Chinese and NA cultivars may have diverged phenotypically via breeder selection pressure. Cluster results here, based on PS, agreed roughly with previous cluster analyses, which were derived from pedigree analysis. The physical distinctness of NA and Chinese cultivars shows that introgression of Chinese cultivars into NA breeding should broaden NA germplasm's agronomic, morphological, and biochemical diversity. Introgression may be accomplished most effectively by avoiding matings of Chinese and NA cultivars from the same phenotypic cluster. DA - 2001/// PY - 2001/// DO - 10.2135/cropsci2001.1954 VL - 41 IS - 6 SP - 1954-1967 SN - 0011-183X ER - TY - JOUR TI - Overexpression of H- and L-ferritin subunits in lens epithelial cells: Fe metabolism and cellular response to UVB irradiation AU - Goralska, M. AU - Holley, B. L. AU - McGahan, M. C. T2 - Investigative Ophthalmology and Visual Science DA - 2001/// PY - 2001/// VL - 42 IS - 8 SP - 1721-1727 ER - TY - PAT TI - Nucleic acids obtained from the envelope coding region of feline immunodeficiency virus molecular clone designated JSY3 AU - Tompkins, W. A. AU - Tompkins, M. AU - Yang, J.-S. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - Muscle regeneration during hindlimb unloading results in a reduction in muscle size after reloading AU - Mozdziak, PE AU - Pulvermacher, PM AU - Schultz, E T2 - JOURNAL OF APPLIED PHYSIOLOGY AB - The hindlimb-unloading model was used to study the ability of muscle injured in a weightless environment to recover after reloading. Satellite cell mitotic activity and DNA unit size were determined in injured and intact soleus muscles from hindlimb-unloaded and age-matched weight-bearing rats at the conclusion of 28 days of hindlimb unloading, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-unloaded rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb unloading, but they were the same (P > 0.05) as those of weight-bearing rats 2 and 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, number of nuclei per millimeter, and DNA unit size were significantly (P < 0.05) smaller for the injured soleus muscles from hindlimb-unloaded rats than for the soleus muscles from weight-bearing rats at each recovery time. Satellite cell mitotic activity was significantly (P < 0.05) higher in the injured soleus muscles from hindlimb-unloaded rats than from weight-bearing rats 2 wk after reloading, but it was the same (P > 0.05) as in the injured soleus muscles from weight-bearing rats 9 wk after reloading. The injured soleus muscles from hindlimb-unloaded rats failed to achieve weight-bearing muscle size 9 wk after reloading, because incomplete compensation for the decrease in myonuclear accretion and DNA unit size expansion occurred during the unloading period. DA - 2001/7// PY - 2001/7// DO - 10.1152/jappl.2001.91.1.183 VL - 91 IS - 1 SP - 183-190 SN - 8750-7587 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0034967920&partnerID=MN8TOARS KW - notexin KW - satellite cell KW - DNA unit KW - myofiber KW - atrophy ER - TY - JOUR TI - Monte Carlo simulations of complete phase diagrams for binary Lennard-Jones mixtures AU - Lamm, MH AU - Hall, CK T2 - FLUID PHASE EQUILIBRIA AB - Vapor–liquid, vapor–solid, liquid–liquid, and liquid–solid coexistence lines are calculated for binary mixtures of Lennard–Jones spheres with diameter ratio σ11/σ22=0.85, well-depth ratio ϵ11/ϵ22=0.45, and binary interaction parameters δ12=1.0, 0.9, and 0.75, using Monte Carlo simulation and the Gibbs–Duhem integration technique. These calculations allow us to construct complete phase diagrams, i.e. showing equilibrium between vapor, liquid, and solid phases. For the mixture with δ12=1, we find a completely miscible vapor–liquid coexistence region with a eutectic solid–liquid coexistence region. These two regions are separated by a completely miscible liquid phase. For the mixtures with δ12<1, we find that the vapor–liquid and solid–liquid coexistence regions interfere. This interference results in a vapor–solid coexistence region bounded above and below by solid–liquid–vapor coexistence lines. We also find that the mixtures with δ12<1 have a region of liquid–liquid immiscibility that is metastable with respect to the solid–fluid phase equilibria. DA - 2001/6/5/ PY - 2001/6/5/ DO - 10.1016/S0378-3812(01)00378-8 VL - 182 IS - 1-2 SP - 37-46 SN - 1879-0224 KW - molecular simulation KW - liquid-liquid equilibria KW - solid-fluid equilibria KW - Lennard-Jones ER - TY - JOUR TI - Intervertebral disk disease in 10 cats AU - Munana, KR AU - Olby, NJ AU - Sharp, NJH AU - Skeen, TM T2 - JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION AB - The medical records of 10 cats diagnosed with intervertebral disk disease were reviewed. No apparent sex or breed predilection was found. The mean age of cats in the study was 9.8 years. Clinical signs included back pain, difficulty ambulating, and incontinence. Radiographs revealed narrowed disk spaces, mineralized intervertebral disks, and evidence of extradural compression on myelography or computed tomography. All intervertebral disk herniations occurred in the thoracolumbar spine, with a peak incidence at the fourth to fifth lumbar (L4-L5) intervertebral disk space. Eight cats had Hansen's type I intervertebral disk herniation. Surgery was performed in seven cats. All cats judged to have an excellent outcome had undergone surgical decompression. DA - 2001/// PY - 2001/// DO - 10.5326/15473317-37-4-384 VL - 37 IS - 4 SP - 384-389 SN - 0587-2871 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0034766968&partnerID=MN8TOARS ER - TY - JOUR TI - Interim monitoring of group sequential trials using spending functions for the type I and type II error probabilities AU - Pampallona, S AU - Tsiatis, A AU - Kim, K T2 - DRUG INFORMATION JOURNAL DA - 2001/// PY - 2001/// DO - 10.1177/009286150103500408 VL - 35 IS - 4 SP - 1113-1121 SN - 0092-8615 KW - group sequential trials KW - information time KW - error spending function ER - TY - JOUR TI - Genetic background and environment influence palmitate content of soybean seed oil AU - Rebetzke, GJ AU - Pantalone, VR AU - Burton, JW AU - Carter, TE AU - Wilson, RF T2 - CROP SCIENCE AB - Dietary concerns over high saturates contained in edible vegetable oils has stimulated development of soybean [ Glycine max (L.) Merr.] cultivars with reduced palmitate content. Little is known of factors that might influence phenotypic expression of palmitate content among soybean populations varying for presence of a major reduced palmitate allele. The objective of this study was to investigate how environment and genetic background influence palmitate content when introducing the reduced palmitate trait into adapted backgrounds. Crosses were made between reduced palmitate germplasm, N87‐2122‐4 (53 g kg −1 palmitate) and normal palmitate cultivars, A3733, Burlison, Kenwood, P9273, and P9341 (103–123 g kg −1 palmitate). For each cross, F 4:6 lines homozygous for major reduced or normal palmitate alleles were bulked separately into Maturity Groups (MG) II, III, IV, and V, and evaluated in 10 contrasting field environments during 1993. Palmitate content varied between 82 and 90 g kg −1 across southern U.S. and Puerto Rican environments. Much of this environmental variation was associated with changes in minimum temperature during the growing season. Genetic background effects were highly significant ( P < 0.01) with cross means for palmitate content ranging between 81 and 93 g kg −1 Across different maturity groups, palmitate content of the progeny was correlated ( r = 0.94–0.99, P < 0.05) with mean content of the normal palmitate parent, such that for every 1 g kg −1 palmitate increase in the normal palmitate parent there was a 0.32 to 0.51 g kg −1 palmitate increase in the progeny. Genetic background effects were presumed to be associated with action of minor alleles transmitted from the normal palmitate parent. Presence of the reduced palmitate allele was associated with significantly ( P < 0.01) lower stearate (−6 to −13%) and higher oleate (+4 to +10%) contents across all maturity groups. Selection of low palmitate, high‐yielding parents should further decrease palmitate content and produce correlated improvements in stearate and oleate contents to improve overall oil quality in progeny containing reduced palmitate alleles. DA - 2001/// PY - 2001/// DO - 10.2135/cropsci2001.1731 VL - 41 IS - 6 SP - 1731-1736 SN - 0011-183X ER - TY - JOUR TI - FTIR and resonance Raman studies of nitric oxide binding to H93G cavity mutants of myoglobin AU - Thomas, MR AU - Brown, D AU - Franzen, S AU - Boxer, SG T2 - BIOCHEMISTRY AB - Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a five- or six-coordinate Fe−NO complex. The H93G mutation eliminates the covalent attachment between the protein and the proximal ligand, allowing NO to bind H93G possibly from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side. The question of whether NO binds on the distal or proximal side was addressed by FTIR spectroscopy of the N−O vibrational frequency ν̄N-O for a set of Mb mutants that perturb the electrostatic environment of the heme pocket. Vibrational spectra of five- and six-coordinate MbNO complexes indicate that ν̄N-O shifts (by as much as 26 cm-1) to higher energies for the distal mutants H64V and H64V/H93G relative to the energies of wild-type and H93G MbNO, while ν̄N-O is not affected by the proximal side mutation S92A/H93G. This result suggests that NO binds on the distal side of heme in the five- and six-coordinate MbNO complexes of H93G. Additionally, values of the Fe−NO vibrational frequency ν̄Fe-NO as measured by resonance Raman spectroscopy are reported for the distal and proximal double mutants of H93G. These results suggest that ν̄Fe-NO is not very sensitive to mutations that perturb the electrostatic environment of the heme pocket, leading to the observation that ν̄N-O and ν̄Fe-NO are not quantitatively correlated for the MbNO complexes presented here. Furthermore, ν̄N-O and ν̄Fe-NO do not correlate well with equilibrium constants for imidazole binding to the five-coordinate MbNO complexes of the H93G double mutants. The data presented here do not appear to support the presence of π-back-bonding or an inverse trans effect of NO binding in Mb mutants that alter the electrostatic environment of the heme pocket. DA - 2001/12/11/ PY - 2001/12/11/ DO - 10.1021/bi011440l VL - 40 IS - 49 SP - 15047-15056 SN - 0006-2960 ER - TY - JOUR TI - Enhanced expression of mucin genes in a guinea pig model of allergic asthma AU - Li, YH AU - Martin, LD AU - Minnicozzi, M AU - Greenfeder, S AU - Fine, J AU - Pettersen, CA AU - Chorley, B AU - Adler, KB T2 - AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY AB - Section:ChooseTop of pageAbstract <2.3.CO;2 VL - 15 IS - 3 SP - 196-199 SN - 0891-6640 KW - alkylating agent KW - cancer KW - chemotherapy KW - feline KW - lymphoma KW - mast cell tumor KW - nitrosourea ER - TY - JOUR TI - Oxygen isotope variability in bones of wild caught and constant temperature reared sub-adult American alligators AU - Stoskopf, MK AU - Barrick, RE AU - Showers, WJ T2 - JOURNAL OF THERMAL BIOLOGY AB - (1) The mean delta18O(BP) ( per thousandSMOW) for any given bone sampled from captive alligators maintained at high constant temperature was lower (indicative of higher temperatures of bone deposition) than that of the same bone from wild alligators caught in Northern Florida, but these differences were only greater than two standard deviations from the mean for the thoracic vertebrae and metatarsal bones. (2) Inter-bone variability of delta18O(BP) ( per thousandSMOW) was similar for captive alligators maintained at constant temperatures and the wild alligators, but intra-bone variability was much greater in wild alligators. (3) The order of mean delta18O(BP) ( per thousandSMOW) of bones (from highest to lowest) differed between treatment groups. However, intra-bone variability obscured the significance of those differences. Nevertheless, the thoracic vertebra had the highest mean delta18O(BP) ( per thousandSMOW), indicative of lower temperatures, and the lowest variability of bones in both groups of alligators. Conversely, the tibia was one of the warmest and more variable bones in both groups of alligators. (4) The pattern of delta18O(BP) ( per thousandSMOW) values across sites within long bones were identical between alligator treatment groups for the femur and humerus but differed between groups for the tibia and metatarsus, and differed between different long bones. The predicted intra-bone pattern for long bones of increasing delta18O(BP) ( per thousandSMOW) indicative of lower temperatures in more distal sampling sites was only obtained from the femurs. (5) Paired cortical and cancellous bone samples from the same site from all individuals in both treatment groups were available for proximal humeri and distal femurs. delta18O(BP) ( per thousandSMOW) values from cortical bone were more variable than those from cancellous bone for both bones. (6) Cortical bone had lower delta18O(BP) ( per thousandSMOW) values indicative of warmer temperatures than cancellous bone at sites sampled on the proximal humeri and distal femurs of all three animals from both treatment groups. DA - 2001/6// PY - 2001/6// DO - 10.1016/S0306-4565(00)00041-3 VL - 26 IS - 3 SP - 183-191 SN - 0306-4565 KW - bone KW - heterothermy KW - oxygen isotopes KW - alligator KW - reptile KW - thermoregulation ER - TY - JOUR TI - Investigation of porphyrin-forming reactions. Part 4. Examination of the reaction course in syntheses of porphyrins via dipyrromethanecarbinols AU - Geier, GR AU - Littler, BJ AU - Lindsey, JS T2 - JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2 AB - The dipyrromethanecarbinol motif is a key component in rational routes to a wide variety of porphyrinic macrocycles. We have investigated the dipyrromethane-monocarbinol self-condensation and the dipyrromethane-dicarbinol + dipyrromethane condensation under four different acid-catalysis conditions and have characterized the oligomer composition (by LD-MS), yield of porphyrin (by UV–Vis), and yield of N-confused porphyrin (by HPLC). Under conditions giving “no-scrambling”, the condensations are rapid and afford an oligomer composition characterized by (1) absence of scrambling, (2) suppression of acidolysis, and (3) a disproportionate amount of long oligomers from which recovery does not occur. The irreversibility of the reaction and lack of recovery from the long oligomers may explain the lower yields of dipyrromethanecarbinol condensations (<30%) compared with reversible reactions of aldehyde + pyrrole (∼50%). Modest levels of acidolysis and scrambling with the dipyrromethanecarbinols are obtained under reaction conditions that cause extensive scrambling in dipyrromethane + aldehyde condensations. Because both reactions give the same porphyrinogen, the scrambling processes must primarily involve the dipyrromethane or oligomer intermediates rather than the porphyrinogen. The dipyrromethane-monocarbinol self-condensation and dipyrromethane-dicarbinol + dipyrromethane condensation afford quite similar oligomer compositions, suggesting that both follow similar pathways to the porphyrinogen. Reaction conditions that suppress scrambling in the dipyrromethanecarbinol condensations were found to dramatically suppress formation of the N-confused porphyrin. Taken together, these experiments show how the greater reactivity of the dipyrromethanecarbinol unit (compared with the pyrrole + aldehyde reaction) facilitates synthesis of meso-substituted porphyrins without scrambling. DA - 2001/// PY - 2001/// DO - 10.1039/b009101o IS - 5 SP - 712-718 SN - 1472-779X ER - TY - JOUR TI - Investigation of porphyrin-forming reactions. Part 3. The origin of scrambling in dipyrromethane plus aldehyde condensations yielding trans-A(2)B(2)-tetraarylporphyrins AU - Geier, GR AU - Littler, BJ AU - Lindsey, JS T2 - JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2 AB - The dipyrromethane + aldehyde condensation is a common method for the synthesis of trans-A2B2-porphyrins, but is often plagued by scrambling processes that lead to a mixture of porphyrins. The problem of scrambling is more pronounced with unhindered dipyrromethanes (e.g., 5-phenyldipyrromethane) than with hindered dipyrromethanes (e.g., 5-mesityldipyrromethane). We have characterized the oligomer composition (by LD-MS), yield of porphyrin (by UV–Vis), yield of N-confused porphyrin (by HPLC), and level of unreacted aldehyde (by TLC) in dipyrromethane + aldehyde condensations leading to trans-A2B2-porphyrins. Reaction conditions known to suppress scrambling in reactions involving 5-phenyldipyrromethane (PDPM) were compared to conditions known to provide extensive scrambling. The low-scrambling conditions were found to suppress scrambling by inhibiting reaction of oligomer fragments generated by acid-induced cleavage of the dipyrromethane, rather than by inhibiting acidolysis itself. However, such reaction conditions were also found to inhibit the condensation, leading to low yields (<10%) of porphyrin. The condensation of PDPM + aldehyde was also compared to reactions involving 5-mesityldipyrromethane (MDPM) to understand why trans-A2B2-porphyrins can be prepared in good yield devoid of scrambling from reactions using MDPM. The absence of scrambling in MDPM + aldehyde condensations was due to the resistance of MDPM to acidolysis. Taken together, these studies provide insight into the origin of scrambling with different types of substrates under different reaction conditions in the dipyrromethane + aldehyde route to trans-A2B2-porphyrins. DA - 2001/// PY - 2001/// DO - 10.1039/b009098k IS - 5 SP - 701-711 SN - 1472-779X ER - TY - JOUR TI - Investigation of porphyrin-forming reactions. Part 2. Examination of the reaction course in two-step, one-flask syntheses of meso-substituted porphyrins AU - Geier, GR AU - Lindsey, JS T2 - JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2 AB - The reaction course leading to meso-substituted porphyrins was examined for reversibility (formation of oligomers, formation of α- and β-pyrrole linkages), inactivation of the acid catalyst, homogeneity of the reaction medium, and the pathway of oligomer formation. The methodology employed enabled characterization of the oligomer composition (LD-MS), yield of porphyrin (UV–Vis), yield of N-confused porphyrin (HPLC), and level of unreacted aldehyde (TLC). Experiments were performed with benzaldehyde and pyrrole with catalysis by TFA or BF3–Et2O. Key observations include the following. (1) Reactions with BF3–Et2O exhibited reversible exchange of oligomers throughout the reaction. With TFA, the oligomer exchange processes were reversible at short reaction times, but became largely irreversible over the course of several hours. (2) The BF3–Et2O activity declined during the course of the reaction, whereas that of TFA was little changed. (3) The reaction medium remained homogeneous at 10 mM pyrrole + aldehyde. (4) Dipyrromethanes comprised of α- but not β-linkages underwent cleavage with either TFA or BF3–Et2O. (5) Condensations with carbinol intermediates (pyrrole-carbinol, dipyrromethane-monocarbinol, dipyrromethane-dicarbinol) provided rapid reactions, lower yields of porphyrin, and longer oligomers than typical in reactions of pyrrole + benzaldehyde. Higher porphyrin yields were obtained with BF3–Et2O than TFA, which is attributed to the more facile recovery from longer oligomers with the former versus the latter catalyst. Collectively, these and other observations lead to a model for the aldehyde + pyrrole condensation comprised of a combination of irreversible and reversible reactions in oligomer formation, irreversible side reactions (formation of dipyrrins, β-linkages), and slow inactivation of the catalyst (BF3–Et2O). DA - 2001/// PY - 2001/// DO - 10.1039/b009092l IS - 5 SP - 687-700 SN - 1472-779X ER - TY - JOUR TI - Investigation of porphyrin-forming reactions. Part 1. Pyrrole plus aldehyde oligomerization in two-step, one-flask syntheses of meso-substituted porphyrins AU - Geier, GR AU - Lindsey, JS T2 - JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2 AB - A deep understanding of the two-step, one-flask synthesis of meso-substituted porphyrins requires characterization of the acyclic oligomers, which typically constitute ≥50% of the products. We have employed laser desorption mass spectrometry (LD-MS) to obtain a qualitative yet high resolution view of the oligomer content of crude oxidized reaction mixtures. This methodology complements other analytical methods which provide information regarding yields of porphyrin, other macrocyclic products, and unreacted aldehyde. Our findings include the following. (1) Crude oxidized porphyrin reaction mixtures provided peaks in the LD-MS spectrum which were readily assigned to oligomers (m/z 100–2000) derived from pyrrole–aldehyde condensation. The oligomers comprised one of four series depending on the ratio of pyrrole and aldehyde units. (2) Quite disparate reaction conditions that gave good yields of porphyrin also afforded a similar oligomer composition. (3) The change in the nature of the oligomers over time was readily monitored. (4) The maximum yield of porphyrin and the maximum diversity of the oligomer composition were attained at similar times. (5) The decline in yield of porphyrin at long reaction time was accompanied by truncation, not elongation, of oligomers. (6) The onset of the truncation of oligomers and the decline in yield of porphyrin were accompanied by a decrease of the aldehyde to low levels. (7) Pyrrole was incorporated into the growing oligomers more rapidly than aldehyde. Taken together, these studies show how various conditions alter the course of the pyrrole–aldehyde condensation. DA - 2001/// PY - 2001/// DO - 10.1039/b009088n IS - 5 SP - 677-686 SN - 1472-779X ER - TY - JOUR TI - Imprint status of M6P/IGF2R and IGF2 in chickens AU - Nolan, CM AU - Killian, JK AU - Petitte, JN AU - Jirtle, RL T2 - DEVELOPMENT GENES AND EVOLUTION DA - 2001/4// PY - 2001/4// DO - 10.1007/s004270000132 VL - 211 IS - 4 SP - 179-183 SN - 0949-944X UR - http://europepmc.org/abstract/med/11455432 KW - genomic imprinting KW - biallelic expression KW - chicken KW - IGF2 KW - M6P/IGF2R ER - TY - JOUR TI - Identification of a gene associated with bit resistance in Heliothis virescens AU - Gahan, LJ AU - Gould, F AU - Heckel, DG T2 - SCIENCE AB - Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used for pest control. Bt-resistant insect strains have been studied, but the molecular basis of resistance has remained elusive. Here, we show that disruption of a cadherin-superfamily gene by retrotransposon-mediated insertion was linked to high levels of resistance to the Bt toxin Cry1Ac in the cotton pest Heliothis virescens. Monitoring the early phases of Bt resistance evolution in the field has been viewed as crucial but extremely difficult, especially when resistance is recessive. Our findings enable efficient DNA-based screening for resistant heterozygotes by directly detecting the recessive allele. DA - 2001/8/3/ PY - 2001/8/3/ DO - 10.1126/science.1060949 VL - 293 IS - 5531 SP - 857-860 SN - 0036-8075 ER - TY - JOUR TI - Histone-like proteins from fish are lethal to the parasitic dinoflagellate Amyloodinium ocellatum AU - Noga, E. J. AU - Fan, Z. Q. AU - Silphaduang, U. T2 - Parasitology DA - 2001/// PY - 2001/// VL - 123 IS - 2001 July SP - 57-65 ER - TY - JOUR TI - Functional equivalence of late gene promoters in bean golden mosaic virus with those in tomato golden mosaic virus AU - Hung, HC AU - Petty, ITD T2 - JOURNAL OF GENERAL VIROLOGY AB - In the bipartite geminivirus tomato golden mosaic virus (TGMV), the activity of late gene promoters is up-regulated by the multifunctional viral protein AL2. Cis-acting sequences required for AL2-mediated promoter responses have not been well characterized. However, nucleotide sequence analysis has implicated a motif termed the conserved late element (CLE). The CLE is present in TGMV and many other begomoviruses, although it is not ubiquitous. Here we analysed the regulation of late gene expression in bean golden mosaic virus (BGMV), one of the begomoviruses which lacks the CLE. Transient reporter gene assays showed that BGMV late gene promoters were trans-activated in Nicotiana benthamiana protoplasts, both by the homologous BGMV AL2 protein and by the heterologous TGMV AL2 protein. The BGMV AL2 protein also trans-activated TGMV late gene promoters. Consistent with these results, we found that hybrid viruses with the late gene promoters exchanged between BGMV and TGMV were viable in planta. DA - 2001/3// PY - 2001/3// DO - 10.1099/0022-1317-82-3-667 VL - 82 SP - 667-672 SN - 0022-1317 ER - TY - JOUR TI - Formation and characterization of the inclusion complexes between poly(dimethylsiloxane) and polyacrylonitrile with gamma-cyclodextrin AU - Porbeni, FE AU - Edeki, EM AU - Shin, ID AU - Tonelli, AE T2 - POLYMER AB - Poly(dimethylsiloxane) and polyacrylonitrile have been observed to form inclusion complexes (ICs) with γ-cyclodextrin. These complexes were prepared by a solution-heating technique. Their structural features were observed with the use of: FTIR, TGA, WAXS, and 13C-NMR. FTIR identifies absorption peaks of the guest polymer molecules in the cyclodextrin. Thermal decomposition shows that the ICs have a higher thermal stability than the pure γ-cyclodextrin. The wide angle X-ray diffraction of the complexes indicates that the ICs form channel structures. CP-MAS 13C-NMR spectra of the ICs show that γ-cyclodextrin, in the presence of a polymer guest, adopts a more symmetric conformation when compared to its pure state. DA - 2001/7// PY - 2001/7// DO - 10.1016/S0032-3861(01)00181-1 VL - 42 IS - 16 SP - 6907-6912 SN - 1873-2291 KW - cyclodextrins KW - poly(dimethylisiloxane) (PDMS) KW - polyacrylonitrile (PAN) ER - TY - JOUR TI - Evaluation of sandbar shiner as a surrogate for assessing health risks to the endangered Cape Fear shiner AU - Chittick, B AU - Stoskopf, M AU - Heil, N AU - Levine, J AU - Law, M T2 - JOURNAL OF AQUATIC ANIMAL HEALTH AB - The health status of the endangered Cape Fear shiner Notropis mekistocholas and the suitability of using the sympatric sandbar shiner N. scepticus as an investigative surrogate were evaluated. Forty Cape Fear shiners from three sites and 50 sandbar shiners from five sites were examined. Findings on gill biopsies, fin biopsies, and skin scrapings were limited to low levels of parasitism and gill aneurysms. Eighty-three bacterial isolates representing 13 aerobic species were cultured from the gastrointestinal tracts. A picornavirus was isolated from one pooled sample of sandbar shiners at one site. Forty-three percent of shiners (12 Cape Fear shiners, 27 sandbar shiners) had granulomas in various tissues of the body, 26% (6 Cape Fear, 17 sandbar) had encysted trematodes, 16% (2 Cape Fear, 12 sandbar) had protozoal aggregates in muscle or connective tissue, and 26% (22 Cape Fear shiners, 1 sandbar shiner) had mild, moderate, or moderately severe hepatic vacuolization. Other microscopic lesions included mild parasitism and degrees of inflammation in various tissues. Sandbar shiners appeared to be suitable surrogates for the Cape Fear shiner in bacteriological sampling; however, parasitic, viral, and nonhepatic histological lesions were more common in sandbar shiners. Findings from this study warrant further investigation of sandbar shiners as a conservative bioindicator species for the presence of potential health risks to Cape Fear shiners. DA - 2001/6// PY - 2001/6// DO - 10.1577/1548-8667(2001)013<0086:EOSSAA>2.0.CO;2 VL - 13 IS - 2 SP - 86-95 SN - 1548-8667 ER - TY - JOUR TI - Characterization and cloning of a major high molecular weight house dust mite allergen (Der f 15) for dogs AU - McCall, C AU - Hunter, S AU - Stedman, K AU - Weber, E AU - Hillier, A AU - Bozic, C AU - Rivoire, B AU - Olivry, T T2 - VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY AB - Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae (D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109 kDa (98/109 kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109 kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2 kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15. DA - 2001/2/10/ PY - 2001/2/10/ DO - 10.1016/S0165-2427(00)00258-0 VL - 78 IS - 3-4 SP - 231-247 SN - 1873-2534 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035086765&partnerID=MN8TOARS KW - atopic dermatitis KW - mite allergen KW - allergen KW - dog KW - IgE ER - TY - JOUR TI - Changes in biosynthesis and degradation of juvenile hormone during breeding by burying beetles: a reproductive or social role? AU - Scott, MP AU - Trumbo, ST AU - Neese, PA AU - Bailey, WD AU - Roe, RM T2 - JOURNAL OF INSECT PHYSIOLOGY AB - Burying beetles, Nicrophorus orbicollis, depend on the location of an unpredictable resource, a small vertebrate carcass, for reproduction. When they discover a carcass, they undergo a correlated rapid rise in titers of juvenile hormone (JH) in the hemolymph and ovarian development. This study investigates the regulation of the changes in JH during breeding in both male and female burying beetles and the role of JH in ovarian development. JH biosynthesis by the corpora allata (CA), measured in vitro, increased in females within an hour of their discovery of a carcass and increased later in males. After returning to low rates as oviposition began, JH biosynthesis rose again 3 days later in females but not in males. Neither the ovaries nor testes synthesized JH. There was a concomitant fall in JH esterase activity within 12 h of discovery of the carcass in both males and females. Although the rise in JH titers and biosynthesis and the fall in JH esterase is correlated with ovarian development, application of methoprene or JH III in the absence of a carcass did not result in vitellogenin uptake by the oocytes. Therefore, we conclude that, in spite of the rapid rise in JH before oviposition, it is not sufficient to regulate vitellogenin synthesis and/or its uptake by the ovaries. We suggest that its role has been preempted to organize social behavior and coordinate parental behavior between mates. DA - 2001/3// PY - 2001/3// DO - 10.1016/S0022-1910(00)00116-5 VL - 47 IS - 3 SP - 295-302 SN - 1879-1611 KW - JH biosynthesis KW - JH esterase KW - vitellogenesis KW - ovarian development KW - Nicrophorus ER - TY - JOUR TI - A spontaneous canine model of mucous membrane (cicatricial) pemphigoid, an autoimmune blistering disease affecting mucosae and mucocutaneous junctions AU - Olivry, T AU - Dunston, SM AU - Schachter, M AU - Xu, LT AU - Nguyen, N AU - Marinkovich, MP AU - Chan, LS T2 - JOURNAL OF AUTOIMMUNITY AB - Mucous membrane pemphigoid (MMP) is a rare autoimmune blistering dermatosis of humans that was previously known as cicatricial pemphigoid. It is characterized by vesicles, ulcers and scarring that affect predominantly mucosae and mucocutaneous junctions. Circulating autoantibodies recognize epitopes on basement membrane proteins such as collagen XVII or laminin-5/6. Herein, we describe the clinico-pathological and immunological characteristics of 17 dogs afflicted with a dermatosis homologous to MMP of humans. Patients exhibited vesicles and erosions predominantly on mucous membranes or mucocutaneous junctions of the mouth, nose, eyes, genitalia or anus. Histopathology revealed subepithelial vesicles with variable dermal inflammation. Direct immunofluorescence demonstrated IgG or complement at the dermoepithelial junction. Indirect immunofluorescence using salt-split epithelia permitted the detection of circulating basement membrane-specific IgG autoantibodies in 15 cases. In 11 patients, autoantibodies recognized the NC16A segment of collagen XVII, as determined by salt-split indirect immunofluorescence, immunoblotting using canine keratinocytes and ELISA with synthetic canine peptides. In one dog, autoantiodies bound to the dermal side of salt-split epithelia and recognized epitopes within the 30 kDa carboxy-terminal segment of human collagen XVII. Canine MMP, like its human counterpart, exhibits distinctive clinical signs and histopathological lesions, yet circulating autoantibodies target different antigenic epitopes. This spontaneous canine model of MMP could prove useful for studies on the pathogenesis or therapy of this human disease. DA - 2001/6// PY - 2001/6// DO - 10.1006/jaut.2001.0510 VL - 16 IS - 4 SP - 411-421 SN - 0896-8411 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0034955036&partnerID=MN8TOARS KW - basement membrane KW - cicatricial KW - dog KW - skin ER - TY - JOUR TI - A multicenter, matched case-control study of risk factors for equine laminitis AU - Alford, P AU - Geller, S AU - Richardson, B AU - Slater, M AU - Honnas, C AU - Foreman, J AU - Robinson, J AU - Messer, M AU - Roberts, M AU - Goble, D AU - Hood, D AU - Chaffin, M T2 - PREVENTIVE VETERINARY MEDICINE AB - Risk factors for equine laminitis were examined in a prospective case-control study of the 258 cases seen at six collaborating veterinary teaching hospitals over a 32-month period. Case-control pairs were matched on institution, clinician, and season of diagnosis. The 90% of case-control pairs (78 acute, 155 chronic) that had complete data for age, gender, and breed were used in separate conditional logistic-regression models for acute and chronic laminitis. There was an increase in risk for horses with acute laminitis from 5 to 7 years of age (OR 4.7, 95% CI 1.3–16) and from 13 to 31 years of age (OR 3.9, 95% CI 1.3–12) (both compared to <5 years); risk was increased for chronic laminitis from 10 to 14 years (OR 3, 95% CI 1.4–6.8) and from 15 to 38 years (OR 2.9, 95% CI 1.4–6.1) (both compared to <6 years). Mares — but not stallions — were more likely than geldings to develop acute laminitis (OR 2.6, 95% CI 1.1–6.2) and chronic laminitis (OR 2.0, 95% CI 1.1–3.6). In the small acute-laminitis data set, the breed variable was collapsed into three categories: Thoroughbred (THB, reference), the Quarter Horse (QH), and other (non-QH-THB). The non-QH-THB group was at increased risk of acute laminitis (OR 3.8, 95% CI 1.2–11.8). For the seven breed-group categories used in the chronic-laminitis model, however, all non-THB breed groups appeared significantly at risk as compared to the THB, with odds ratios ranging from 3.3 (95% CI 1.3–8.30) for the QH to 9.1 (95% CI 2.1–39.3) for ponies. DA - 2001/5/1/ PY - 2001/5/1/ DO - 10.1016/S0167-5877(01)00188-X VL - 49 IS - 3-4 SP - 209-222 SN - 0167-5877 KW - laminitis KW - horse KW - age influence KW - breed influence KW - gender ER - TY - JOUR TI - beta-Mannosidase from Thermotoga species AU - Parker, K. N. AU - Chhabra, S. AU - Lam, D. AU - Snead, M. A. AU - Mathur, E. J. AU - Kelly, R. M. T2 - Hyperthermophilic enzymes. Part A CN - QP601 .M49 vol. 330 DA - 2001/// PY - 2001/// VL - 330 SP - 238-246 ER - TY - JOUR TI - beta-Mannanases from Thermotoga species AU - Chhabra, S. AU - Parker, K. N. AU - Lam, D. AU - Callen, W. AU - Snead, M. A. AU - Mathur, E. J. AU - Short, J. M. AU - Kelly, R. M. T2 - Hyperthermophilic enzymes. Part A CN - QP601 .M49 vol. 330 DA - 2001/// PY - 2001/// VL - 330 SP - 224-238 ER - TY - JOUR TI - beta-Endoglucanase from Pyrococcus furiosus AU - Cady, S. G. AU - Bauer, M. W. AU - Callen, W. AU - Snead, M. A. AU - Mathur, E. J. AU - Short, J. M. AU - Kelly, R. M. T2 - Hyperthermophilic enzymes. Part A CN - QP601 .M49 vol. 330 DA - 2001/// PY - 2001/// VL - 330 SP - 346-354 ER - TY - JOUR TI - alpha-Glucosidase from Pyrococcus furiosus AU - Chang, S. T. AU - Parker, K. N. AU - Bauer, M. W. AU - Kelly, R. M. T2 - Hyperthermophilic enzymes. Part A CN - QP601 .M49 vol. 330 DA - 2001/// PY - 2001/// VL - 330 SP - 260-269 ER - TY - JOUR TI - alpha-D-galactosidases from Thermotoga species AU - Miller, E. S. AU - Parker, K. N. AU - Liebl, W. AU - Lam, D. AU - Callen, W. AU - Snead, M. A. AU - Mathur, E. J. AU - Short, J. M. AU - Kelly, R. M. T2 - Hyperthermophilic enzymes. Part A CN - QP601 .M49 vol. 330 DA - 2001/// PY - 2001/// VL - 330 SP - 246-260 ER - TY - JOUR TI - Xylose isomerases from Thermotoga AU - Vieille, C. AU - Sriprapundh, D. AU - Kelly, R. M. AU - Zeikus, J. G. T2 - Hyperthermophilic enzymes. Part A CN - QP601 .M49 vol. 330 DA - 2001/// PY - 2001/// VL - 330 SP - 215-224 ER - TY - JOUR TI - Vanadium stimulates human bronchial epithelial cells to produce heparin-binding epidermal growth factor-like growth factor - A mitogen for lung fibroblasts AU - Zhang, LM AU - Rice, AB AU - Adler, K AU - Sannes, P AU - Martin, L AU - Gladwell, W AU - Koo, JS AU - Gray, TE AU - Bonner, JC T2 - AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY AB - Section:ChooseTop of pageAbstract <15 kg) with osteosarcoma (OSA) of the axial skeleton were reviewed to determine prevalence of metastasis and survival associated with this neoplasm. All dogs were treated with more than 1 mode of therapy including palliative radiation (n = 12), definitive radiation (n = 8), surgery (n = 7), chemotherapy (n = 12), or some combination of these therapies. Metastasis was documented in 10 of 22 dogs (46%), and the median survival for all dogs was 137 days. Primary cause of death was local tumor recurrence (54%). Breed (retriever versus purebred versus mixed-breed survival was 100, 182, and 264 days, respectively) and radiation therapy protocol (survival in dogs treated with palliative radiation therapy versus those treated with definitive radiation therapy was 79 and 265 days, respectively) were significantly related to survival (P < .05). Prevalence of metastasis and median survival for large-breed dogs with axial skeleton OSA seems to be similar to that reported for large-breed dogs with appendicular skeleton OSA. Definitive radiation therapy may have a role in the treatment of axial skeleton osteosarcoma. DA - 2001/// PY - 2001/// DO - 10.1892/0891-6640(2001)015<0120:RAOASO>2.3.CO;2 VL - 15 IS - 2 SP - 120-124 SN - 0891-6640 KW - canine KW - metastasis KW - radiation therapy KW - retriever ER - TY - JOUR TI - Resolving domains of interdigitated phospholipid membranes with 95 GHz spin labeling EPR AU - Smirnov, AI AU - Smirnova, TI T2 - APPLIED MAGNETIC RESONANCE DA - 2001/// PY - 2001/// DO - 10.1007/BF03162420 VL - 21 IS - 3-4 SP - 453-467 SN - 1613-7507 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035528064&partnerID=MN8TOARS ER - TY - JOUR TI - Polymer-cyclodextrin inclusion compounds: Toward new aspects of their inclusion mechanism AU - Rusa, CC AU - Luca, C AU - Tonelli, AE T2 - MACROMOLECULES AB - α-Cyclodextrin inclusion compounds were prepared in the presence of a polymer and a small molecule model for the polymer repeat unit. By means of this technique, we are able to demonstrate that α-cyclodextrin prefers the inclusion of the longer molecular chain guest. Comparison of the cyclodextrin inclusion compounds formed with poly(ε-caprolactone) and hexanoic acid, separately and from solution containing both poly(ε-caprolactone) and hexanoic acid in varying amounts, enables us to draw certain conclusions concerning both the thermodynamic and kinetic aspects of poly(ε-caprolactone)−α-cyclodextrin inclusion compound formation. Differential scanning calorimetry, Fourier transform infrared, and wide-angle X-ray diffraction have been used to verify the formation and successfully characterize all inclusion compounds. DA - 2001/2/27/ PY - 2001/2/27/ DO - 10.1021/ma001868c VL - 34 IS - 5 SP - 1318-1322 SN - 0024-9297 ER - TY - JOUR TI - Multiple imputation methods for estimating regression coefficients in the competing risks model with missing cause of failure AU - Lu, KF AU - Tsiatis, AA T2 - BIOMETRICS AB - We propose a method to estimate the regression coefficients in a competing risks model where the cause-specific hazard for the cause of interest is related to covariates through a proportional hazards relationship and when cause of failure is missing for some individuals. We use multiple imputation procedures to impute missing cause of failure, where the probability that a missing cause is the cause of interest may depend on auxiliary covariates, and combine the maximum partial likelihood estimators computed from several imputed data sets into an estimator that is consistent and asymptotically normal. A consistent estimator for the asymptotic variance is also derived. Simulation results suggest the relevance of the theory in finite samples. Results are also illustrated with data from a breast cancer study. DA - 2001/12// PY - 2001/12// DO - 10.1111/j.0006-341X.2001.01191.x VL - 57 IS - 4 SP - 1191-1197 SN - 0006-341X KW - cause-specific hazard KW - missing at random KW - partial likelihood ER - TY - JOUR TI - Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter - Basal transcription is mediated by Sp1 and Sp3 AU - Baek, SJ AU - Horowitz, JM AU - Eling, TE T2 - JOURNAL OF BIOLOGICAL CHEMISTRY AB - Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is known to be associated with anti-tumorigenic activity and belongs to the transforming growth factor-β superfamily. In the present study, we cloned the promoter region (−3500 to +41) and investigated the transcriptional regulatory mechanisms of the basal expression of the human NAG-1 gene. Several potential transcription factor-binding sites in this region were identified. Based on the results from clones of nested deletions, the construct between −133 and +41 base pairs contains three Sp1-binding sites (Sp1-A, Sp1-B, and Sp1-C), which confer basal transcription specific activity of NAG-1 expression. When the Sp1-C site was mutated (GG to TT), a 60–80% decrease in promoter activity was observed in HCT-116 cells. Gel shift, co-transfection, and chromatin immunoprecipitation assays showed that the Sp transcription factors bind to the Sp1-binding sites and transactivate NAG-1 expression. In addition, chicken ovalbumin upstream promoter-transcription factor 1 can interact with the C-terminal region of Sp1 and Sp3 proteins and induce NAG-1 promoter activity through Sp1 and Sp3 transcription factors. These results identify the critical regulatory regions for the human NAG-1 basal promoter. Furthermore, the results suggest that the level of expression of the NAG-1 gene will depend on the availability of Sp proteins and on co-factors such as chicken ovalbumin upstream promoter-transcription factor 1. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is known to be associated with anti-tumorigenic activity and belongs to the transforming growth factor-β superfamily. In the present study, we cloned the promoter region (−3500 to +41) and investigated the transcriptional regulatory mechanisms of the basal expression of the human NAG-1 gene. Several potential transcription factor-binding sites in this region were identified. Based on the results from clones of nested deletions, the construct between −133 and +41 base pairs contains three Sp1-binding sites (Sp1-A, Sp1-B, and Sp1-C), which confer basal transcription specific activity of NAG-1 expression. When the Sp1-C site was mutated (GG to TT), a 60–80% decrease in promoter activity was observed in HCT-116 cells. Gel shift, co-transfection, and chromatin immunoprecipitation assays showed that the Sp transcription factors bind to the Sp1-binding sites and transactivate NAG-1 expression. In addition, chicken ovalbumin upstream promoter-transcription factor 1 can interact with the C-terminal region of Sp1 and Sp3 proteins and induce NAG-1 promoter activity through Sp1 and Sp3 transcription factors. These results identify the critical regulatory regions for the human NAG-1 basal promoter. Furthermore, the results suggest that the level of expression of the NAG-1 gene will depend on the availability of Sp proteins and on co-factors such as chicken ovalbumin upstream promoter-transcription factor 1. transforming growth factor-β nonsteroidal antiinflammatory drug-activated gene-1 base pair polymerase chain reaction electrophoretic mobility shift assay chicken ovalbumin upstream promoter-transcription factor 1 growth and differentiation factor kilobase(s) glutathioneS-transferase The TGF-β1 superfamily genes play roles in adult and embryonic growth and development, in inflammation, and in repair including angiogenesis (1Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1730) Google Scholar). This superfamily includes bone morphogenetic proteins, cartilage-derived morphogenetic proteins, Mullerrian inhibiting substance, activins, inhibins, growth and differentiation factors (GDFs), and TGF-β (1Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1730) Google Scholar). Multiple lines of evidence suggest that the TGF-β signaling pathway is a potent tumor suppressor of human colorectal carcinogenesis (2Markowitz S. Roberts A. Cytokine Growth Factor Rev. 1996; 7: 93-102Crossref PubMed Scopus (396) Google Scholar). In addition, overexpression of TGF-β in arterial endothelium results in apoptosis (3Schulick A.H. Taylor A.J. Zuo W. Qiu C.B. Dong G. Woodward R.N. Agah R. Roberts A.B. Virmani R. Dichek D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6983-6988Crossref PubMed Scopus (158) Google Scholar) and alteration of the TGF-β signal transduction pathway results in a reduction of tumorigenecity (4Zhu Y. Richardson J.A. Parada L.F. Graff J.M. Cell. 1998; 94: 703-714Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 5Wang J. Sun L. Myeroff L. Wang X. Gentry L.E. Yang J. Liang J. Zborowska E. Markowitz S. Willson J.K. Brattain M.G. J. Biol. Chem. 1995; 270: 22044-22049Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Thus, signaling and transcriptional regulation of TGF-β play an important role in tumor development. Several promoters for members of the TGF-β superfamily have been described. The TGF-β isoforms exhibit great diversity in their promoter structure (6Malipiero U. Holler M. Werner U. Fontana A. Biochem. Biophys. Res. Commun. 1990; 171: 1145-1151Crossref PubMed Scopus (30) Google Scholar), whereas the promoter organization of the bone morphogenetic proteins appears to be highly conserved (7Gitelman S.E. Kobrin M. Lee A. Fet V. Lyons K. Hogan B.L.M. Derynck R. Mamm. Genome. 1997; 8: 212-214Crossref PubMed Scopus (16) Google Scholar). NAG-1 was identified as a pro-apoptotic and anti-tumorigenic protein (8Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar). The human cDNA has been cloned by six different groups (also known as MIC-1, PDF, GDF-15, PLAB, and PTGFB) and encodes a secreted protein with homology to members of the TGF-β superfamily (8Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar, 9Bootcov M.R. Bauskin A.R. Valenzuela S.M. Moore A.G. Bansal M. He X.Y. Zhang H.P. Donnellan M. Mahler S. Pryor K. Walsh B.J. Nicholson R.C. Fairlie W.D. Por S.B. Robbins J.M. Breit S.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11514-11519Crossref PubMed Scopus (880) Google Scholar, 10Lawton L.N. Bonaldo M.F. Jelenc P.C. Qiu L. Baumes S.A. Marcelino R.A. Jesus G.M. Wellington S. Knowles J.A. Warburton D. Brown S. Bento-Soares M. Gene (Amst.). 1997; 203: 17-26Crossref PubMed Scopus (152) Google Scholar, 11Paralkar V.M. Vail A.L. Grasser W.A. Brown T.A. Xu H. Vukicevic S. Ke H.Z. Qi H. Owen T.A. Thompson D.D. J. Biol. Chem. 1998; 273: 13760-13767Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 12Yokoyama-Kobayashi M. Saeki M. Sekine S. Kato S. J. Biochem. (Tokyo). 1997; 122: 622-626Crossref PubMed Scopus (95) Google Scholar, 13Hromas R. Hufford M. Sutton J. Xu D. Li Y. Lu L. Biochim. Biophys. Acta. 1997; 1354: 40-44Crossref PubMed Scopus (195) Google Scholar). Moreover, NAG-1 expression is up-regulated in human colorectal cancer cells by several nonsteroidal anti-inflammatory drugs that are known to have anti-tumorigenic and pro-apoptotic activities (8Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar). It is also induced by the tumor suppressor gene p53 (14Li P.X. Wong J. Ayed A. Ngo D. Brade A.M. Arrowsmith C. Austin R.C. Klamut H.J. J. Biol. Chem. 2000; 275: 20127-20135Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 15Tan M. Wang Y. Guan K. Sun Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 109-114Crossref PubMed Scopus (230) Google Scholar). Although abundant expression of NAG-1 is present in the placenta and prostate, significant expression is also observed in colon and kidney (11Paralkar V.M. Vail A.L. Grasser W.A. Brown T.A. Xu H. Vukicevic S. Ke H.Z. Qi H. Owen T.A. Thompson D.D. J. Biol. Chem. 1998; 273: 13760-13767Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). At present, no information on the tissue-specific and/or basal transcriptional regulation is available. A recent publication reporting the partial promoter sequences of the human NAG-1 (also known as PTGFB) gene provide little information into the regulatory mechanisms of NAG-1 expression (10Lawton L.N. Bonaldo M.F. Jelenc P.C. Qiu L. Baumes S.A. Marcelino R.A. Jesus G.M. Wellington S. Knowles J.A. Warburton D. Brown S. Bento-Soares M. Gene (Amst.). 1997; 203: 17-26Crossref PubMed Scopus (152) Google Scholar). Therefore, further characterization of the NAG-1 promoter is required to elucidate the mechanisms for regulation of anti-tumorigenic TGF-β family proteins. In the present study, we isolated and characterized the NAG-1 promoter region and identified several cis-acting elements. In the proximal region of the NAG-1 promoter, Sp1-binding sites regulate basal NAG-1 expression. In addition, we have shown a difference between Sp1 and Sp3, with regards to the binding of Sp1 sites as well as to transactivation of NAG-1 gene. We also show that Sp1 and/or Sp3 proteins interact with COUP-TF1 transcription factor to transactivate NAG-1 promoter activity. These data provide a link between Sp1 transcription factors and anti-tumorigenic protein expression. Recombinant bacteriophage clones were isolated by the plaque hybridization method (16Benton W.D. Davis R.W. Science. 1977; 196: 180-182Crossref PubMed Scopus (2924) Google Scholar) from human genomic chromosome-19 specific library (American Type Culture Collection, Manassas, VA). The library was screened using a DNA probe containing 966 bp of the NAG-1 promoter, which was labeled by random priming (Ambion, Austin, TX) in the presence of [α-32P]dCTP (PerkinElmer Life Sciences; 3,000 Ci/mmol). After three rounds of screening, large scale phage DNA was prepared according to the procedures of Helms (1987) using DEAE-cellulose chromatography. One of the positive clones (λNAG61) was purified and identified to contain the 9-kb NAG-1 promoter and the full-length NAG-1 gene. The insert of λNAG61 clone was digested with EcoRI restriction enzyme and subcloned into plasmid vector (pBlueScript II) for sequencing analysis. In addition, the 3.5-kb SmaI fragment containing the NAG-1 promoter was cloned into pGLBasic3 luciferase reporter vector (Promega, Madison, WI) and assayed for luciferase activity. A −3500 to +41-bp human NAG-1 promoter construct (pNAG3500/LUC) was generated as follows. λNAG61 clone was digested with SmaI restriction enzyme, and the 3.5-kb fragment was isolated and ligated into pGLBasic3 luciferase vector (Promega) digested with SmaI. The deletion clones were generated from pNAG3500/LUC using ExoIII nuclease. COUP-TF1 in expression vector (pRSCOUP-TF1) and in pGEM7 vector (pGEM7-COUP-TF1) were generously provided by Dr. Tsai (Baylor College of Medicine, Houston, TX). Sp1 (pCMV4-Sp1flu) and Sp3 (pCMV4-Sp3flu) in expression vectors were described previously (17Udvadia A.J. Templeton D.J. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3953-3957Crossref PubMed Scopus (198) Google Scholar, 18Udvadia A.J. Rogers K.T. Higgins P.D. Murata Y. Martin K.H. Humphrey P.A. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3265-3269Crossref PubMed Scopus (187) Google Scholar). HNF-4α in an expression vector (pCMV-HNF4α) was generated by reverse transcriptase-PCR from HCT-116 cells using two primers: sense, 5′-CGGAGACGGACAAAGTCCGGGGAC-3′, and antisense, 5′-GTCCCCGGACTTTGTCCGTCTCCG-3′. The Sp1-binding sites (Sp1-A, Sp1-B, and Sp1-C) were deleted or mutated using the Quick Change site-mutagenesis kit (Stratagene). For the deletion of Sp1-A or Sp1-BC sites on the −1086, −474, and −133 promoter regions, the following primers were used: ΔSP1-A sense, 5′-CCCCCTAAATACACCCCCAGACTGTGGTCATTGG-3′; ΔSP1-A antisense, 5′-AAACACTCCAATGACCACAGTCTGGGGGTGTATTTAG-3′; ΔSP1-BC sense, 5′-ACTCTGCAGGCAGGGGGAGGAAGACGGACAAAG-3′; and ΔSP1-BC antisense, 5′-CCCCGGACTTTGTCCGTCTTCCTCCCCCTGCC-3′. For the point mutation of Sp1-BC sites on the −133 promoter region, the following primers were used: mut1 sense, 5′-GGAGTT CGGGACTGAGCAGGCGGAGACGGA-3′; mut1 antisense, 5′-TCCGTCTCCGCCTGCTCAGTCCCGAACTCC-3′; mut2 sense, 5′-GGAGGGCGGGACTGAGCATT CGGAGACGGA-3′; mut2 antisense, 5′-TCCGTCTCCGAATGCTCAGTCCCGCCCTCC-3′; mut12 sense, 5′-GGAGTT CGGGACTGAGCATT CGGAGACGGA-3′; and mut12 antisense, 5′-TCCGTCTCCGAATGCTCAGTCCCGAACTCC-3′. Site-specific mutations (underlined) were confirmed by DNA sequencing. HCT-116 cells were plated in 6-well plates at 2 × 105cells/well in McCoy's 5A medium supplemented with 10% fetal bovine serum. After growth for 16 h, plasmid mixtures containing 1 μg of NAG-1 promoter linked to luciferase and 0.1 μg of pRL-TK (Promega) were transfected by LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocol. After 48 h transfection, the cells were harvested in 1× luciferase lysis buffer, and luciferase activity was determined and normalized to the pRL-TK luciferase activity using a dual luciferase assay kit (Promega). Nuclear extracts were prepared as described previously (19Kim Y. Fischer S.M. J. Biol. Chem. 1998; 273: 27686-27694Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) with the following modification. In detail, exponentially growing cells were washed with cold phosphate-buffered saline. The cells were pelleted in a microcentrifuge tube for 10 s and incubated in two packed cell volumes of buffer A (10 mmHEPES, pH 8.0, 0.5% Nonidet P-40, 1.5 mmMgCl2, 10 mm KCl, 0.5 mmdithiothreitol, and 200 mm sucrose) for 5 min at 4 °C with flicking of the tube. The crude nuclei were collected by microcentrifugation for 15 s, and the pellets were rinsed with buffer A. After centrifugation, the pellets were resuspended in one packed cell volume of buffer B (20 mm HEPES, pH 7.9, 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, and 1.0 mm dithiothreitol) and incubated on a rocking platform for 30 min at 4 °C. The crude nuclear extracts were clarified by microcentrifugation for 5 min, and the supernatants were diluted 1:1 with buffer C (20 mmHEPES, pH 7.9, 100 mm KCl, 0.2 mm EDTA, 20% glycerol, and 1 mm dithiothreitol). Protease inhibitor mixture (Sigma; catalog number P8340) was added to each type of buffer. Nuclear extracts were frozen in liquid nitrogen and kept at −80 °C until use. For the gel shift assay, double-stranded oligonucleotides (Life Technologies, Inc.) were end-labeled with [γ-32P]ATP by T4 polynucleotide kinase (New England Biolabs). Assays were performed by incubating 4 μg of nuclear extracts in the binding buffer (Promega) containing 200,000 cpm of labeled probe for 20 min at room temperature. To assure the specific binding of transcription factors to the probe, the probe was chased by 1-, 10-, and 50-fold molar excesses of cold wild type or mutant oligonucleotide. For the supershift experiments, antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with nuclear extracts on ice for 30 min before adding to the binding reaction. The samples were then electrophoresed on 5% nondenaturing polyacrylamide gels with 0.5× TBE, and the gels were dried and subjected to autoradiography. Exponentially growing HCT-116 cells in 150 mm plates were fixed by the addition of 1% formaldehyde to the medium for 10 min. The cells were scraped and collected by centrifugation. After washing with phosphate-buffered saline, the cell pellets were resuspended in 300 μl of lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris, pH 8.1) containing proteinase inhibitor (Sigma). The cells were then sonicated 10 times for 10 s each time, and the lysates were cleared by centrifugation and diluted in immunoprecipitation buffer (0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mm NaCl) containing proteinase inhibitor. Chromatin solution was precleared for 1 h at 4 °C on protein A-Sepharose 4B beads, and 20 μg of sonicated salmon sperm DNA, 50 μg of bovine serum albumin, and 10 μg of Sp1 or Sp3 antibody (Santa Cruz, CA) were added for overnight. The beads were washed four times with 1 ml of washing buffer (0.1% Triton X-100, 20 mm Tris, pH 8.0, 150 mm NaCl, 2 mm EDTA) and eluted by three successive 5-min incubations with 150 μl of elution buffer (1% SDS, 100 mmNaHCO3). After combining the three eluted solutions, 1 μl of RNase (10 mg/ml) was added, and NaCl was adjusted to 0.3m. Cross-links were reversed by heating at 65 °C for 4 h, and DNA was extracted by adding 10 μl of 2 mTris, pH 6.8, 10 μl of 0.5 m EDTA, 2 μl of proteinase K (20 mg/ml) at 45 °C for 2 h. After phenol/chlorform extraction and ethanol precipitation, the pellet was resuspended in 50 μl of H2O. For the PCR reaction (25 cycles), 4 μl of DNA and 0.1 μg of each primer were added into the PCR mixture solution (Sigma). After electrophoresis, the gel was transferred onto Nylon membrane and subjected to Sourthern analysis using32P-labeled oligonucleotide (5′-GGAGGGCGGGACTGAGCAGGCGGA-3′) as a probe. The GST-Sp1 clones were described previously (20Murata Y. Kim H.G. Rogers K.T. Udvadia A.J. Horowitz J.M. J. Biol. Chem. 1994; 269: 20674-20681Abstract Full Text PDF PubMed Google Scholar). The full-length Sp3 was amplified from pBSK-Sp3/flu (21Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar) by use of PCR and 5′ primer (5′-GGGGGATCCGCCACCATGAATTCCGGGCCATCGCCG-3′) and 3′ primer (5′-GGAATTCCTCCATTGTCTCATTTCCAG-3′). Amplified Sp3 product was subcloned into pCR-Blunt-TOPO (Invitrogen, CA) and subsequently subcloned into pGEX-2TK (Amersham Pharmacia Biotech) at theBamHI and EcoRI sites to create pGEX-Sp3-ALL. The pGEX-Sp3-B/C/Zn/D clone was generated by subcloning directly from pCR-M2/flu (21Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar) into the BamHI site of pGEX-2TK. The pGEX-Sp3-A/B was generated by cleaving pBSKSp3/flu (21Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar) withBamHI and BpmI. A 1300-bp DNA fragment was then isolated, treated with mung bean nuclease to create blunt ends, and subcloned into the SmaI site of pGEX-2TK. COUP-TF1 coding sequence was transcribed with Sp6 polymerase, followed by translation with rabbit reticulocyte and [35S]methionine. Fusion proteins were synthesized inEscherichia coli BL21 (Stratagene). Expression was induced by isopropthiogalactopyranoside (0.1 mm), and whole cell extracts were prepared by sonication and centrifugation. GST or GST fusion proteins were incubated for 30 min at 4 °C with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) in NETN buffer (20 mm Tris, pH 8.0, 100 mm NaCl, 1 mm EDTA, and 0.5% Nonidet P-40). Subsequently, the beads were washed four times with NETN buffer and purified. GST alone or GST fusion proteins were run on SDS-polyacrylamide gel electrophoresis to determine the concentrations of these proteins after purification. Equal amounts of GST or GST fusion proteins were incubated with35S-labeled COUP-TF1 that had been produced in a rabbitin vitro translation kit (Promega) in NETN buffer. Finally, the beads were washed five times with NETN buffer and analyzed on 12% SDS-polyacrylamide gel electrophoresis. The gel was dried and exposed to x-ray film. The human NAG-1 gene has been mapped to chromosome 19p12–13.1 using fluorescence in situhybridization (10Lawton L.N. Bonaldo M.F. Jelenc P.C. Qiu L. Baumes S.A. Marcelino R.A. Jesus G.M. Wellington S. Knowles J.A. Warburton D. Brown S. Bento-Soares M. Gene (Amst.). 1997; 203: 17-26Crossref PubMed Scopus (152) Google Scholar). To clone and investigate transcriptional regulation of the NAG-1 gene, human chromosome 19-specific library constructed in λ charon 40 (American Type Culture Collection number 57766) was screened with a radiolabeled 966-bp fragment corresponding to the 5′ end of the human NAG-1 gene (PTGFB). One million plaques were screened, and eight positive clones were isolated and subjected to plaque purification. Southern blot and sequencing analysis indicated that one clone, λNAG61, contained the full-length of NAG-1 including two exons and a 9-kb 5′-flanking region (Fig. 1). The 9-kb promoter region was subcloned into a plasmid vector, sequenced on both strands by standard sequencing methods, and deposited into GenBank (accession number AF305420). Alternatively, the BAC clone (BAC182K4) containing NAG-1 gene was purchased from Research Genetics and digested with several restriction enzymes, and the NAG-1 gene was identified by Southern blot analysis (data not shown). Putative cis-acting elements were examined in the human NAG-1 sequences within the 3.5-kb promoter using the TESS (www.cbil.upenn.edu/cgi-bin/tess/tess33?_if=1&RQ= WELCOME) and TFsearch (www.cbrc.jp/research/db/TFSEARCH.html) programs. Numerous potential cis-acting elements were identified including but not limited to binding sites for Sp1, AP-1 (activator protein), AP-2, GR (glucocorticoid receptor), NF-κB, H4TF-1 (H4 histone gene-inducing element), HiNF-A (cell cycle-regulated human H1 histone gene), c-Myc, and MIG-1 (GC-box binding zinc finger protein) (Fig. 2). At this point, it was unclear which of these putative elements played a role in transcriptional regulation of NAG-1. However, Sp1-binding sites in the proximal promoter region of NAG-1 were interesting, because many TGF-β family members are regulated by Sp1 transcription factors (22Feng Z.M. Bardin C.W. Chen C.L. Mol. Endocrinol. 1989; 3: 939-948Crossref PubMed Scopus (59) Google Scholar, 23Kim Y. Ratziu V. Choi S.G. Lalazar A. Theiss G. Dang Q. Kim S.J. Friedman S.L. J. Biol. Chem. 1998; 273: 33750-33758Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 24Lafyatis R. Lechleider R. Kim S.J. Jakowlew S. Roberts A.B. Sporn M.B. J. Biol. Chem. 1990; 265: 19128-19136Abstract Full Text PDF PubMed Google Scholar). During evolution, the important transcriptional binding sites are conserved between species. Therefore, the human and mouse NAG-1 promoters were compared with the conserved cis-acting elements. Mouse NAG-1 gene (also known as mouse GDF-15) has been reported (25Hsiao E.C. Koniaris L.G. Zimmers-Koniaris T. Sebald S.M. Huynh T.V. Lee S.J. Mol. Cell. Biol. 2000; 20: 3742-3751Crossref PubMed Scopus (225) Google Scholar). Sequence comparison between the human and mouse NAG-1 promoter in the ∼700-bp region revealed a 39% homology with considerable gapping (Fig. 3). Thus, significant homology is not apparent in the NAG-1 promoter between human and mouse, implying that human and mouse NAG-1 may be regulated in different ways. However, the major potential transcriptional binding sites such as TATA, Sp1, AP-1, and Nkx-2 in the human and mouse NAG-1 promoters are present in the same sequential order, indicating the significance of these sites on NAG-1 expression.Figure 3Alignment of the ∼700-bp region of human and mouse NAG-1 promoters by the GCG program. The top strand represents human NAG-1 promoter, whereas the bottom strand represents mouse NAG-1 promoter (also known as GDF-15; GenBankTM accession number AJ011967). Theunderlined sequences represent several specific sequences with high homology between the two promoters.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To evaluate the importance of cis-acting elements in conferring basal NAG-1 expression, the 3.5-kbSmaI fragment was released from λNAG61 (Fig. 1) and ligated to pGLBasic3 luciferase vector (pNAG3500/LUC). In addition, we identified and confirmed a transcription initiation site using primer extension and 5′-rapid amplification of cDNA end experiments (data not shown), which is consistent with a previous report showing putative transcription initiation site (10Lawton L.N. Bonaldo M.F. Jelenc P.C. Qiu L. Baumes S.A. Marcelino R.A. Jesus G.M. Wellington S. Knowles J.A. Warburton D. Brown S. Bento-Soares M. Gene (Amst.). 1997; 203: 17-26Crossref PubMed Scopus (152) Google Scholar). Deletion analysis of the 3.5-kb promoter sequence was performed using ExoIII nuclease digestion to generate the deletion clones pNAG1739/LUC, pNAG1086/LUC, pNAG474/LUC, and pNAG133/LUC. The properly deleted DNA fragment without introducing any mutations was confirmed by sequencing the junction sequences. Each construct was transfected into HCT-116 human colorectal cancer cells, which have been reported to express endogenous NAG-1 (8Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar). As an internal control, the plasmid pRL-TK (Promega) was used for adjusting transfection efficiency. As shown in Fig.4, the results obtained from the luciferase studies demonstrated a large increase in luciferase activity between constructs pNAG474/LUC and pNAG1086/LUC. In contrast, there was a decrease in luciferase activity between pNAG1086/LUC and pNAG1739/LUC. A similar decrease was found between pNAG1739/LUC and pNAG3500/LUC. These data suggest that there is a positive regulator between −474 and −1086. As a negative control, pGLBasic3 promoterless vector was also transfected into HCT-116 cells and resulted in no significant luciferase activity. The construct pNAG133/LUC showed around 17-fold induction of luciferase activity compared with pGLBasic3, suggesting that this region may contain basal transcription machinery. The promoter region within −133 bp was further investigated to find any specific cis-acting elements conferring basal expression. From the sequencing analysis, we found three potential Sp1 binding sites (Sp1-A, Sp1-B, and Sp1-C) (Fig. 5), and one of them (Sp1-C) is conserved in the mouse promoter (Fig. 3). In addition, many TGF-β superfamily genes are regulated by Sp1 transcription factors, which are usually associated with basal promoter activity. Therefore, the proximal region containing Sp1 sites were further examined to elucidate basal promoter activity. First, three Sp1 sites (Sp1-A, Sp1-B, and Sp1-C) were deleted to see whether these sites were important for basal transcription of NAG-1. As shown in Fig.5 A, the transfection of constructs with deleted Sp1-A sites resulted in ∼50% reduction of luciferase activity compared with the appropriate wild type constructs, pNAG1086/LUC, pNAG474/LUC, and pNAG133/LUC, respectively. In contrast, the constructs with deleted Sp1-BC sites resulted in much greater reduction (∼60–80%) compared with wild type constructs. Interestingly, the pNAG1086 construct showed the highest luciferase activity, and the deletion of Sp1-BC sites resulted in significant reduction of luciferase activity, indicating that Sp1-BC sites are crucial sites to regulate the basal level of NAG-1 promoter activity. It also suggests that a positive regulator is located in the region between −474 and −1086, which may independently work to the Sp1-BC site. Further examination was performed using point mutation. Sp1-BC sites were point-mutated using a site-directed mutagenesis kit. In the Sp1-BC sites, GG was mutated to TT in either one site or both sites (Fig. 5 B). The point mutation in Sp1-B site resulted in only ∼30% reduction of luciferase activity, whereas the mutation of either the Sp1-C site or both sites showed ∼70% reduction of luciferase activity. This suggests that the Sp1-C site is more crucial than Sp1-B site in terms of basal NAG-1 expression (Fig. 5 B). The point mutation of the Sp1-C site resulted in almost the same reduction as the deletion mutation as shown in Fig.5 A. Because the deletion and point mutation of Sp1 sites (Sp1-B and Sp1-C) resulted in a reduction of luciferase activity, the Sp1 sites in this promoter should bind the Sp family of transcription factors. First, gel shift assays were performed to address whether the HCT-116 cells contained binding activities for these regions. Sp isoforms (Sp1, Sp2, and Sp3) are expressed in HCT-116 cells as measured by Western analysis (data not shown). The results using nuclear extracts of HCT-116 cells and a probe corresponding to the Sp1-BC element at positions −73 to −44 (Fig.6 A, top panel) show multiple DNA-protein complexes with a mobility (Fig. 6 A,bottom panel, arrows α, β, andγ), similar to the previous reports using Sp1 consensus oligonucleotides (21Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 26Suske G. Gene (Amst.). 1999; 238: 291-300Crossref PubMed Scopus (985) Google Scholar). These bands represent a specific protein binding to the Sp1 sequence elements, because complex formation was diminished by the addition of 10 or 50 molar excess of nonradiolabeled identical competitor, but not by addition of the identical oligonucleotide in which the Sp1-BC sites were point-mutated (Fig.6 A). Furthermore, to determine which base pairs are important for the binding of these complexes, we performed mutational analysis of the Sp1 sites. The mutations that we generated changed selected pairs of guanidine to pairs of thymidine residues (Fig.6 A, top panel). An oligonucleotide containing mut12 was unable to compete for the binding of any of the complexes at 50-fold molar excess (Fig. 6 A, compare lane 5with lane 8). Interestingly, incubation with mut1 oligonucleotide did not fully compete with labeled oligonucleotide (Fig. 6 A, lane 6), indicating that Sp1-B site has probably ∼50% activity with regard to DNA binding. In contrast, incubation with mut2 oligonucleotide competes in bands α and β but not band γ, indicating that at least one protein preferentially binds to the Sp1-C site. To confirm that Sp1 binds to these sites, we performed a gel shift assay in the presence of Sp family antibodies to demonstrate supershifting. We also examined COUP-TF1 and HNF-4α antibodies for binding to this site. The DA - 2001/9/7/ PY - 2001/9/7/ DO - 10.1074/jbc.M101814200 VL - 276 IS - 36 SP - 33384-33392 SN - 0021-9258 ER - TY - PAT TI - Method of producing an undifferentiated avian cell culture using avian primordial germ cells AU - Petitte, J. N. AU - Chang, I.-K. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - Infection with Bartonella weissii and Detection of Nanobacterium Antigens in a North Carolina Beef Herd AU - Breitschwerdt, E. B. AU - Sontakke, S. AU - Cannedy, A. AU - Hancock, S. I. AU - Bradley, J. M. T2 - Journal of Clinical Microbiology AB - Very recently, Bartonella organisms have been isolated from large ruminants (deer, elk, and dairy and beef cattle) located in the United States and in France. In this study, we report the serologic, microbiologic, and molecular findings related to the isolation of a Bartonella species in North Carolina beef cattle and the detection of nanobacterial antigen using a commercially available enzyme-linked immunosorbent assay. Between August 1998 and September 1999, blood was collected from 38 cattle ranging in age from 1 month to 6.5 years. After a 1-month incubation period, a Bartonella sp. was isolated on a 5% rabbit blood agar plate from three of six EDTA blood samples. PCR amplification of the 16S rRNA gene from all three isolates resulted in a DNA sequence that was 100% identical to that of B. weissii 16S rRNA (GenBank no. AF199502). By IFA testing, 36 of 38 cattle had antibodies (> or =1:64) to Bartonella weissii (bovine origin) antigens. Nanobacterial antigen was detected in 22 of 22 serum samples. We conclude that infection with an organism similar or closely related to B. weissii can occur in North Carolina cattle and that although their actual existence is still controversial Nanobacterium antigens were detected with a commercially available test kit. The epidemiology, vector biology, and potential pathogenicity of these organisms in cattle deserve future consideration. DA - 2001/3/1/ PY - 2001/3/1/ DO - 10.1128/JCM.39.3.879-882.2001 VL - 39 IS - 3 SP - 879-882 J2 - Journal of Clinical Microbiology LA - en OP - SN - 0095-1137 UR - http://dx.doi.org/10.1128/JCM.39.3.879-882.2001 DB - Crossref ER - TY - JOUR TI - In vitro studies of the fate of sulfadimethoxine and ormetoprim in the aquatic environment AU - Bakal, RS AU - Stoskopf, MK T2 - AQUACULTURE AB - These studies showed sulfadimethoxine and ormetoprim to be stable at salinities of 0 and 30 ppt and at pHs of 2, 7, and 12 for a period of 1 year. Sulfadimethoxine was stable at 25°C and 37°C, but showed a marked decrease in concentration at 4°C. Warming of the 4°C sample resulted in a return to original drug levels indicating that the drug had redistributed out of the aquatic phase at the lower temperature. Ormetoprim concentrations were stable at all temperatures evaluated. The concentrations of both sulfadimethoxine and ormetoprim were unaffected by the presence of silica sand, high density poly-ethylene, or poly-vinyl chloride. The presence of bentonite clay caused a reduction in ormetoprim concentrations while sulfadimethoxine was unchanged by this substrate. Acidification of the sample containing the bentonite clay resulted in a return of ormetoprim concentration to original levels. From these studies, it is apparent that the potential environmental half-lives for these drugs must exceed 1 year and are likely to be several years in duration. DA - 2001/4/2/ PY - 2001/4/2/ DO - 10.1016/S0044-8486(00)00539-1 VL - 195 IS - 1-2 SP - 95-102 SN - 0044-8486 KW - sulfadimethoxine KW - ormetoprim KW - sediments KW - marine pollution KW - therapeutic drugs ER - TY - JOUR TI - In utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by Streptococcus suis type II AU - Feng, WH AU - Laster, SM AU - Tompkins, M AU - Brown, T AU - Xu, JS AU - Altier, C AU - Gomez, W AU - Benfield, D AU - McCaw, MB T2 - JOURNAL OF VIROLOGY AB - ABSTRACT Porcine reproductive and respiratory syndrome (PRRS) consistently elevates the frequency of disease and mortality in young pigs. Many different secondary bacterial diseases occur in PRRS virus (PRRSV)-infected pigs. However, to date, establishing a reproducible experimental model of PRRSV infection in weaned pigs, with subsequent clinical disease following secondary bacterial challenge, has been difficult. PRRSV is frequently isolated during outbreaks from weak-born piglets affected by secondary bacterial diseases. This study was performed to investigate the potential role of intrauterine PRRSV infection on piglet susceptibility to secondary bacterial infection. PRRSV-free pregnant sows were intranasally infected at 98 days of gestation with PRRSV strain SD 23983. All piglets born to the PRRSV-infected sows were viremic. Piglets were removed from the sows at birth and deprived of colostrum. Piglets from PRRSV-infected and noninfected sows were randomly assigned to Streptococcus suis challenge or control subgroups. At 5 days of age, piglets were challenged intranasally with strain MN 87555 of S. suis type II. Total and differential leukocyte counts were performed on blood samples collected at 3 days of age. The numbers of leukocytes, lymphocytes, and monocytes were significantly reduced in the PRRSV-infected piglets. Lesions were observed in bone marrow, brain, lung, heart, spleen, lymph node, tonsil, and thymus of PRRSV-infected piglets. Thymus/body weight ratios of in utero PRRSV-infected piglets were significantly reduced compared to those of non-PRRSV-infected piglets, and thymic lesions were characterized by severe cortical depletion of thymocytes. Lesions were not observed in piglets born to PRRSV-free sows. Overall, 20 out of 22 piglets in the PRRSV- S. suis dual-infection group died within 1 week after challenge with S. suis (10 of 11 in each of two trials). This contrasts with 1 of 18 piglets in the PRRSV-infection-only group and 5 of 23 piglets in the S. suis -challenge-only group (1 of 12 in trial 1 and 4 of 11 in trial 2). No piglets died in the uninfected control groups. Most of the piglets in the PRRSV- S. suis dual-infection group developed suppurative meningitis. S. suis type II was recovered from their brains and joints. These results indicate that in utero infection by PRRSV makes piglets more susceptible to infection and disease following challenge by S. suis type II. In utero infection by PRRSV may provide a useful model to study the interaction between PRRSV and bacterial coinfections in piglets. DA - 2001/5// PY - 2001/5// DO - 10.1128/JVI.75.10.4889-4895.2001 VL - 75 IS - 10 SP - 4889-4895 SN - 1098-5514 ER - TY - JOUR TI - Homomultimeric protease and putative bacteriocin homolog from Thermotoga maritima AU - Hicks, P. M. AU - Chang, L. S. AU - Kelly, R. M. T2 - Hyperthermophilic enzymes. Part A CN - QP601 .M49 vol. 330 DA - 2001/// PY - 2001/// VL - 330 SP - 455-460 ER - TY - JOUR TI - Evaluation of the dystrophin-glycoprotein complex, alpha-actinin, dysferlin and calpain 3 in an autosomal recessive muscular dystrophy in Labrador retrievers AU - Olby, NJ AU - Sharp, NJH AU - Anderson, LVB AU - Kunkel, LM AU - Bonnemann, CG T2 - NEUROMUSCULAR DISORDERS AB - Labrador retrievers suffer from an autosomal recessive muscular dystrophy of unknown aetiology. Dogs affected with this disease develop generalized weakness associated with severe, generalized skeletal muscle atrophy and mild elevations in creatine kinase in the first few months of life. The severity of signs tends to progress over the first year of life but can vary from mild exercise intolerance to non-ambulatory tetraparesis. Beyond 1 year of age, the signs usually stabilize and although muscle mass does not increase, affected dogs' strength may improve slightly. The pathological changes present on muscle biopsy include marked variation in muscle fibre size with hypertrophied and round atrophied fibres present. There is an increased number of fibres with central nuclei and split fibres can be seen. It has been suggested that the disorder is a model for limb-girdle muscular dystrophy. In recent years, mutations in genes encoding the proteolytic enzyme, calpain 3, a novel protein named dysferlin, and components of the dystrophin-glycoprotein complex have been identified as causes of autosomal recessive limb-girdle muscular dystrophy. We have evaluated these proteins in normal dogs and in three Labrador retrievers with autosomal recessive muscular dystrophy using immunohistochemistry and Western blot analysis on frozen skeletal muscle. The results demonstrate that dystrophin, the sarcoglycans, alpha-actinin, dysferlin and calpain 3 are present in the normal and affected dogs. We conclude that this autosomal recessive muscular dystrophy is not due to a deficiency of alpha-actinin, or any of the known autosomal recessive limb-girdle muscular dystrophy proteins, although we cannot rule out a malfunction of any of these proteins. DA - 2001/1// PY - 2001/1// DO - 10.1016/S0960-8966(00)00166-8 VL - 11 IS - 1 SP - 41-49 SN - 0960-8966 KW - autosomal recessive muscular dystrophy in Labrador retrievers KW - limb-girdle muscular dystrophy KW - congenital muscular dystrophy KW - sarcoglycan KW - merosin KW - calpain 3 KW - dysferlin ER - TY - JOUR TI - Dynamics of isolated polycaprolactone chains in their inclusion complexes with cyclodextrins AU - Lu, J AU - Mirau, PA AU - Tonelli, AE T2 - MACROMOLECULES AB - Solid-state carbon NMR with magic-angle spinning has been used to study the structure and dynamics of semicrystalline polycaprolactone (PCL) and its inclusion complexes formed with α- and γ-cyclodextrins (α- and γ-CDs), which are shown to have channel structures occupied by single and two parallel, side-by-side chains, respectively. Guest−host magnetization exchange has been observed, but the results differ substantially from those observed in semicrystalline polymers and blends. The conventional relaxation experiments and 2D wide-line separation NMR with windowless isotropic mixing have been used to measure the chain dynamics. The results suggest that the intermolecular interactions restrict the dynamics of some atoms more than others, but that the chains in the complex are more mobile than in semicrystalline PCL. These results are compared with the inclusion compound formed between the model compound valeric acid and α-CD that is also a channel complex structure. DA - 2001/5/8/ PY - 2001/5/8/ DO - 10.1021/ma001820z VL - 34 IS - 10 SP - 3276-3284 SN - 0024-9297 ER - TY - JOUR TI - Developmental profile, isolation, and biochemical characterization of a novel lipoglycoheme-carrier protein from the American dog tick, Dermacentor variabilis (Acari : Ixodidae) and observations on a similar protein in the soft tick, Ornithodoros parkeri (Acari : Argasidae) AU - Gudderra, NP AU - Neese, PA AU - Sonenshine, DE AU - Apperson, CS AU - Roe, RM T2 - INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY AB - A novel lipoglycoheme-carrier protein (CP) in the American dog tick, Dermacentor variabilis (Say) has been purified and characterized. CP was purified by native–PAGE from partially fed virgin females. CP has a density of 1.25 g/ml with a molecular weight of 200 K by native–PAGE and 340 K by gel filtration chromatography. CP is comprised of two majour subunits, 98 K and 92 K in molecular weight by SDS–PAGE. Separate amino acid composition of the two subunits indicated high contents of As(x), Gl(x) and leucine. However, the N-terminal amino acid sequence of the two subunits was only 13% identical. The lower molecular weight subunit showed 61% identity to artemocyanin (biliprotein) in fairy shrimps, 46% identity to minor vitellogenin in chickens and 13% identity to vitellin of the black-legged tick. No similarity match was found for the other subunit. CP is a lipoglycoheme-protein as indicated by selective staining of native–PAGE gel for lipids, carbohydrates and heme. Lipid analysis by thin layer chromatography revealed the presence of cholesterol, phospholipids, monoacylglycerides, triacylglycerides and free fatty acids. Heme associated with purified CP demonstrated a λmax of 397.5 nm while the λmax of crude hemolymph plasma was 402.5 nm. The presence of CP in whole body homogenates of eggs, unfed and fed larvae and fed nymphs as well as in the plasma of unfed and fed adults including vitellogenic females was demonstrated by native–PAGE. Although a protein of analogous size was not found in the soft tick, Ornithodoros parkeri Cooley, a high molecular weight protein (500 K) is the predominant plasma protein in both unfed and fed male and female adults of that species as determined by native–PAGE. Also, CP appears to function as a biliprotein which sequesters heme. DA - 2001/3/15/ PY - 2001/3/15/ DO - 10.1016/s0965-1748(00)00122-3 VL - 31 IS - 4-5 SP - 299-311 SN - 0965-1748 KW - Acari KW - Ixodidae KW - Argasidae KW - carrier protein KW - artemocyanin ER - TY - JOUR TI - Continuous cultivation of hyperthermophiles AU - Pysz, M.A. AU - Rinker, K.D. AU - Shockley, K.R. AU - Kelly, R.M. T2 - Methods in Enzymology AB - This chapter describes a continuous culture system that can generate biomass from hyperthermophiles on a scale suitable for enzyme purification. Hightemperature chemostats have several advantages over large-scale batch systems. Long-term, stable, steady-state operation (arising from minimal problems with contamination) can provide biomass generated from exponential growth phase—that is, balanced growth. Because of the smaller operating volumes, continuous systems are inexpensive to construct and minimize problems with handling toxic and explosive gas substrates and products—for example, H2S, He, CH4. Small operating volumes also minimize problems associated with the growth of sulfide-producing anaerobes and thermoacidophiles in terms of choosing a proper material for reactor construction—for example, glass and gold. Continuous cultivation has also been useful for studying the bioenergetics and physiology of hyperthermophiles and for developing media formulations that induce enzyme expression. CN - QP601 .M49 vol. 330 DA - 2001/// PY - 2001/// DO - 10.1016/S0076-6879(01)30369-5 VL - 330 SP - 31–40 ER - TY - PAT TI - Compositions for fracturing subterranean formations AU - Kelly, R. M. AU - Khan, S. A. AU - Leduc, P. AU - Tayal, A. AU - Prud'homme, R. K. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - Causal inference on the difference of the restricted mean lifetime between two groups AU - Chen, PY AU - Tsiatis, AA T2 - BIOMETRICS AB - When comparing survival times between two treatment groups, it may be more appropriate to compare the restricted mean lifetime, i.e., the expectation of lifetime restricted to a time L, rather than mean lifetime in order to accommodate censoring. When the treatments are not assigned to patients randomly, as in observational studies, we also need to account for treatment imbalances in confounding factors. In this article, we propose estimators for the difference of the restricted mean lifetime between two groups that account for treatment imbalances in prognostic factors assuming a proportional hazards relationship. Large-sample properties of our estimators based on martingale theory for counting processes are also derived. Simulation studies were conducted to compare these estimators and to assess the adequacy of the large-sample approximations. Our methods are also applied to an observational database of acute coronary syndrome patients from Duke University Medical Center to estimate the treatment effect on the restricted mean lifetime over 5 years. DA - 2001/12// PY - 2001/12// DO - 10.1111/j.0006-341X.2001.01030.x VL - 57 IS - 4 SP - 1030-1038 SN - 0006-341X KW - causal inference KW - Cox's proportional hazard model KW - martingale process KW - observational study KW - restricted lifetime KW - stochastic integral KW - survival analysis ER - TY - JOUR TI - Carboxylesterase from Sulfolobus solfataricus P1 AU - Sehgal, A. C. AU - Callen, W. AU - Mathur, E. J. AU - Short, J. M. AU - Kelly, R. M. T2 - Hyperthermophilic enzymes. Part A CN - QP601 .M49 vol. 330 DA - 2001/// PY - 2001/// VL - 330 SP - 461-471 ER - TY - JOUR TI - Bunyavirus infections in North Carolina white-tailed deer (Odocoileus virginianus) AU - Nagayama, J. N. AU - Komar, N. AU - Levine, J. F. AU - Biggerstaff, B. AU - Apperson, C. S. T2 - Vector Borne and Zoonotic Diseases (Larchmont, N.Y.) DA - 2001/// PY - 2001/// VL - 1 IS - 2 SP - 169-172 ER - TY - JOUR TI - Antiviral efficacy and pharmacokinetics of oral adefovir dipivoxil in chronically woodchuck hepatitis virus-infected woodchucks AU - Cullen, JM AU - Ll, DH AU - Brown, C AU - Eisenberg, EJ AU - Cundy, KC AU - Wolfe, J AU - Toole, J AU - Gibbs, C T2 - ANTIMICROBIAL AGENTS AND CHEMOTHERAPY AB - The antiviral efficacy of orally administered adefovir dipivoxil was evaluated in an 18-week study (12 weeks of treatment and 6 weeks of recovery) conducted with woodchucks chronically infected with woodchuck hepatitis virus (WHV). Adefovir dipivoxil is a prodrug of adefovir designed to enhance its oral bioavailability. Following administration of 15 mg of adefovir dipivoxil per kg of body weight in four WHV-infected animals, the mean maximum concentration of adefovir in serum was 0.462 microg/ml, with an elimination half-life of 10.2 h, and the oral bioavailability of adefovir was estimated to be 22.9% (+/-11.2%). To study antiviral efficacy, the animals were divided into three groups. There were six animals each in a high-dose group (15 mg/kg/day) and a low-dose group (5 mg/kg/day). A vehicle control group consisted of five animals because WHV DNA was detectable only by PCR at the time of the study in one of the original six animals. Efficacy was evaluated by determining the levels of WHV DNA in serum. The geometric mean WHV DNA level for the high-dose group diminished by >40-fold (>1.6 log(10)) after 2 weeks of treatment and >300-fold (>2.5 log(10)) at 12 weeks. There was a >10-fold reduction in five of six low-dose animals by 2 weeks, but levels were unchanged in one animal. By 12 weeks of treatment there was a >45-fold (>1.6 log(10)) reduction of WHV DNA levels, and serum WHV DNA levels were below the limit of quantification in three of six animals. Viral DNA levels returned to pretreatment levels during the 6-week recovery period. There were no clinically significant changes in body weight, hematology, or serum chemistry values, including bicarbonate or lactate, in any of the treated animals. No histologic evidence of liver injury was apparent in the biopsies. Under the conditions of this study, adefovir dipivoxil was an effective antihepadnaviral agent. DA - 2001/10// PY - 2001/10// DO - 10.1128/AAC.45.10.2740-2745.2001 VL - 45 IS - 10 SP - 2740-2745 SN - 1098-6596 ER - TY - JOUR TI - Airway epithelium and mucus - Intracellular signaling pathways for gene expression and secretion AU - Adler, KB AU - Li, YH T2 - AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY DA - 2001/10// PY - 2001/10// DO - 10.1165/ajrcmb.25.4.f214 VL - 25 IS - 4 SP - 397-400 SN - 1044-1549 ER - TY - JOUR TI - Use of an intravitreal sustained-release cyclosporine delivery device for treatment of equine recurrent uveitis AU - Gilger, BC AU - Wilkie, DA AU - Davidson, MG AU - Allen, JB T2 - AMERICAN JOURNAL OF VETERINARY RESEARCH AB - Abstract Objective —To evaluate the use of an intravitreal sustained-release cyclosporine (CsA) delivery device for treatment of horses with naturally occurring recurrent uveitis. Animals —16 horses with recurrent uveitis. Procedures —Horses with frequent recurrent episodes of uveitis or with disease that was progressing despite appropriate medication were selected for this study. Additional inclusion criteria included adequate retinal function as determined by use of electroretinography, lack of severe cataract formation, and no vision-threatening ocular complications (eg, retinal detachment, severe retinal degeneration, and posterior synechia). Sustained-release CsA delivery devices (4 µg of CsA/d) were implanted into the vitreous through a sclerotomy at the pars plana. Reexaminations were performed 1, 3, 6, and 12 months after implantation, then continued annually. Ophthalmic changes, number of recurrent episodes of uveitis, and vision were recorded. Results —The rate of recurrent episodes after device implantation (0.36 episodes/y) was less than prior to surgery (7.5 episodes/y). In addition, only 3 horses developed episodes of recurrent uveitis after surgery. Vision was detected in 14 of 16 affected eyes at a mean follow-up time of 13.8 months (range, 6 to 24 months). Conclusions and Clinical Relevance —This intravitreal sustained-release CsA delivery device may be a safe and important tool for long-term treatment of horses with chronic recurrent uveitis. ( Am J Vet Res 2001;62:1892–1896) DA - 2001/12// PY - 2001/12// DO - 10.2460/ajvr.2001.62.1892 VL - 62 IS - 12 SP - 1892-1896 SN - 0002-9645 ER - TY - JOUR TI - Tissue disposition and depletion of penicillin G after oral administration with milk in unweaned dairy calves AU - Musser, JMB AU - Anderson, KL AU - Boison, JO T2 - JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION AB - To determine tissue depletion of penicillin G in calves after oral ingestion with milk replacer and estimate a withdrawal period.Longitudinal controlled trial.26 Holstein calves.Once daily, 24 calves were fed milk replacer containing procaine penicillin G (0.68 mg/kg [0.31 mg/lb] of body weight); 2 calves served as controls. After 1 feeding, 12 calves were euthanatized in groups of 3 each 4, 6.5, 9.5, and 13 hours after feeding. After 14 days, 12 calves were euthanatized in groups of 3 each 4, 6.5, 9.5, and 13 hours after the final feeding. Concentrations of penicillin G were determined in tissues, blood, and urine by use of high-performance liquid chromatography.Penicillin G was not detected in muscle samples of treated calves. The highest concentrations of penicillin G in plasma, kidney, and liver were 13 ng/ml, 92 ng/g, and 142 ng/g, respectively. Thirteen carcasses had violative drug residues; 12 had violative residues in the liver only, and 1 had violative residues in the liver and kidney. A 21-hour withdrawal period was estimated.Liver had the highest concentration of penicillin G and was most likely to have violative residues. Feeding calves milk containing penicillin G has the potential to cause violative drug residues in tissues. It is recommended to observe an appropriate withdrawal time prior to slaughter if calves are fed milk from cows treated with penicillin G. DA - 2001/8/1/ PY - 2001/8/1/ DO - 10.2460/javma.2001.219.346 VL - 219 IS - 3 SP - 346-350 SN - 0003-1488 ER - TY - JOUR TI - The effects of genetic selection on production parameters of single comb white leghorn hens AU - Jones, DR AU - Anderson, KE AU - Davis, GS T2 - POULTRY SCIENCE AB - Four commercial table egg genetic stocks consisting of the Ottawa Control Strains 5, 7, and 10 (CS5, CS7, and CS10) and the 1993 H&N "Nick Chick" (CCS) were housed in the same environment and compared for production characteristics. These birds were housed in an environmentally controlled laying facility with trideck cages. Feed consumption, egg production, and mortality were monitored daily and compiled every 28 d. The study was conducted for two egg production cycles, including the molt period. Body weight was progressively lower for the more modern strains with CS5 being the heaviest and CCS maintaining the smallest body weight throughout the production periods. The CCS had the highest (P < 0.0001) hen-day production rate, which resulted in the greatest daily egg mass among the strains. The CCS consumed the greatest amount of feed and exhibited the highest gross egg income among the strains. We concluded that genetic selection has improved production parameters in commercial layers as determined by measurements in this study. DA - 2001/8// PY - 2001/8// DO - 10.1093/ps/80.8.1139 VL - 80 IS - 8 SP - 1139-1143 SN - 0032-5791 KW - genetic selection KW - egg production ER - TY - JOUR TI - The ACVD task force on canine atopic dermatitis (V): biology and role of inflammatory cells in cutaneous allergic reactions AU - Hill, P.B. AU - Olivry, T. T2 - Veterinary Immunology and Immunopathology AB - Numerous inflammatory cells are thought to play a role in the pathogenesis of canine atopic dermatitis (AD) although, in the past, mast cells were considered the most important. However, evidence for this assumption is lacking. In this paper, we review the literature concerning the role of inflammatory cells in allergic reactions and conclude that a complex interplay exists between a wide variety of cell types. Thus, on the basis of the available evidence, the cells that appear to be the most important in the pathogenesis of canine AD are Langerhans' cells and dermal dendritic cells (both responsible for antigen processing and presentation), B-lymphocytes (responsible for reaginic antibody production), allergen-specific helper T-lymphocytes (responsible for cytokine production leading to activation of B-cells and other inflammatory cells) and mast cells (production of inflammatory mediators leading to inflammation). DA - 2001/9/20/ PY - 2001/9/20/ DO - 10.1016/S0165-2427(01)00310-5 VL - 81 IS - 3-4 SP - 187-198 SN - 0165-2427 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035922038&partnerID=MN8TOARS KW - allergic disease KW - atopy KW - dendritic cells KW - dog KW - lymphocytes KW - mast cells ER - TY - CONF TI - T cell apoptosis in FIV-infected cats is blocked by the addition of antibodies to B7.1 and B7.2 AU - Bull, M. E. AU - Vahlenkamp, T. W. AU - Tompkins, M. B. AU - Tompkins, W. A. F. C2 - 2001/// C3 - Journal of Leukocyte Biology DA - 2001/// SP - 256 M1 - 2001 ER - TY - JOUR TI - Survival of clinical and poultry-derived isolates of Campylobacter jejuni at a low temperature (4 degrees C) AU - Chan, KF AU - Tran, HL AU - Kanenaka, RY AU - Kathariou, S T2 - APPLIED AND ENVIRONMENTAL MICROBIOLOGY AB - ABSTRACT Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans, and contamination of poultry has been implicated in illness. The bacteria are fastidious in terms of their temperature requirements, being unable to grow below ca. 31°C, but have been found to be physiologically active at lower temperatures and to tolerate exposure to low temperatures in a strain-dependent manner. In this study, 19 field isolates of C. jejuni (10 of clinical and 9 of poultry origin) were studied for their ability to tolerate prolonged exposure to low temperature (4°C). Although substantial variability was found among different strains, clinical isolates tended to be significantly more likely to remain viable following cold exposure than poultry-derived strains. In contrast, the relative degree of tolerance of the bacteria to freezing at −20°C and freeze-thawing was strain specific but independent of strain source (poultry versus clinical) and degree of cold (4°C) tolerance. DA - 2001/9// PY - 2001/9// DO - 10.1128/AEM.67.9.4186-4191.2001 VL - 67 IS - 9 SP - 4186-4191 SN - 1098-5336 ER - TY - JOUR TI - Species diagnosis and Bacillus thuringiensis resistance monitoring of Heliothis virescens and Helicoverpa zea (Lepidoptera : noctuidae) field strains from the southern United States using feeding disruption bioassays AU - Bailey, WD AU - Brownie, C AU - Bacheler, JS AU - Gould, F AU - Kennedy, GG AU - Sorenson, CE AU - Roe, RM T2 - JOURNAL OF ECONOMIC ENTOMOLOGY AB - Validation of a feeding disruption bioassay for the detection of resistance to Bacillus thuringiensis toxin and species identification is reported using field strains of Heliothis virescens and Helicoverpa zea collected from the southern United States in 1998. Feeding disruption is measured by a lack of fecal production from larvae exposed to a diagnostic concentration of CryIAc in a blue indicator diet. The bioassay provided rapid (24 h) diagnosis of the species composition of larvae tested and also monitored for the presence of resistance in H. virescens. An additional diagnostic concentration was established for monitoring resistance in H. zea. A probit model was used to compare the fecal production responses of insect strains over a range of CryIAc doses. Probability calculations, derived from our assay results, are also presented to aid in the interpretation of future results from field trials. Integration of the feeding disruption bioassay into integrated pest management programs is discussed. DA - 2001/2// PY - 2001/2// DO - 10.1603/0022-0493-94.1.76 VL - 94 IS - 1 SP - 76-85 SN - 1938-291X KW - Heliothis viresccns KW - Helicoverpa zea KW - Bacillus thuringiensis KW - tobacco budworm KW - bollworm KW - insecticide resistance KW - cotton integrated pest management ER - TY - JOUR TI - Skin mast cell histamine release following stem cell factor and high-affinity immunoglobulin E receptor cross-linking in dogs with atopic dermatitis AU - Hammerberg, B AU - Olivry, T AU - Orton, SM T2 - VETERINARY DERMATOLOGY AB - Stem cell factor (SCF) influences mast cell activation and inflammatory mediator release, and is elevated in tissues undergoing allergic inflammation. Wheal formation in response to the injection of SCF or anti-immunoglobulin (Ig)E antibody injection was compared between normal (n = 10) and nonlesional atopic (n = 10) canine skin. In situ SCF secretion was compared between lesional and nonlesional skin using immunohistochemistry. Histamine release by skin cell suspensions after stimulation with SCF, concanavalin A (ConA) or rabbit anticanine IgE antibodies was compared between normal and atopic dogs. All dogs exhibited strong responses to intradermal SCF injection at 10 and 50 ng mL(-1). Atopic dogs had significantly (P = 0.002) larger wheal responses to anti-IgE than normal dogs; but there was no difference in numbers of skin mast cells bearing IgE as detected by immunohistochemistry. Only atopic dogs exhibited interstitial deposition of SCF in both lesional and nonlesional skin specimens. Median histamine release stimulated by SCF in the absence of IgE from lesional skin cells was higher in atopic than normal dogs (P = 0.04). These experiments suggest that dermal SCF secretion could potentiate histamine release following IgE receptor cross-linking and thus, could be one of the explanations for the inherent mast cell hyperexcitability observed in canine atopic dermatitis. DA - 2001/12// PY - 2001/12// DO - 10.1046/j.0959-4493.2001.00273.x VL - 12 IS - 6 SP - 339-346 SN - 0959-4493 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035749975&partnerID=MN8TOARS KW - atopic dermatitis KW - histamine KW - mast cell KW - stem cell factor ER - TY - JOUR TI - Skin biopsy site selection in small animal dermatology with an introduction to histologic pattern-analysis of inflammatory skin lesions AU - Linder, KE T2 - CLINICAL TECHNIQUES IN SMALL ANIMAL PRACTICE AB - The skin biopsy is an invaluable diagnostic tool in veterinary dermatology. Biopsy site selection and interpretation of the biopsy report significantly influence the value of this procedure for diagnosing inflammatory skin diseases and are discussed in this article. Skin diseases often present with several different recognizable lesions that change significantly during their evolution. Individual lesions are typically heterogenous--some areas are diagnostic and some are not. Understanding which skin lesions to biopsy, and when and where to sample them, can significantly improve the value of information collected. To increase the information returned to clinicians for a biopsy, veterinary dermatopathologists have adopted the pattern-analysis method of classifying inflammatory skin lesions. This approach is based on recognizing morphologically distinct inflammatory patterns in skin biopsies and their association with particular sets of diseases. A basic knowledge of the pattern-analysis method is essential for maximizing the interpretation of skin biopsy reports. DA - 2001/11// PY - 2001/11// DO - 10.1053/svms.2001.27595 VL - 16 IS - 4 SP - 207-213 SN - 1096-2867 ER - TY - JOUR TI - Serologic and molecular evidence of coinfection with multiple vector-borne pathogens in dogs from Thailand AU - Suksawat, J AU - Yu, XJ AU - Hancock, SI AU - Hegarty, BC AU - Nilkumhang, P AU - Breitschwerdt, EB T2 - JOURNAL OF VETERINARY INTERNAL MEDICINE AB - Forty-nine dogs from Thailand were evaluated for serologic evidence of exposure or polymerase chain reaction (PCR) evidence of infection with vectorborne pathogens, including Ehrlichia sp. (Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, and Ehrlichia risticii), Bartonella vinsonii subsp. berkhoffi (Bvb), spotted fever group (SFG) rickettsiae (Rickettsia rickettsii), Typhus group (TG) rickettsiae (Rickettsia canada, Rickettsia prowazekii, and Rickettsia typhi), and Babesia sp. (Babesia canis and Babesia gibsonii). All study dogs had at least 1 of 3 entry criteria: fever, anemia, or thrombocytopenia. By immunofluorescence antibody (IFA) testing, seroreactivity was most prevalent to E chaffeensis (74%) and E canis (71%) antigens, followed by E equi (58%), Bvb (38%), E risticii (38%), R prowazekii (24%), B canis (20%), R rickettsii (12%), R canada (4%), and B gibsonii (4%) antigens. There was 100% concordance between E canis IFA and Western blot immunoassay (WI) for 35 of 35 samples; 2 samples were IFA and WI reactive only to E equi antigens. By PCR amplification, 10 dogs were found to be infected with E canis, 5 with Ehrlichia platys, and 3 with B canis. Sequencing of PCR products was undertaken to compare Ehrlichia strains from Thailand to strains originating from the United States. Partial DNA sequence analysis confirmed infection with E canis and E platys, with identical 16S rRNA sequence alignment to E canis (U26740) and to E platys (M83801), as reported in GenBank. Partial E canis P28.1 and P28.2 amino acid sequences from Thai dogs were divergent from analogous sequences derived from North American E canis (AF082744) strains, suggesting that the Thai dogs were infected with a geographically distinct strain of E canis compared to North American strains. The results of this study indicate that dogs in Thailand have substantial exposure to vectorborne diseases and that coinfection with these pathogens may be common. DA - 2001/// PY - 2001/// DO - 10.1892/0891-6640(2001)015<0453:SAMEOC>2.3.CO;2 VL - 15 IS - 5 SP - 453-462 SN - 1939-1676 KW - canine KW - diseases KW - polymerase chain reaction KW - serology KW - ticks ER - TY - JOUR TI - Robust two-stage estimation in hierarchical nonlinear models AU - Yeap, BY AU - Davidian, M T2 - BIOMETRICS AB - Summary. Hierarchical models encompass two sources of variation, namely within and among individuals in the population; thus, it is important to identify outliers that may arise at each sampling level. A two‐stage approach to analyzing nonlinear repeated measurements naturally allows parametric modeling of the respective variance structure for the intraindividual random errors and interindividual random effects. We propose a robust two‐stage procedure based on Huber's (1981, Robust Statistics ) theory of M‐estimation to accommodate separately aberrant responses within an experimental unit and subjects deviating from the study population when the usual assumptions of normality are violated. A toxicology study of chronic ozone exposure in rats illustrates the impact of outliers on the population inference and hence the advantage of adopting the robust methodology. The robust weights generated by the two‐stage M‐estimation process also serve as diagnostics for gauging the relative influence of outliers at each level of the hierarchical model. A practical appeal of our proposal is the computational simplicity since the estimation algorithm may be implemented using standard statistical software with a nonlinear least squares routine and iterative capability. DA - 2001/3// PY - 2001/3// DO - 10.1111/j.0006-341X.2001.00266.x VL - 57 IS - 1 SP - 266-272 SN - 0006-341X KW - M-estimation KW - mixed effects KW - outliers KW - repeated measurements ER - TY - JOUR TI - Propane and propylene sorption in solid polymer electrolytes based on poly(ethylene oxide) and silver salts AU - Sunderrajan, S AU - Freeman, BD AU - Hall, CK AU - Pinnau, I T2 - JOURNAL OF MEMBRANE SCIENCE AB - The sorption of propylene and propane in solid polymer electrolytes based on blends of poly(ethylene oxide) (PEO) and silver nitrate, AgNO3, silver triflate, AgCF3SO3, silver trifluoroacetate, AgCF3CO2, or silver tetrafluoroborate, AgBF4, are reported. These solid polymer electrolytes exhibit preferential sorption for propylene over propane due to complexation of propylene with silver ions in the polymer matrix and reduced propane solubility in the solid polymer electrolyte films relative to that in PEO alone. The order of olefin solubility in blends containing 1 mol of silver ions per mole of ethylene oxide (EO) units is: AgBF4⪢AgCF3SO3>AgCF3CO2>AgNO3. At 35°C and 40 cmHg pressure, a PEO/AgBF4 film containing 1 mol of silver ions per mole of EO sorbed 5.3 g propylene but only 0.083 g propane per 100 g of solid polymer electrolyte. AgBF4 is highly soluble in PEO and completely disrupts polymer crystallinity even at low salt concentration (approximately 10 wt.%) without significantly altering the glass transition temperature. Other silver salts (e.g. AgNO3) do not dissolve completely in PEO and are much less effective at promoting enhanced olefin solubility. DA - 2001/2/15/ PY - 2001/2/15/ DO - 10.1016/S0376-7388(00)00569-X VL - 182 IS - 1-2 SP - 1-12 SN - 0376-7388 KW - olefin/paraffin separation KW - solid polymer electrolyte KW - poly(ethylene oxide) KW - facilitated transport ER - TY - JOUR TI - Nontraumatic rupture of an adrenal gland tumor causing intra-abdominal or retroperitoneal hemorrhage in four dogs AU - Whittemore, JC AU - Preston, CA AU - Kyles, AE AU - Hardie, EM AU - Feldman, EC T2 - JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION AB - Diagnosis and surgical management of intra-abdominal or retroperitoneal hemorrhage in 4 dogs with rupture of an adrenal gland tumor were determined. All 4 dogs were lethargic and weak with pale mucous membranes on initial examination. Three dogs did not have any history of clinical signs of hyperadrenocorticism or pheochromocytoma prior to examination. In 3 of the dogs, a mass in the area of the adrenal gland was identified with ultrasonography prior to surgery. All dogs developed ventricular premature contractions before or during anesthesia. Three dogs survived adrenalectomy; 1 dog was euthanatized during surgery because of an inability to achieve adequate hemostasis. The remaining 3 dogs all survived more than 5 months after surgery; 1 was euthanatized 9 months after surgery because of rupture of a hepatic mass. On the basis of these results, we suggest that hemodynamic stabilization followed by adrenalectomy is the treatment of choice for dogs with nontraumatic rupture of an adrenal gland tumor and resulting life-threatening hemorrhage. DA - 2001/8/1/ PY - 2001/8/1/ DO - 10.2460/javma.2001.219.329 VL - 219 IS - 3 SP - 329-333 ER - TY - JOUR TI - New auto-immune diseases of dogs and cats, first part: diseases targeting hair follicle and basal keratinocytes AU - Rivierre, C. AU - Olivry, T. T2 - Pratique Medicale et Chirurgicale de L'animal de Compagnie DA - 2001/// PY - 2001/// VL - 36 IS - 6 SP - 635-644 ER - TY - JOUR TI - Mixture component effects on the in vitro dermal absorption of pentachlorophenol AU - Riviere, J. E. AU - Qiao, G. L. AU - Baynes, R. E. AU - Brooks, J. D. AU - Mumtaz, M. T2 - Archives of Toxicology DA - 2001/// PY - 2001/// DO - 10.1007/s002040100242 VL - 75 IS - 6 SP - 329-334 ER - TY - JOUR TI - Investigation of rational syntheses of heteroleptic porphyrinic lanthanide (europium, cerium) triple-decker sandwich complexes AU - Gross, T AU - Chevalier, F AU - Lindsey, JS T2 - INORGANIC CHEMISTRY AB - The use of lanthanide triple-decker sandwich molecules containing porphyrins and phthalocyanines in molecular information storage applications requires the ability to attach monomeric triple deckers or arrays of triple deckers to electroactive surfaces. Such applications are limited by existing methods for preparing triple deckers. The reaction of a lanthanide porphyrin half-sandwich complex ((Por)M(acac)) with a dilithium phthalocyanine (PcLi2) in refluxing 1,2,4-trichlorobenzene (bp 214 degrees C) affords a mixture of triple deckers of composition (Pc)M(Pc)M(Por), (Por)M(Pc)M(Por), and (Pc)M(Por)M(Pc). We have investigated more directed methods for preparing triple deckers of a given type with distinct metals in each layer. Application of the method of Weiss, which employs reaction of a (Por)M(acac) species with a lanthanide double decker in refluxing 1,2,4-trichlorobenzene, afforded the desired triple decker in some cases but a mixture of triple deckers in others. The approach we developed employs in situ formation of the lanthanide reagent EuCl[N(SiMe3)2]2 or CeI[N(SiMe3)2]2, which upon reaction with a porphyrin affords the half-sandwich complex (Por)EuX or (Por)CeX' (X = Cl, N(SiMe3)2; X' = I, N(SiMe3)2). Subsequent reaction with PcLi2 gives the double decker (Por)M(Pc). The (Por(1))EuX half-sandwich complex gave the desired triple decker upon reaction with (Pc)Eu(Pc) but little of the desired product upon reaction with (Por(2))Eu(Pc). The (Por(1))CeX' half-sandwich complex reacted with europium double deckers (e.g., (tBPc)Eu(Por(2)), (tBPc)2Eu) to give the triple deckers (Por(1))Ce(tBPc)Eu(Por(2)) and (Por(1))Ce(tBPc)Eu(tBPc) in a rational manner (tB = tetra-tert-butyl). The reactions yielding the half-sandwich, double-decker, and triple-decker complexes were performed in refluxing bis(2-methoxyethyl) ether (bp 162 degrees C). The porphyrins incorporated in the various triple deckers include meso-tetrapentylporphyrin, meso-tetra-p-tolylporphyrin, octaethylporphyrin, and meso-tetraarylporphyrins bearing iodo, ethynyl, or iodo and ethynyl substituents. The triple deckers bearing iodo and/or ethynyl substituents constitute useful building blocks for information storage applications. DA - 2001/8/27/ PY - 2001/8/27/ DO - 10.1021/ic0101634 VL - 40 IS - 18 SP - 4762-4774 SN - 0020-1669 ER - TY - JOUR TI - Images from the 2001 ACVR certifying examination - CT/MR elective section AU - Thrall, DE T2 - VETERINARY RADIOLOGY & ULTRASOUND AB - Veterinary Radiology & UltrasoundVolume 42, Issue 6 p. 594-594 IMAGES FROM THE 2001 ACVR CERTIFYING EXAMINATION CT/MR Elective Section Donald E. Thrall, Donald E. Thrall College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606. Reprints not available.Search for more papers by this author Donald E. Thrall, Donald E. Thrall College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606. Reprints not available.Search for more papers by this author First published: 19 May 2005 https://doi.org/10.1111/j.1740-8261.2001.tb00990.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume42, Issue6November 2001Pages 594-594 RelatedInformation DA - 2001/// PY - 2001/// DO - 10.1111/j.1740-8261.2001.tb00990.x VL - 42 IS - 6 SP - 594-594 SN - 1058-8183 ER - TY - JOUR TI - Histone-like protein: a novel method for measuring stress in fish AU - Robinette, DW AU - Noga, EJ T2 - DISEASES OF AQUATIC ORGANISMS AB - We assessed the effect of chronic stress using a group of potent, broad-spectrum antimicrobial polypeptides, called histone-like proteins (HLPs), which appear to be an important component of non-specific immunity in channel catfish Ictalurus punctatus skin. An enzyme-linked immunosorbent assay (ELISA) was developed to measure the predominant HLP (HLP-1) in channel catfish skin. Catfish were then exposed to a chronic stress consisting of overcrowding and elevated ammonia. Healthy unstressed fish had consistently high HLP-1 levels, but fish that had been stressed for 1 wk had significantly depressed HLP-1 levels; HLP-1 levels declined further in fish stressed for 3 or 4 wk. The time-dependent decline in HLP-1 levels was not accompanied by any gross signs of disease. In contrast to HLP-1 levels, antibacterial activity in the skin was significantly greater in fish stressed for 1 wk compared with unstressed fish; in addition, antibacterial activity was the same in fish that were unstressed or stressed for 3 or 4 wk. This suggests that other antibiotics besides HLP-1 may be induced in the skin, especially during early stages of stress, that may compensate for depressed HLP-1 levels. Our results indicate that chronic stress has a significant suppressive effect on HLP-1 levels in channel catfish skin. The reduction of HLP-1 in the absence of clinical signs of disease, combined with evidence that its levels are not affected by the acute stressors of capture or sampling, suggests that HLP levels may be a promising indicator for monitoring fish health. DA - 2001/3/9/ PY - 2001/3/9/ DO - 10.3354/dao044097 VL - 44 IS - 2 SP - 97-107 SN - 1616-1580 KW - endobiotics KW - non-specific immunity KW - diagnostic stress test KW - ELISA ER - TY - PAT TI - High-density non-volatile memory devices incorporating thiol-derivatized porphyrin trimers AU - Clausen, P. C. AU - Lindsey, J. S. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - Feline retrovirus testing and management AU - Levy, J. AU - Richards, J. AU - Edwards, D. AU - Elston, T. AU - Hartmann, K. AU - Rodan, I. AU - Thayer, V. AU - Tompkins, M. AU - Wolf, A. T2 - Compendium on Continuing Education for the Practicing Veterinarian DA - 2001/// PY - 2001/// VL - 23 IS - 7 SP - 652- ER - TY - CONF TI - Feline immunodeficiency virus (FIV) infection induces B7(+)CTLA4(+) T cell apoptosis: A model for T cell depletion and immunodeficiency AU - Tompkins, M. B. AU - Bull, M. E. AU - Dow, J. L. AU - Tompkins, W. A. F. C2 - 2001/// C3 - Journal of Leukocyte Biology DA - 2001/// SP - 254 M1 - 2001 ER - TY - JOUR TI - Examining the effects of prestorage incubation of turkey breeder eggs on embryonic development and hatchability of eggs stored for four or fourteen days AU - Fasenko, GM AU - Christensen, VL AU - Wineland, MJ AU - Petitte, JN T2 - POULTRY SCIENCE AB - Thirty-six hundred British United Turkey hatching eggs were used in two separate trials to test whether prestorage incubation (PRESI) treatments of 0, 6, and 12 h (Trial 1) or 0, 7, and 14 h (Trial 2) could improve the hatchability of eggs stored (17 C) for 14 versus 4 d. The development of the embryos (n = 30) was staged before and after exposing eggs to the various PRESI treatments. Embryonic development was also established after storage to ascertain whether embryonic development was occurring during storage. The remaining eggs in each trial were split into three groups (n = 500) and incubated for 28 d to examine embryonic mortality and hatchability. No changes were observed in embryonic development due to egg storage. Embryos were significantly more developed as the number of PRESI h increased; therefore, embryos from different PRESI treatments were placed in storage at different stages of development. Early mortality (1 to 7 d of incubation), mortality at internal and external pipping, and hatchability of fertile eggs were significantly reduced in eggs stored for 14 versus 4 d. The various PRESI treatments did not significantly affect the mortality or hatchability of eggs stored for 4 d. However, the hatchability of eggs incubated prior to storage for 12 h and then stored for 14 d was restored to the levels reported for eggs subjected to the treatment that represents the industry norm (0 h of PRESI and 4 d storage). These results indicate that embryos of eggs stored for 14 d, which have developmentally advanced to the stage of complete hypoblast formation (PRESI for 12 h), have a survival advantage over eggs stored for 14 d that have not been subjected to any PRESI. DA - 2001/2// PY - 2001/2// DO - 10.1093/ps/80.2.132 VL - 80 IS - 2 SP - 132-138 SN - 0032-5791 UR - http://europepmc.org/abstract/med/11232999 KW - egg storage KW - embryonic development KW - hatchability KW - prestorage incubation ER - TY - JOUR TI - Electrostatic and conformational effects on the electronic structures of distortional isomers of a mixed-valence binuclear Cu complex AU - Franzen, S AU - Miskowski, VM AU - Shreve, AP AU - Wallace-Williams, SE AU - Woodruff, WH AU - Ondrias, MR AU - Barr, ME AU - Moore, L AU - Boxer, SG T2 - INORGANIC CHEMISTRY AB - The electronic structure of the binuclear copper complex [Cu(2)(L)](3+) [L = N(CH(2)CH(2)N(H)CH(2)CH(2)N(H)CH(2)CH(2))(3)N] has been investigated by resonance Raman and electroabsorption spectroscopy. Crystallographic Cu(2) distances of 2.364(1) and 2.415(1) A determined for the nitrate and acetate salts, respectively, are consistent with a substantial metal-metal interaction. The Cu-Cu bonding interaction in the binuclear complex is modulated both in the solid state and in solution by the ligand environment through coupling to ligand torsional modes that are, in turn, stabilized by hydrogen bonding. Electroabsorption data on the three major visible and near-infrared electronic transitions of Cu(2)L, lambda(max) (epsilon(max)) = 1000 nm ( approximately 1200 M(-1) cm(-1)), 748 nm (5600 M(-1) cm(-1)), and 622 nm (3350 M(-1) cm(-1)), reveal a difference dipole moment between the ground and excited states (Deltamu(A)) because of symmetry breaking. The difference polarizability for all three of the transitions is negative, indicating that the ground state is more polarizable than the excited state. A general model to explain this behavior in terms of the proximity of accessible transitions involving copper d electrons is proposed to explain the larger polarizability of the ground state. Raman excitation profiles (REPs) provide evidence for multiple conformational states of [Cu(2)(L)](3+). Separate REPs were obtained for each of the components of the two major Raman bands for nu(1) (a Cu-Cu stretching mode) and nu(2) (a Cu-Cu-N(eq) bending mode). The Raman data along with quantum chemical ZINDO/S CI calculations provide evidence for isomeric forms of Cu(2)L with strong coupling between the conformation of L and the Cu-Cu bond length. DA - 2001/12/3/ PY - 2001/12/3/ DO - 10.1021/ic010494g VL - 40 IS - 25 SP - 6375-6382 SN - 0020-1669 ER - TY - JOUR TI - Differentiation of Ehrlichia platys and E. equi Infections in Dogs by Using 16S Ribosomal DNA-Based PCR AU - Hancock, S. I. AU - Breitschwerdt, E. B. AU - Pitulle, C. T2 - Journal of Clinical Microbiology AB - We have encountered a previously unrecognized specificity problem when using the small-subunit ribosomal DNA (16S rDNA)-based PCR primers recommended for use in the identification of Ehrlichia equi in clinical samples. These PCR primers annealed to E. platys 16S rDNA in blood samples containing high levels of E. platys organisms. Therefore, we designed and tested new PCR primers for the identification of E. equi. DA - 2001/12/1/ PY - 2001/12/1/ DO - 10.1128/JCM.39.12.4577-4578.2001 VL - 39 IS - 12 SP - 4577-4578 J2 - Journal of Clinical Microbiology LA - en OP - SN - 0095-1137 UR - http://dx.doi.org/10.1128/JCM.39.12.4577-4578.2001 DB - Crossref ER - TY - JOUR TI - Differential aluminum tolerance in soybean: An evaluation of the role of organic acids AU - Silva, IR AU - Smyth, TJ AU - Raper, CD AU - Carter, TE AU - Rufty, TW T2 - PHYSIOLOGIA PLANTARUM AB - The role of organic acids in aluminum (Al) tolerance has been the object of intensive research. In the present work, we evaluated the roles of organic acid exudation and concentrations at the root tip on Al tolerance of soybean. Exposing soybean seedlings to Al3+ activities up to 4.7 &mgr;M in solution led to different degrees of restriction of primary root elongation. Al tolerance among genotypes was associated with citrate accumulation and excretion into the external media. Citrate and malate efflux increased in all genotypes during the first 6 h of Al exposure, but only citrate efflux in Al-tolerant genotypes was sustained for an extended period. Tolerance to Al was correlated with the concentration of citrate in root tips of 8 genotypes with a range of Al sensitivities (r2=0.75). The fluorescent stain lumogallion indicated that more Al accumulated in root tips of the Al-sensitive genotype Young than the Al-tolerant genotype PI 416937, suggesting that the sustained release of citrate from roots of the tolerant genotype was involved in Al exclusion. The initial stimulation of citrate and malate excretion and accumulation in the tip of all genotypes suggested the involvement of additional tolerance mechanisms. The experiments included an examination of Al effects on lateral root elongation. Extension of lateral roots was more sensitive to Al than that of tap roots, and lateral root tips accumulated more Al and had lower levels of citrate. DA - 2001/6// PY - 2001/6// DO - 10.1034/j.1399-3054.2001.1120208.x VL - 112 IS - 2 SP - 200-210 SN - 1399-3054 ER - TY - JOUR TI - Comparison of subcutaneous ivermectin and oral moxidectin for the treatment of notoedric acariasis in hamsters AU - Beco, L AU - Petite, A AU - Olivry, T T2 - VETERINARY RECORD AB - Thirty hamsters diagnosed with a Notoedres infestation on the basis of their clinical signs and skin scrapings were allocated to three matched groups. The hamsters in group 1 received ivermectin at 400 microg/kg subcutaneously once a week for eight weeks, those in group 2 were treated with moxidectin at 400 microg/kg orally once a week, and those in group 3 were treated with moxidectin at the same dosage, but twice a week. The hamsters' skin lesions were scored weekly on the basis of the severity of crusting, erythema, scaling and excoriations at various sites. In all three groups the lesion scores were significantly lower after four and eight weeks, and there was no significant difference between the efficacy of the treatments. However, at the end of the treatment, skin scrapings were negative in only 60 to 70 per cent of the animals in each group. DA - 2001/9/15/ PY - 2001/9/15/ DO - 10.1136/vr.149.11.324 VL - 149 IS - 11 SP - 324-327 SN - 2042-7670 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035883763&partnerID=MN8TOARS ER - TY - JOUR TI - Case presentation - Dysphagia caused by squamous cell carcinoma in two horses AU - Jones, S. L. AU - Zimmel, D. AU - Tate, L. P. AU - Campbell, N. AU - Redding, W. R. AU - Carlson, G. P. T2 - Compendium on Continuing Education for the Practicing Veterinarian DA - 2001/// PY - 2001/// VL - 23 IS - 11 SP - 1020-1024 ER - TY - JOUR TI - Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase AU - Vieille, C AU - Epting, KL AU - Kelly, RM AU - Zeikus, JG T2 - EUROPEAN JOURNAL OF BIOCHEMISTRY AB - Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile amino-acid residues (i.e. N and Q) in hyperthermophilic proteins. The N + Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperthermophiles was examined. It was found to correlate inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus licheniformis DSM13 (BLXI), which had an N + Q content comparable to that of homologs from much more thermophilic sources. To determine whether BLXI behaves as a thermostable enzyme, it was expressed in Escherichia coli, and the thermostability and activity properties of the recombinant enzyme were studied. Indeed, it was optimally active at 70-72 degrees C, which is significantly higher than the optimal growth temperature (37 degrees C) of B. licheniformis. The kinetic properties of BLXI, determined at 60 degrees C with glucose and xylose as substrates, were comparable to those of other class II XIs. The stability of BLXI was dependent on the metallic cation present in its two metal-binding sites. The enzyme thermostability increased in the order apoenzyme < Mg2+-enzyme < Co2+-enzyme approximately Mn2+-enzyme, with melting temperatures of 50.3 degrees C, 53.3 degrees C, 73.4 degrees C, and 73.6 degrees C. BLXI inactivation was first-order in all conditions examined. The energy of activation for irreversible inactivation was also strongly influenced by the metal present, ranging from 342 kJ x mol(-1) (apoenzyme) to 604 kJ x mol(-1) (Mg2+-enzyme) to 1166 kJ x mol(-1) (Co2+-enzyme). These results suggest that the first irreversible event in BLXI unfolding is the release of one or both of its metals from the active site. Although N + Q content was an indicator of thermostability for class II XIs, this pattern may not hold for other sets of homologous enzymes. In fact, the extremely thermostable alpha-amylase from B. licheniformis was found to have an average N + Q content compared with homologous enzymes from a variety of mesophilic and thermophilic sources. Thus, it would appear that protein thermostability is a function of more complex molecular determinants than amino-acid content alone. DA - 2001/12// PY - 2001/12// DO - 10.1046/j.0014-2956.2001.02587.x VL - 268 IS - 23 SP - 6291-6301 SN - 0014-2956 KW - Bacillus licheniformis KW - metal binding KW - thermostability KW - xylose isomerase ER - TY - JOUR TI - Using auxiliary time-dependent covariates to recover information in nonparametric testing with censored data AU - Murray, S AU - Tsiatis, AA T2 - LIFETIME DATA ANALYSIS AB - Murrayand Tsiatis (1996) described a weighted survival estimate thatincorporates prognostic time-dependent covariate informationto increase the efficiency of estimation. We propose a test statisticbased on the statistic of Pepe and Fleming (1989, 1991) thatincorporates these weighted survival estimates. As in Pepe andFleming, the test is an integrated weighted difference of twoestimated survival curves. This test has been shown to be effectiveat detecting survival differences in crossing hazards settingswhere the logrank test performs poorly. This method uses stratifiedlongitudinal covariate information to get more precise estimatesof the underlying survival curves when there is censored informationand this leads to more powerful tests. Another important featureof the test is that it remains valid when informative censoringis captured by the incorporated covariate. In this case, thePepe-Fleming statistic is known to be biased and should not beused. These methods could be useful in clinical trials with heavycensoring that include collection over time of covariates, suchas laboratory measurements, that are prognostic of subsequentsurvival or capture information related to censoring. DA - 2001/// PY - 2001/// DO - 10.1023/A:1011392622173 VL - 7 IS - 2 SP - 125-141 SN - 1380-7870 KW - Kaplan-Meier estimate KW - longitudinal KW - missing data KW - survival KW - two-sample test ER - TY - JOUR TI - Treatment of canine atopic dermatitis with cyclosporine: a pilot clinical study AU - Fontaine, J AU - Olivry, T T2 - VETERINARY RECORD AB - Veterinary RecordVolume 148, Issue 21 p. 662-663 Short Communication Treatment of canine atopic dermatitis with cyclosporine: a pilot clinical study J. Fontaine DrMedVet, DipECVD, J. Fontaine DrMedVet, DipECVD Clinique Wterinaire, Avenue Brugmann, 425, B-1 180, Brussels, BelgiumSearch for more papers by this authorT. Olivry DrVet, PhD, DipACVD, DipECVD, T. Olivry DrVet, PhD, DipACVD, DipECVD Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USASearch for more papers by this author J. Fontaine DrMedVet, DipECVD, J. Fontaine DrMedVet, DipECVD Clinique Wterinaire, Avenue Brugmann, 425, B-1 180, Brussels, BelgiumSearch for more papers by this authorT. Olivry DrVet, PhD, DipACVD, DipECVD, T. Olivry DrVet, PhD, DipACVD, DipECVD Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USASearch for more papers by this author First published: 26 May 2001 https://doi.org/10.1136/vr.148.21.662Citations: 25Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume148, Issue21May 2001Pages 662-663 RelatedInformation DA - 2001/5/26/ PY - 2001/5/26/ DO - 10.1136/vr.148.21.662 VL - 148 IS - 21 SP - 662-663 SN - 0042-4900 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035954203&partnerID=MN8TOARS ER - TY - JOUR TI - Synthesis and excited-state photodynamics of perylene-porphyrin dyads. 1. Parallel energy and charge transfer via a diphenylethyne linker AU - Prathapan, S AU - Yang, SI AU - Seth, J AU - Miller, MA AU - Bocian, DF AU - Holten, D AU - Lindsey, JS T2 - JOURNAL OF PHYSICAL CHEMISTRY B AB - The photophysical properties of a perylene−porphyrin dyad have been examined with the aim of using this construct for molecular photonics applications. The dyad consists of a perylene-bis(imide) dye (PDI) connected to a zinc porphyrin (Zn) via a diphenylethyne linker (pep). In both polar and nonpolar solvents, the photoexcited perylene unit (PDI*) decays very rapidly (lifetimes of 2.5 (toluene) and 2.4 ps (acetonitrile)) by energy transfer to the porphyrin, forming PDI−pep−Zn* in high yield (80%, toluene; 70% acetonitrile), and hole transfer to the porphyrin, forming PDI-−pep−Zn+ in lesser yield (20%, toluene; 30% acetonitrile). In both toluene and acetonitrile, the Zn* excited state subsequently decays with a lifetime of 0.4 ns primarily (80%) by electron transfer to the perylene (forming PDI-−pep−Zn+). In the nonpolar solvent (toluene), the PDI-−pep−Zn+ charge-transfer product has a lifetime of >10 ns and decays by charge recombination primarily to the ground state but also by thermal repopulation of the Zn* excited state. The occurrence of the latter process provides a direct experimental measure of the energy of the charge-separated state. In the polar solvent (acetonitrile), the PDI-−pep−Zn+ charge-separated state decays much more rapidly (<0.5 ns) and exclusively to the ground state. In general, the complementary perylene and porphyrin absorption properties together with very fast and efficient PDI*−pep−Zn → PDI−pep−Zn* energy transfer suggest that perylenes have significant potential as accessory pigments in porphyrin-based arrays for light-harvesting and energy-transport applications. Furthermore, the finding of fast energy transfer initiated in PDI*, charge-transfer reactions that can be elicited either in PDI* or Zn*, and a charge-separated state (PDI-−pep−Zn+) that can be long- or short-lived depending on solvent polarity, indicates the versatility of the perylene−porphyrin motif for a variety of applications in molecular photonics. DA - 2001/8/30/ PY - 2001/8/30/ DO - 10.1021/jp010335i VL - 105 IS - 34 SP - 8237-8248 SN - 1089-5647 ER - TY - JOUR TI - Synthesis and excited-state photodynamics in perylene-porphyrin dyads 2. Effects of porphyrin metalation state on the energy-transfer, charge-transfer, and deactivation channels AU - Yang, SI AU - Prathapan, S AU - Miller, MA AU - Seth, J AU - Bocian, DF AU - Lindsey, JS AU - Holten, D T2 - JOURNAL OF PHYSICAL CHEMISTRY B AB - The photophysical properties of two perylene−porphyrin dyads have been examined in detail with the aim of expanding the functional utility of these constructs for molecular optoelectronics applications. The dyads consist of a perylene-bis(imide) dye (PDI) connected to either a magnesium porphyrin (Mg) or a free base porphyrin (Fb) via a diphenylethyne (pep) linker. The photophysical behavior of these two dyads show both similarities and differences to one another and to the dyad containing a zinc porphyrin (Zn) that was examined in the previous paper in this series. In the case of both PDI−pep−Fb and PDI−pep−Mg in toluene, the excited perylene unit (PDI*) decays rapidly (Fb = 2.9 ps; Mg = 2.5 ps) by energy transfer to the porphyrin forming PDI−pep−Por* in relatively high yield (Fb ∼ 85%; Mg ∼ 50%) and hole transfer to the porphyrin forming PDI-−pep−Por+ (Fb ∼ 15%; Mg ∼ 50%). This behavior parallels that observed for PDI−pep−Zn, for which rapid (2.5 ps) decay of PDI* affords PDI−pep−Zn* and PDI-−pep−Por+ with yields of 80% and 20%, respectively. The subsequent behavior of the Fb-containing dyad is distinctly different in two ways from that of the Zn or Mg porphyrin-containing dyads. (1) Charge recombination within PDI-−pep−Fb+ primarily forms PDI−pep−Fb*, thereby complementing the formation of the latter species from PDI*−pep−Fb. (2) PDI−pep−Fb* subsequently decays to the ground state via fluorescence emission with a rate and yield that are nearly identical to those of an isolated Fb porphyrin. In contrast, for both PDI−pep−Mg and PDI−pep−Zn, the predominant decay process for PDI−pep−Por* is electron-transfer yielding PDI-−pep−Por+ (Zn ∼ 80%; Mg >99%). The rapid electron-transfer quenching of PDI−pep−Por* and the nonemissive character of PDI-−pep−Por+ leads to negligible fluorescence from the two metalloporphyrin-containing dyads after photoexcitation. The PDI-−pep−Por+ charge-separated product with Por = Mg or Zn is very long-lived (>10 ns) in toluene but decays much more rapidly (<0.5 ns) in acetonitrile. The differences in the rates of the various charge-transfer and charge-recombination processes of all of the dyads are consistent with a rate versus free-energy-gap profile (based on the relative redox potentials of the porphyrin constituents) that is in qualitative accord with electron-transfer theory. Collectively, the studies reported in this and the previous paper indicate that PDI−pep−Fb has the greatest potential utility in photonics applications wherein light harvesting by an accessory pigment, energy transport to an output chromophore, and emission (or energy transfer to another chromophore) are desired. On the other hand, PDI−pep−Mg (like PDI−pep−Zn) would be most useful as an all-optical gating element in which excited-state energy in an appended chromophore chain can be quenched by the charge-separated state of the perylene-porphyrin dyad, thereby shunting the light output or flow of energy. DA - 2001/8/30/ PY - 2001/8/30/ DO - 10.1021/jp010336a VL - 105 IS - 34 SP - 8249-8258 SN - 1089-5647 ER - TY - JOUR TI - Risk factors for development of status epilepticus in dogs with idiopathic epilepsy and effects of status epilepticus on outcome and survival time: 32 cases (1990-1996) AU - Saito, M AU - Munana, KR AU - Sharp, NJH AU - Olby, NJ T2 - JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION AB - To identify risk factors for episodes of status epilepticus (SE) in dogs with idiopathic epilepsy and determine how SE affects long-term outcome and survival time.Retrospective study.32 dogs with idiopathic epilepsy.Information on signalment, seizure onset, initiation of treatment, anticonvulsants administered, number of episodes of SE, overall seizure control, and long-term outcome was obtained from medical records and through telephone interviews. Differences between dogs that did and did not have episodes of SE were evaluated statistically.19 (59%) dogs had 1 or more episodes of SE. Body weight was the only variable significantly different between dogs that did and did not have episodes of SE. Thirteen dogs (9 that did not have episodes of SE and 4 that did) were still alive at the time of the study and were > or = 10 years old. Six of the 19 (32%) dogs that had episodes of SE died of causes directly attributed to the seizure disorder. Mean life spans of dogs that did and did not have episodes of SE were 8.3 and 11.3 years, respectively. Survival time was significantly different between groups.Results suggest that a substantial percentage of dogs with idiopathic epilepsy will have episodes of SE. Dogs with greater body weights were more likely to have episodes of SE, and early appropriate seizure treatment did not appear to decrease the risk that dogs would have episodes. Most dogs with idiopathic epilepsy had an expected life span, but survival time was shorter for dogs that had episodes of SE. DA - 2001/9/1/ PY - 2001/9/1/ DO - 10.2460/javma.2001.219.618 VL - 219 IS - 5 SP - 618-623 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0342902123&partnerID=MN8TOARS ER - TY - JOUR TI - Protein kinase C-alpha coordinately regulates cytosolic phospholipase A(2) activity and the expression of cyclooxygenase-2 through different mechanisms in mouse keratinocytes AU - Wang, HQ AU - Kim, MP AU - Tiano, HF AU - Langenbach, R AU - Smart, RC T2 - MOLECULAR PHARMACOLOGY AB - Transgenic mice (K5-PKCα) in which the keratin 5 promoter directs the expression of protein kinase C-α (PKCα) to epidermal keratinocytes display a 10-fold increase in PKCα protein in their epidermis and alterations in phorbol ester-induced cutaneous inflammation [J Cell Science 1999;112:3497–3506]. In the current study, we have used these K5-PKCα mice to examine the role of PKCα in keratinocyte phospholipid metabolism/eicosanoid production and cutaneous inflammation. Primary keratinocytes from wild-type and transgenic mice were prelabeled in culture with [3H]arachidonic acid (AA) and subsequently treated with TPA. Compared with wild-type keratinocytes, K5-PKCα keratinocytes displayed a 2-fold increase in AA release. TPA treatment resulted in the phosphorylation of cPLA2. PKC inhibitors GF-109203X or H7, but not mitogen-activated protein/extracellular signal-regulated protein kinase (MEK) inhibitor PD 98059, could inhibit phosphorylation and AA release. Topical 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of K5-PKCα mice resulted in a 5-fold increase in epidermal COX-2 induction and a 2- to 3-fold increase in prostaglandin (PG) E2 levels above that observed in TPA-treated wild-type mice. PD 98059, GF-109203X, or H7 could block cyclooxygenase-2 (COX-2) induction by TPA. Because C/EBPβ, a basic leucine zipper transcription factor, can be activated via a PKCα/mitogen-activated protein kinase pathway and can influence COX-2 expression, we examined whether C/EBPβ is involved in TPA-induced epidermal COX-2 expression. TPA-induced COX-2 expression was similar in C/EBPβ nullizygous and wild-type mice. In summary, our results indicate that epidermal PKCα coordinately regulates cPLA2 activity and COX-2 expression resulting in increased levels of AA and PGE2. Furthermore, PKCα-induced AA release and cPLA2phosphorylation are independent of MEK, whereas PKCα-induced COX-2 expression and PGE2 production are MEK-dependent and C/EBPβ-independent events. DA - 2001/4// PY - 2001/4// DO - 10.1124/mol.59.4.860 VL - 59 IS - 4 SP - 860-866 SN - 0026-895X ER - TY - JOUR TI - Predictors of outcome after dorsal decompressive laminectomy for degenerative lumbosacral stenosis in dogs: 69 cases (1987-1997) AU - De Risio, L AU - Sharp, NJH AU - Olby, NJ AU - Munana, KR AU - Thomas, WB T2 - JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION AB - Abstract Objective —To identify predictive factors of long-term outcome after dorsal decompressive laminectomy for the treatment of degenerative lumbosacral stenosis (DLSS) in dogs. Design —Retrospective study. Sample Population —69 client-owned dogs. Procedure —Medical records of dogs that had undergone dorsal laminectomy at North Carolina State University and the University of Tennessee between 1987 and 1997 were reviewed. Dogs with diskospondylitis, traumatic lesions, or neoplasia of the lumbosacral region were excluded. All dogs had evidence of cauda equina compression on myelography, epidurography, computed tomography, or magnetic resonance imaging, along with subsequent confirmation of the lesion at surgery. Follow-up was performed by telephone inquiries to the referring veterinarian, the owner, or both, using a detailed questionnaire. Results —The outcome was excellent or good in 54 of 69 (78%) dogs over a mean follow-up period of 38 ± 22 months. Five of these 54 dogs had been incontinent for a median of 2 weeks prior to surgery. Six of the 15 dogs with a poor outcome had been incontinent for a median of 8 weeks before surgery. A significant correlation was detected between the presence of urinary and fecal incontinence prior to surgery and outcome. When duration of signs was considered, urinary incontinence was the only variable that significantly affected outcome. Conclusions and Clinical Relevance —Decompressive laminectomy is an effective treatment for DLSS, although dogs with urinary or fecal incontinence have a worse prognosis than dogs that are continent before surgery. Chronic urinary incontinence is a predictor of poor outcome for dogs with DLSS. ( J Am Vet Med Assoc 2001;219:624–628) DA - 2001/9/1/ PY - 2001/9/1/ DO - 10.2460/javma.2001.219.624 VL - 219 IS - 5 SP - 624-628 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035462838&partnerID=MN8TOARS ER - TY - JOUR TI - Neodymium: yttrium aluminum garnet laser ablation of a urethral web to relieve urinary outflow obstruction in a horse AU - Blikslager, A.T. AU - Tate, L.P. AU - Jones, S. T2 - Journal of the American Veterinary Medical Association AB - An 8-year-old Hanoverian gelding was examined because of urine dribbling and urethral obstruction. Mild proprioceptive deficits of the left hind limb were evident during neurologic examination. Ultrasonography per rectum revealed dilatation of the pelvic portion of the urethra. Endoscopy of the urethra revealed 2 webs of tissue: 1 was located 10 cm proximal to the external urethral opening; the other was located 65 cm proximal to the external urethral opening and prevented passage of the endoscope into the urinary bladder. The mass was ablated with a neodymium:yttrium-aluminum-garnet laser, using a transendoscopic noncontact technique. On follow-up examination 6 months after laser surgery, an endoscope could easily be passed into the bladder, and no urethral web was seen. The horse was able to void a stream of urine but continued to dribble urine intermittently. The proximal location of the urethral lesion in this horse would have made use of traditional surgical methods problematic, whereas transendoscopic laser photoablation was easy and effective. DA - 2001/6// PY - 2001/6// DO - 10.2460/javma.2001.218.1970 VL - 218 IS - 12 SP - 1970–1972 J2 - Journal of the American Veterinary Medical Association LA - en OP - SN - 0003-1488 UR - http://dx.doi.org/10.2460/javma.2001.218.1970 DB - Crossref ER - TY - JOUR TI - Molecular epidemiologic features and antimicrobial susceptibility profiles of various ribotypes of Pseudomonas aeruginosa isolated from humans and ruminants AU - Rivas, AL AU - Bodis, M AU - Bruce, JL AU - Anderson, KL AU - Klein, RF AU - Gonzalez, RN AU - Quimby, FW AU - Batt, CA AU - Lein, DH T2 - AMERICAN JOURNAL OF VETERINARY RESEARCH AB - Abstract Objectives —To assess automated ribotyping for characterization of Pseudomonas aeruginosa isolates and to identify their type prevalence and geographic distribution. Sample Population —39 human and 56 ruminant P aeruginosa isolates. Procedures —Isolates were identified by use of bacteriologic techniques and automated PvuII -based ribotyping. Susceptibility to antimicrobials was tested in vitro. Data were analyzed for index of discrimination; prevalence ratio; geographic distribution of ribotypes found only in humans, only in cows, or only in goats (single-host ribotypes); and geographic distribution of ribotypes found in humans and ruminants (multihost ribotypes). Results —All isolates were typeable (45 ribotypes, 35 single-host ribotypes). Ribotyping index of discrimination was 0.976. More isolates (45.3%) than expected yielded multihost ribotypes (22% of all ribotypes). Although 8.6% of single-host ribotypes were found in 4 or more isolates, 60% of multihost ribotypes were found in 4 or more isolates. Ninety percent of multihost ribotypes were isolated from different geographic areas, whereas 3.0% of singlehost ribotypes were isolated from different geographic areas. All ruminant isolates were susceptible to gentamicin and polymyxin B. In contrast, antibiogram profiles differed for human isolates from different geographic areas. Susceptibility to antimicrobials differentiated 6 isolates not distinguished by ribotyping. Conclusions and Clinical Relevance —Automated ribotyping with Pvu II discriminated more isolates than in vitro antimicrobial susceptibility. In combination, both tests provided more information than either test alone. Given the greater prevalence and geographic distribution of multihost ribotypes, immunocompromised humans and lactating ruminants may have a greater risk for disease if exposed to multihost P aeruginosa ribotypes, compared with single-host ribotypes. ( Am J Vet Res 2001;62:864–870) DA - 2001/6// PY - 2001/6// DO - 10.2460/ajvr.2001.62.864 VL - 62 IS - 6 SP - 864-870 SN - 0002-9645 ER - TY - JOUR TI - Investigating magnetically aligned phospholipid bilayers with EPR spectroscopy at 94 GHz AU - Mangels, ML AU - Harper, AC AU - Smirnov, AI AU - Howard, KP AU - Lorigan, GA T2 - JOURNAL OF MAGNETIC RESONANCE AB - In this paper, we report our initial results on studying magnetically aligned phospholipid bilayers (bicelles) at high magnetic fields (approximately 3.4 T) with electron paramagnetic resonance (EPR) spectroscopy at 95 GHz (W-band). In order to characterize this system for W-band EPR studies, we have utilized the nitroxide spin probe 3beta-doxyl-5alpha-cholestane to demonstrate the effects of macroscopic bilayer alignment. At W-band due to the increase in magnetic field strength (when compared to X-band studies at 9.5 GHz) (S. M. Garber et al., J. Am. Chem. Soc. 121, 3240-3241 (1999)), we were able to examine magnetically aligned phospholipid bilayers at two orientations with the bilayer normal oriented either perpendicular or parallel (upon addition of YbCl3) with respect to the direction of the static magnetic field. Additionally, at a magnetic field of 3.4 T (g=2 resonance at W-band), we were able to study the parallel alignment with a lower concentration of Yb3+, thereby eliminating the possible unwanted effects associated with lanthanide-protein interactions and paramagnetic shifts and/or line broadening induced by the lanthanide ions. The development of this new spin label alignment technique will open up a whole new area of investigation for phospholipid bilayer systems and membrane protein EPR studies at high magnetic fields. DA - 2001/8// PY - 2001/8// DO - 10.1006/jmre.2001.2368 VL - 151 IS - 2 SP - 253-259 SN - 1096-0856 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035743313&partnerID=MN8TOARS KW - model membrane KW - nitroxide spin label KW - EPR spectroscopy KW - high-field EPR KW - orientation ER - TY - JOUR TI - Design and synthesis of porphyrin-based optoelectronic gates AU - Ambroise, A AU - Wagner, RW AU - Rao, PD AU - Riggs, JA AU - Hascoat, P AU - Diers, , JR AU - Seth, J AU - Lammi, RK AU - Bocian, DF AU - Holten, D AU - Lindsey, JS T2 - CHEMISTRY OF MATERIALS AB - Two porphyrin-based optoelectronic gates and several prototypical redox-switching components of gates have been synthesized for studies in molecular photonics. Linear and T-shaped molecular optoelectronic gates contain a boron-dipyrrin (BDPY) dye as the input unit, a zinc (Zn) porphyrin as the transmission unit, a free base (Fb) porphyrin as the output unit, and a magnesium (Mg) porphyrin as the redox-switching unit. The linear gate and T gate were synthesized using a molecular building block approach. In the linear gate synthesis, a BDPY−Zn porphyrin dyad was coupled with a Fb porphyrin−Mg porphyrin dimer. The synthesis of the T gate utilized a Zn porphyrin bearing four different meso substituents: mesityl, 4-iodophenyl, 4-[2-(trimethylsilyl)ethynyl]phenyl, and 4-[2-triisopropyl)ethynyl]phenyl. Attachment of the three different groups to the Zn porphyrin was accomplished using successive Pd-mediated coupling reactions in the following sequence: Fb porphyrin (output unit), BDPY dye (input unit), and Mg porphyrin (redox-switching unit). Both the linear gate and T gate syntheses introduce the Mg porphyrin at the final step to minimize demetalation of the Mg porphyrin. Refinements to various components of these gates were investigated through the preparation of a ferrocene−porphyrin, a ferrocene−phthalocyanine, and a ferrocene−porphyrin−phthalocyanine. A dyad motif for studies of optically based redox switching was prepared that contains a derivative of Ru(bpy)3X2 coupled to a porphyrin. From these and related studies have emerged a number of design considerations for the development of refined optoelectronic gates. DA - 2001/3// PY - 2001/3// DO - 10.1021/cm000773m VL - 13 IS - 3 SP - 1023-1034 SN - 1520-5002 ER - TY - JOUR TI - Deep digital flexor tenotomy for treatment of severe laminitis in a cow AU - Gayle, JM AU - Burrell, GA AU - Anderson, KL AU - Redding, WR AU - Blikslager, AT T2 - JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION AB - A first-calf Guernsey cow was referred for evaluation of severe udder edema, mastitis, metritis, and ketosis. During the course of treatment, the cow became recumbent and was unable to rise. Intensive treatment resulted in the cow being able to stand for short periods with the aid of a sling. However, severe pressure necrosis of the udder and ongoing mastitis made performance of a complete mastectomy necessary. After surgery, the cow's condition improved, although assistance in standing was still required. Radiography of the distal phalanges revealed severe rotation in the right lateral and left medial digits of the hind limbs. The laminitis was nonresponsive to medical management; therefore, a deep digital flexor tenotomy was performed in the affected claws. The procedure provided almost immediate relief of signs of foot pain and resulted in ability to stand without assistance. Deep digital flexor tenotomy should be considered when treating cows with severe laminitis. DA - 2001/9/1/ PY - 2001/9/1/ DO - 10.2460/javma.2001.219.644 VL - 219 IS - 5 SP - 644-646 SN - 0003-1488 ER - TY - JOUR TI - Constitutive expression of types 1 and 2 cytokines by alveolar macrophages from feline immunodeficiency virus-infected cats AU - Ritchey, JW AU - Levy, JK AU - Bliss, SK AU - Tompkins, WAF AU - Tompkins, MB T2 - VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY AB - Evidence suggests that feline immunodeficiency virus (FIV), causes pulmonary immunodeficiency. The overall objective of this study was to explore FIV-induced alterations in cell counts and cytokine gene expression in the pulmonary compartment during the acute stage infection. Bronchoalveolar lavage (BAL) cells were collected from FIV-infected and control cats at 0, 4, 10, and 16 weeks post-FIV infection for phenotype and cytokine analysis. The major change in BAL cellular populations following FIV-infection was the development of a neutrophilia. Total BAL cell counts and relative numbers of alveolar macrophages (AM), eosinophils, and lymphocytes remained similar in both groups. The RT-qcPCR analyses of AM purified from BAL showed constitutive expression of TNFα, IL6 and IL10 mRNAs that peaked during the acute stage of infection then declined. The TNFα and IL6 bioactive protein secretion showed a similar response. In contrast, IFNγ expression increased progressively with time after infection and paralleled a progressive increase in FIV-gag mRNA in AM. The IL12 p40 expression also differed from the other cytokines in that there was a progressive decrease in the number of cats with AM IL12 expression following FIV infection. Infection of AM in vitro with FIV also caused an increase in TNFα and IL6 mRNA and bioactive protein suggesting that the increased cytokine response by AM following infection of cats with FIV is an intrinsic characteristic of FIV-infected AM. In summary, pulmonary immune changes seen in FIV-infected cats are similar to those seen in HIV-infected human patients. DA - 2001/5/10/ PY - 2001/5/10/ DO - 10.1016/S0165-2427(01)00250-1 VL - 79 IS - 1-2 SP - 83-100 SN - 0165-2427 KW - FIV KW - macrophages KW - interleukins ER - TY - JOUR TI - Computed tomographic imaging of infiltrative lipoma in 22 dogs AU - McEntee, MC AU - Thrall, DE T2 - VETERINARY RADIOLOGY & ULTRASOUND AB - Twenty two dogs with an infiltrative lipoma had computed tomographic (CT) images acquired to evaluate the extent of local disease. Ten dogs had undergone at least one cytoreductive surgical procedure (range = 1-3; median = 2) prior to imaging. Twenty dogs had measurable disease on CT images; 2 dogs had diffuse disease at a previous surgical site that could not be measured. Tumor volume (n = 20) ranged from 20 to 5,632 cm3 (median = 345 cm3; mean = 996 cm3). None of the dogs had evidence of bone involvement on the CT images; 2 of the 22 dogs had tumors that did not come into direct contact with osseous structures. All dogs with measurable disease had evidence of a fat opacity mass with variable degrees of muscle infiltration. Eleven of 22 dogs were given intravenous contrast medium prior to image acquisition and there was not evidence of enhancement of the infiltrative lipoma in any dog. Based on CT images, tumors were classified as well-defined in 9 dogs, moderately well-defined in 4, not well-defined in 3 and a mix of well-defined and not well-defined in 6 dogs. Tumors tended to be less well-defined in regions where the infiltrative lipoma interdigitated with normal body fat. It appears CT imaging allows adequate discrimination of tumor with the caveat that differentiation of normal fat from infiltrative lipoma can be problematic. DA - 2001/// PY - 2001/// DO - 10.1111/j.1740-8261.2001.tb00928.x VL - 42 IS - 3 SP - 221-225 SN - 1740-8261 KW - infiltrative or infiltrating lipoma KW - computed tomography KW - canine ER - TY - JOUR TI - Compatiblization of polymers via coalescence from their common cyclodextrin inclusion compounds AU - Wei, M AU - Tonelli, AE T2 - MACROMOLECULES AB - We have found, when inherently immiscible polymers are included as guests in the narrow channels of their common inclusion compounds (ICs) formed with host cyclodextrins (CDs) and then these polymer-1/polymer-2-CD-IC crystals are washed with hot water to remove the host CD lattice and coalesce the guest polymers, that intimately mixed blends of the polymers are obtained. This behavior had been observed previously by us for the crystallizable poly(ε-caprolactone) (PCL)/poly(l-lactic acid) (PLLA) pair, where in the coaelsced blend PCL and PLLA crystallinity was completely and nearly completely suppressed, respectively. Here we report similar observations made on the polycarbonate (PC)/poly(methyl methacrylate) (PMMA) pair, which are respectively difficult to crystallize and amorphous. PC/PMMA blends coalesced from their common γ-CD-ICs are amorphous and generally exhibit single glass transitions at temperatures (Tg) between those of pure PC and PMMA. Interestingly, a 1:4 molar PC:PMMA blend coalesced from its common γ-CD-IC is characterized by a Tg lower than that of pure PMMA. FTIR spectroscopy suggests an intimate mixing of and possible specific interactions between PC and PMMA chains in the coalesced blends as reflected by substantial shifts in the frequencies of the PMMA and PC CO vibrations. In addition, repeated DSC scanning or annealing of the coalesced PC/PMMA blends for up to 2 h at T ≥ 200 °C did not lead to phase separation as judged from the constancy of the single Tg's observed by DSC. Though not described in detail here, very similar results were also obtained for the PC/polystyrene (PS) pair, including a Tg for a PS-rich blend that is below the Tg for pure PS. We are currently studying the molecular-level mixing in and thermal stabilities of these blends. DA - 2001/6/5/ PY - 2001/6/5/ DO - 10.1021/ma010235a VL - 34 IS - 12 SP - 4061-4065 SN - 0024-9297 ER - TY - JOUR TI - Characterization of Escherichia coli type 1 pilus mutants with altered binding specificities AU - Harris, SL AU - Spears, PA AU - Havell, EA AU - Hamrick, TS AU - Horton, , JR AU - Orndorff, PE T2 - JOURNAL OF BACTERIOLOGY AB - ABSTRACT PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH , the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket. DA - 2001/7// PY - 2001/7// DO - 10.1128/JB.183.13.4099-4102.2001 VL - 183 IS - 13 SP - 4099-4102 SN - 0021-9193 ER - TY - JOUR TI - An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa AU - Shaw, L. C. AU - Skold, A. AU - Wong, F. AU - Petters, R. AU - Hauswirth, W. AU - Lewin, A. S. T2 - Molecular Vision DA - 2001/// PY - 2001/// VL - 7 IS - 2 SP - 6-13 ER - TY - JOUR TI - Ulcerative dermatosis of the Shetland sheepdog and rough collie dog may represent a novel vesicular variant of cutaneous lupus erythematosus AU - Jackson, HA AU - Olivry, T T2 - VETERINARY DERMATOLOGY AB - A syndrome of ulcerative dermatitis (UDSSC) previously has been described as unique to the Shetland sheepdog and rough collie dog. The pathogenesis of this disease is poorly understood and it has been suggested that it may be a variant of canine dermatomyositis (DM) which is also seen in these breeds. Information on the clinical presentation and previous medical history was collected from five Shetland sheepdogs and three rough collie dogs previously diagnosed with UDSSC. Characteristic features of the disease were adult onset in the summer months with annular, polycyclic and serpiginous ulcerations distributed over sparsely haired areas of the body. Skin biopsies taken from active lesions were compared in a blinded fashion with histological sections from seven Shetland sheepdogs and one rough collie with DM. Dermatomyositis was characterized histologically as a cell poor interface dermatitis associated with follicular atrophy. In contrast, the lesional pattern of UDSSC is that of a lymphocyte-rich interface dermatitis and folliculitis with vesiculation at the dermal-epidermal junction. The authors conclude that these represent two distinct diseases and that UDSSC may be a vesicular form of cutaneous lupus erythematosus seen in the adult rough collie dog and Shetland sheepdog. DA - 2001/2// PY - 2001/2// DO - 10.1046/j.1365-3164.2001.00212.x VL - 12 IS - 1 SP - 19-27 SN - 0959-4493 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035261489&partnerID=MN8TOARS KW - canine KW - cutaneous lupus erythematosus KW - dermatomyositis ER - TY - JOUR TI - Studies related to the design and synthesis of a molecular octal counter AU - Gryko, D AU - Li, JZ AU - Diers, , JR AU - Roth, KM AU - Bocian, DF AU - Kuhr, WG AU - Lindsey, JS T2 - JOURNAL OF MATERIALS CHEMISTRY AB - An approach to the storage of multiple bits of information at the molecular level employs molecules with a large number of distinct oxidation states. Europium triple-decker sandwich molecules composed of porphyrins and phthalocyanines afford four cationic states and are very attractive for molecular information-storage applications. A larger number of states can be achieved by combinations of triple deckers that afford interleaved oxidation potentials. In order to identify suitable candidates for effective interleaving of oxidation potentials, a library of 19 new triple-decker complexes was prepared. Electron-donating groups have been attached to the porphyrin and/or phthalocyanine moieties in order to achieve oxidation states in the low potential regime. The triple deckers are of three different types: (Pc)Eu(Pc)Eu(Por), (Pc)Eu(Por)Eu(Pc), and (Por)Eu(Pc)Eu(Por). The solution electrochemistry of each member of the library was examined. These studies revealed suitable pairs of triple deckers that provide effective interleaving of oxidation potentials. Six triple deckers of type (Pc)Eu(Pc)Eu(Por) were derivatized with a thioacetyl or thiocyanate group on the porphyrin unit for attachment to an electroactive surface. Each of the S-(acetylthio)-derivatized triple deckers forms a self-assembled monolayer (SAM) on Au viain situ cleavage of the thiol protecting group. The SAM of each triple decker is electrochemically robust and exhibits four, well-resolved reversible oxidation waves. Upon disconnection from the source of applied potential, the triple-decker SAMs retain charge for tens to hundreds of seconds. The exact value of the charge-retention time depends on the specific porphyrin/phthalocyanine in the triple decker and the particular oxidation state of the molecules in the SAM (e.g., mono- vs. di- vs. tri- vs. tetracation). For all of the triple-decker SAMs, the charge-retention time monotonically increases as the oxidation state of the molecules in the SAM increases. Collectively, the studies suggest that the triple-decker complexes are excellent candidates for multibit molecular information storage elements. DA - 2001/// PY - 2001/// DO - 10.1039/b008224o VL - 11 IS - 4 SP - 1162-1180 SN - 0959-9428 ER - TY - JOUR TI - Screening method for identification of beta-lactams in bovine urine by use of liquid chromatography and a microbial inhibition test AU - Musser, JMB AU - Anderson, KL AU - Moats, WA T2 - AMERICAN JOURNAL OF VETERINARY RESEARCH AB - Abstract Objective —To develop a multiple-residue screening method for the detection of β-lactams in bovine urine. Animals —6 clinically normal Holstein cows and 6 calves. Procedure —Pooled urine obtained from cows was used as a negative-control sample or spiked with varying concentrations of 6 β-lactam antibiotics. Urine samples were prepared for liquid chromatography by diluting 1 ml of urine with 9 ml of 0.01 M KH 2 PO 4 , 0.01 M Na 2 PO 4 , and filtering. Filtrate (2,000 ml) was eluted with a mobile phase in a gradient program. A fraction corresponding to each β-lactam of interest was collected and evaporated to < 1 ml, and water then was added to achieve a 1ml volume. The collected fraction was tested, using a microbial inhibition test. Then, calves were fed milk spiked with a mixture of 5 β-lactam antibiotics at a concentration 40X the FDA tolerance in milk. Three hours following the feeding, urine samples were obtained from the calves and tested, as described for the urine samples for the cows. Results —The lowest concentrations of amoxicillin, ampicillin, cephapirin, cloxacillin, desfuroylceftiofurcysteine, and penicillin G that were consistently detected in urine were 100, 10, 100, 250, 1,000, and 10 ng/ml, respectively. Amoxicillin, ampicillin, cephapirin, cloxacillin, desacetylcephapirin, and penicillin G were detected in urine samples of 6/6, 5/6, 0/6, 6/6, 2/6, and 3/6 calves respectively, fed antibiotic- spiked milk. Conclusions and Clinical Relevance —The integrated method described can be used to detect or identify β-lactam antibiotics in bovine urine. This method can be used to test cattle for β-lactam residues. ( Am J Vet Res 2001;62:326–330) DA - 2001/3// PY - 2001/3// DO - 10.2460/ajvr.2001.62.326 VL - 62 IS - 3 SP - 326-330 SN - 0002-9645 ER - TY - JOUR TI - Ovarian follicular development following administration of progesterone or aspiration of ovarian follicles in Holstein cows AU - Cavalieri, J AU - Farin, PW AU - Kinder, JE AU - Van Camp, SD AU - Whitacre, MD AU - Washburn, SP AU - Britt, JH T2 - THERIOGENOLOGY AB - The objective of this study was to compare the effects of administration of a single injection of progesterone (P4) and follicle aspiration on Day 7 of the estrous cycle on the timing and synchrony of follicular wave emergence, time of ovulation, and concentrations of P4, estradiol and FSH in Holstein cows. Twenty cows were assigned to 4 groups (n=5 cows per group) in a 2 by 2 factorial arrangement. Cows were treated on Day 7 (Day 0 = estrus) of the estrous cycle with either sham follicular aspiration and an oil vehicle administered intramuscularly (control), aspiration of ovarian follicles (aspiration), 200 mg of P4 im, or aspiration and 200 mg of P4 im (aspiration + P4). On Day 11, PGF(2alpha)(25mg) was administered to all groups. Synchrony of ovulation was less variable in each of the treatment groups compared with the control group (P<0.05), whereas ovulation was delayed in cows in the P4 group (P<0.05). Day of follicular wave emergence was delayed in the cows of the P4 group compared with cows in the aspiration and aspiration + P4 groups (P<0.01), whereas variability in wave emergence was less among both groups of aspirated cows compared with the cows in the control group (P<0.01). More follicles 4 to 7 mm in diameter were detected in the 2 aspiration groups compared with the cows in the control and P4 group (P<0.05). No difference was detected among groups in the maximum concentration of FSH associated with follicular wave emergence. We conclude that both the administration of P4 and the aspiration of follicles on Day 7 of the estrous cycle improves the synchrony of ovulation when luteolysis is induced on Day 11 and results in similar concentrations of FSH at the time of follicular wave emergence, but the timing of wave emergence and the number of follicles post-emergence differ. DA - 2001/2/1/ PY - 2001/2/1/ DO - 10.1016/S0093-691X(01)00445-9 VL - 55 IS - 3 SP - 805-821 SN - 1879-3231 KW - ovarian follicle aspiration KW - FSH KW - progesterone KW - synchronization KW - ultrasonography ER - TY - JOUR TI - Mechanisms of excited-state energy-transfer gating in linear versus branched multiporphyrin arrays AU - Lammi, RK AU - Wagner, RW AU - Ambroise, A AU - Diers, , JR AU - Bocian, DF AU - Holten, D AU - Lindsey, JS T2 - JOURNAL OF PHYSICAL CHEMISTRY B AB - We have investigated electrochemical switching of excited-state electronic energy migration in two optoelectronic gates with different architectures. Each gate consists of diarylethyne-linked subunits: a boron-dipyrrin (BDPY) input unit, a Zn-porphyrin transmission unit, a free-base-porphyrin (Fb-porphyrin) output unit, and a Mg-porphyrin redox-switched site connected either to the Fb porphyrin (linear gate) or to the Zn porphyrin (branched, T gate). Both the linear and branched architectures show Fb-porphyrin emission when the Mg porphyrin is neutral and nearly complete quenching when the Mg porphyrin is oxidized to the π-cation radical. To determine the mechanism of gating, we undertook a systematic photophysical study of the gates and their dyad and triad components in neutral and oxidized forms, using static and time-resolved optical spectroscopy. Two types of photoinduced energy-transfer (and/or charge-transfer) processes are involved in gate operation: transfer between adjacent subunits and transfer between nonadjacent subunits. All of the individual energy-transfer steps that funnel input light energy to the fluorescent output element in the neutral systems are highly efficient, occurring primarily by a through-bond mechanism. Similarly efficient energy-transfer processes occur between the BDPY and the Zn and Fb porphyrins in the oxidized systems, but are followed by rapid and efficient energy/charge transfer to the redox-switched site and consequent nonradiative deactivation. Energy/charge transfer between nonadjacent porphyrins, which occurs principally by superexchange, is crucial to the operation of the T gate. Collectively, our studies elucidate the photophysics of gating and afford great flexibility and control in the design of more elaborate arrays for molecular photonics applications. DA - 2001/6/7/ PY - 2001/6/7/ DO - 10.1021/jp010857y VL - 105 IS - 22 SP - 5341-5352 SN - 1520-5207 ER - TY - JOUR TI - Heme photolysis occurs by ultrafast excited state metal-to-ring charge transfer AU - Franzen, S AU - Kiger, L AU - Poyart, C AU - Martin, JL T2 - BIOPHYSICAL JOURNAL AB - Ultrafast time-resolved resonance Raman spectra of carbonmonoxy hemoglobin (Hb), nitroxy Hb, and deoxy Hb are compared to determine excited state decay mechanisms for both ligated and unligated hemes. Transient absorption and Raman data provide evidence for a sequential photophysical relaxation pathway common to both ligated and unligated forms of Hb* (photolyzed heme), in which the excited state 1Q decays sequentially: 1Q-->Hb*I-->Hb*II-->Hb ground state. Consistent with the observed kinetics, the lifetimes of these states are <50 fs, approximately 300 fs, and approximately 3 ps for 1Q, Hb*I, and Hb*II, respectively. The transient absorption data support the hypothesis that the Hb*I state results from an ultrafast iron-to-porphyrin ring charge transfer process. The Hb*II state arises from porphyrin ring-to-iron back charge transfer to produce a porphyrin ground state configuration a nonequilibrium iron d-orbital population. Equatorial d-pi* back-bonding of the heme iron to the porphyrin during the lifetime of the Hb*II state accounts for the time-resolved resonance Raman shifts on the approximately 3 ps time scale. The proposed photophysical pathway suggests that iron-to-ring charge transfer is the key event in the mechanism of photolysis of diatomic ligands following a porphyrin ring pi-pi* transition. DA - 2001/5// PY - 2001/5// DO - 10.1016/S0006-3495(01)76207-8 VL - 80 IS - 5 SP - 2372-2385 SN - 0006-3495 ER - TY - JOUR TI - Genetic analysis of larval survival and larval growth of two populations of Leptinotarsa decemlineata on tomato AU - Lu, WH AU - Kennedy, GG AU - Gould, F T2 - ENTOMOLOGIA EXPERIMENTALIS ET APPLICATA AB - Abstract The genetics of adaptation to tomato in Leptinotarsa decemlineata (Say) were investigated in reciprocal F 1 , F 2 , and backcross populations generated from crosses between beetles from a tomato adapted population and from a population that was poorly adapted to tomato. Larvae from the parent and test populations were reared on tomato for four days, after which survivorship and larval weights were recorded. Most results indicate that differences in larval growth and survival on tomato between the parent populations are largely determined by autosomal, polygenic mechanisms, the inheritance of which involves a significant dominance component. However, results from F 2 crosses are not consistent with this conclusion. A significant difference in larval weights, but not in survival, between reciprocal F 1 populations in an analysis of combined data from four separate experiments suggests that maternal cytoplasmic effects may contribute to differences in larval performance on tomato between the adapted and unadapted populations. The unusual results obtained from F 2 crosses in this study are not atypical of results from previous studies of the genetics of adaptation to host plants by the Colorado potato beetle. Host plant adaptation by Colorado potato beetles may therefore involve unusual genetic mechanisms that are not easily assessed by classical Mendelian analysis. DA - 2001/5// PY - 2001/5// DO - 10.1046/j.1570-7458.2001.00812.x VL - 99 IS - 2 SP - 143-155 SN - 1570-7458 KW - Leptinotarsa decemlineata KW - genetic variation KW - adaptation on tomato KW - inheritance ER - TY - JOUR TI - Egg marketing in national supermarkets: Specialty eggs - Part 2 AU - Patterson, PH AU - Koelkebeck, KW AU - Bell, DD AU - Carey, JB AU - Anderson, KE AU - Darre, MJ T2 - POULTRY SCIENCE AB - Large eggs promoted as having one or more features beyond conventional white or brown shell eggs (specialty eggs) were evaluated for quality and price in a national retail study. Subtypes of specialty eggs included: nutritionally altered eggs, organic eggs, fertile eggs, eggs from welfare-managed hens, or hens fed all-vegetable diets. Extension Poultry Specialists in California (CA), Connecticut, Illinois, North Carolina, Pennsylvania and Texas conducted a survey of egg quality and price and compared 246 dozen specialty eggs with 390 dozen conventional white shell eggs during the summer of 1996. Age of the eggs based on carton dating indicated specialty eggs were older (16.5 d) than white eggs (11.7 d). Average egg weights for specialty compared to white were 60.2 and 59.6 g, respectively. Interior egg quality evaluations including albumen height, Haugh units (HU), and percentage HU, <55, indicated white eggs were superior (5.0 mm, 67.5, and 10.6%, respectively) compared to specialty eggs (4.7 mm, 63.8, and 16.3%). Although the percentage of cracked eggs was similar between specialty and white eggs (5.4 and 5.7%), the percentage of leakers was threefold higher for the specialty eggs (1.0 vs. 0.3%). Egg price was substantially higher for the specialty eggs, averaging $2.18/dozen with a range from 0.88 to $4.38, compared to white eggs, averaging $1.23/dozen and ranging from 0.39 to $2.35. DA - 2001/4// PY - 2001/4// DO - 10.1093/ps/80.4.390 VL - 80 IS - 4 SP - 390-395 SN - 0032-5791 KW - specialty eggs KW - organic KW - egg quality KW - age KW - price ER - TY - JOUR TI - Egg marketing in national supermarkets: Products, packaging, and prices - Part 3 AU - Koelkebeck, KW AU - Bell, DD AU - Carey, JB AU - Anderson, KE AU - Darre, MJ T2 - POULTRY SCIENCE AB - As part of a national retail egg quality study, the variety of shell eggs and egg products offered for sale, type of packaging, and price relationships were compared in five major metropolitan regions. A total of 81 stores in 28 cities were sampled in California (CA), Illinois (IL), North Carolina (NC), Texas (TX), and New England (NE). Data were recorded for the variety of brands, sizes, white or brown shell eggs, specialty eggs, liquid or frozen eggs, carton sizes, package labeling and coding, and price relationships of shell eggs, liquid, and frozen egg products displayed for sale. The total variety of shell eggs displayed per store was the greatest for CA and NE stores. Stores in CA and TX offered more (P < 0.05) variety of white shell eggs than did stores in the other states, whereas stores in NE displayed the greatest variety (P < 0.05) of brown shell eggs. The average number of liquid and frozen egg products was highest (P < 0.05) for NC stores. Packaging type, USDA labeling, and carton coding differed somewhat among states. The price per one dozen cartons of all white shell egg sizes was highest (P < 0.05) in CA stores, and the average liquid plus frozen egg product prices were higher in CA and NE stores compared to the other states. However, the ratio of liquid and frozen product prices to all large shell egg prices was among the lowest for CA and NC stores. These data indicate that product selection, packaging, and consumer prices for shell eggs and egg products varied considerably across five separate regions of the country. DA - 2001/4// PY - 2001/4// DO - 10.1093/ps/80.4.396 VL - 80 IS - 4 SP - 396-400 SN - 0032-5791 KW - egg marketing KW - shell egg varieties KW - egg product varieties KW - egg packaging KW - consumer egg price ER - TY - JOUR TI - Egg marketing in national supermarkets: Egg quality - Part 1 AU - Bell, DD AU - Patterson, PH AU - Koelkebeck, KW AU - Anderson, KE AU - Darre, MJ AU - Carey, JB AU - Kuney, DR AU - Zeidler, G T2 - POULTRY SCIENCE AB - Two surveys were conducted to determine the quality of eggs offered to consumers in large supermarkets in various regions of the US. The first survey was conducted in California (CA) in 1994 and included 38 samples of large (L) and extra large (XL) white eggs in 15 markets. Individual eggs were weighed, candled, and broken out for Haugh unit (HU) determination. Regional differences in age of eggs, the number of eggs below 55 HU, and the percentage of cracked eggs were observed. The second survey was conducted in California (CA), Illinois (IL), Pennsylvania (PA), Texas (TX), North Carolina (NC), and New England (NE). This study included brown and white eggs and samples from 115 stores in 38 cities. Significant age, egg weight, HU, and cracked egg differences were observed between states. Brown and white eggs were different relative to age and HU, but egg weights and cracked eggs were statistically the same. The two surveys, 1994 and 1996, within CA demonstrated very similar measurements when L-white eggs were compared. DA - 2001/4// PY - 2001/4// DO - 10.1093/ps/80.4.383 VL - 80 IS - 4 SP - 383-389 SN - 0032-5791 KW - egg marketing KW - retail KW - egg quality KW - egg breakage ER - TY - JOUR TI - Comparative efficacy of tricaine methanesulfonate and clove oil for use as anesthetics in red pacu (Piaractus brachypomus) AU - Sladky, KK AU - Swanson, CR AU - Stoskopf, MK AU - Loomis, MR AU - Lewbart, GA T2 - AMERICAN JOURNAL OF VETERINARY RESEARCH AB - Abstract Objective —To compare the anesthetic efficacy and physiologic changes associated with exposure to tricaine methanesulfonate and clove oil (100% eugenol). Animals —15 adult cultured red pacu ( Piaractus brachypomus ). Procedure —Fish were exposed to each of 6 anesthetic concentrations in a within-subjects complete crossover design. Stages of anesthesia and recovery were measured, and physiologic data were collected before and during anesthesia. Results —Interval to induction was more rapid and recovery more prolonged in fish exposed to eugenol, compared with those exposed to tricaine methanesulfonate. The margin of safety for eugenol was narrow, because at the highest concentration, most fish required resuscitation. Mixed venous-arterial PO 2 consistently decreased with anesthesia, while PCO 2 consistently increased with anesthesia in all fish regardless of anesthetic agent. The increase in PCO 2 was accompanied by a decrease in pH, presumably secondary to respiratory acidosis. Anesthesia was associated with increased blood glucose, potassium, and sodium concentrations as well as Hct and hemoglobin. Fish anesthetized with eugenol were more likely to react to a hypodermic needle puncture than fish anesthetized with tricaine methanesulfonate. Conclusions and Clinical Relevance —Anesthesia induced with tricaine methanesulfonate or eugenol contributes to hypoxemia, hypercapnia, respiratory acidosis, and hyperglycemia in red pacu. Similar to tricaine methanesulfonate, eugenol appears to be an effective immobilization compound, but eugenol is characterized by more rapid induction, prolonged recovery, and a narrow margin of safety. Care must be taken when using high concentrations of eugenol for induction, because ventilatory failure may occur rapidly. In addition, analgesic properties of eugenol are unknown. ( Am J Vet Res 2001;62:337–342) DA - 2001/3// PY - 2001/3// DO - 10.2460/ajvr.2001.62.337 VL - 62 IS - 3 SP - 337-342 SN - 1943-5681 ER - TY - JOUR TI - Characterization of the inflammatory infiltrate during IgE-mediated late phase reactions in the skin of normal and atopic dogs AU - Olivry, T AU - Dunston, SM AU - Murphy, KM AU - Moore, PF T2 - VETERINARY DERMATOLOGY AB - In canine and human atopic patients, the intracutaneous injection of offending allergens is followed by the development of both immediate and late-phase reactions. The present study was performed to expand on the characterization and dynamics of inflammatory cell subsets during IgE-mediated late-phase reactions in canine skin. Three normal dogs and three Dermatophagoides farinae-allergic dogs were selected for this experiment. All dogs were challenged intradermally with mite allergen, purified anticanine IgE antibodies (positive control) or phosphate-buffered saline (negative control). Skin biopsies were obtained before and 6, 12 and 24 h post-injection. Sections were stained with metachromatic and eosinophil-specific histological stains. Additionally, we used an immunohistochemical method with antibodies specific for canine leukocyte antigens. This study confirmed the occurrence of a late-phase reaction in atopic skin following allergen challenge, and in normal and atopic canine skin after intradermal injection of IgE-specific antibodies. Whereas early emigrating dermal cells were composed chiefly of neutrophil and activated eosinophil granulocytes, there was an influx of alpha beta T-lymphocytes and dermal dendritic cells in later stages of the late-phase reactions. Because IgE-mediated late-phase reactions resemble spontaneous atopic canine skin lesions, both at macroscopic and microscopic levels, we propose the use of similar challenges to study the anti-inflammatory effects of anti-allergic drugs in a pre-clinical setting. DA - 2001/2// PY - 2001/2// DO - 10.1046/j.1365-3164.2001.00230.x VL - 12 IS - 1 SP - 49-58 SN - 1365-3164 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035260986&partnerID=MN8TOARS KW - allergy KW - atopic dermatitis KW - dog KW - late-phase reaction KW - skin ER - TY - JOUR TI - Baculovirus expression of turkey coronavirus nucleocapsid protein AU - Breslin, JJ AU - Smith, LG AU - Guy, JS T2 - AVIAN DISEASES AB - The nucleocapsid (N) gene of turkey coronavirus (TCV) was amplified by reverse transcriptase-polymerase chain reaction, cloned, and expressed in the baculovirus expression system. A recombinant baculovirus containing the TCV N gene (rBTCV/N) was identified by polymerase chain reaction and expression of TCV N protein as determined by western immunoblot analysis. Two TCV-specific proteins, 52 and 43 kDa, were expressed by rBTCV/N; one of these proteins, p52, was comparable in size to native TCV N protein. Baculovirus-expressed N proteins were used as antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for detection of TCV-specific antibodies. The ELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related avian coronavirus, but did not detect antibodies specific for other avian viruses (avian influenza, avian reovirus, avian paramyxovirus 3, avian adenovirus 1, or Newcastle disease virus). These findings indicate that baculovirus-expressed TCV N protein is a suitable source of antigen for ELISA-based detection of TCV-specific antibodies in turkeys. DA - 2001/// PY - 2001/// DO - 10.2307/1593020 VL - 45 IS - 1 SP - 136-143 SN - 0005-2086 KW - turkey coronavirus KW - baculovirus KW - enzyme-linked immunosorbent assay ER - TY - JOUR TI - Assessing the association between the geographic distribution of deer ticks and seropositivity rates to various tick-transmitted disease organisms in dogs AU - Hinrichsen, Virginia L. AU - Whitworth, Ulysses G. AU - Breitschwerdt, Edward B. AU - Hegarty, Barbara C. AU - Mather, Thomas N. T2 - Journal of the American Veterinary Medical Association AB - To determine whether the geographic distribution of deer ticks (Ixodes scapularis) was associated with the distribution of dogs seropositive for various tick-transmitted disease organisms (ie, Borrelia burgdorferi, Rickettsia rickettsii, the human granulocytic ehrlichiosis [HGE] agent, Ehrlichia canis, and Bartonella vinsonii subsp berkhoffii).Serologic survey.Serum samples from 277 dogs in animal shelters and veterinary hospitals in Rhode Island.Overall, 143 (52%) dogs were seropositive for B burgdorferi, 59 (21.3%) were seropositive for R rickettsii, 40 (14.4%) were seropositive for the HGE agent, 8 (2.9%) were seropositive for E canis, and 6 (2.2%) were seropositive for B vinsonii. Regression analysis indicated that the natural logarithm of nymphal deer tick abundance was correlated with rate of seropositivity to the HGE agent and to B burgdorferi but not to rate of seropositivity to R rickettsii, E canis, or B vinsonii. Percentages of samples seropositive for B burgdorferi, R rickettsii, the HGE agent, and E canis were significantly higher for samples from the southwestern part of the state where ticks in general and deer ticks in particular are abundant than for samples from the northern and eastern portions of the state, where ticks are relatively rare.Results suggested that all 5 disease agents are in Rhode Island and pose a risk to dogs and humans. Knowledge concerning tick distributions may be useful in predicting the pattern of disease associated with particular tick species and may aid diagnostic, prevention, and control efforts. DA - 2001/4// PY - 2001/4// DO - 10.2460/javma.2001.218.1092 VL - 218 IS - 7 SP - 1092-1097 J2 - Journal of the American Veterinary Medical Association LA - en OP - SN - 0003-1488 UR - http://dx.doi.org/10.2460/javma.2001.218.1092 DB - Crossref ER - TY - JOUR TI - alpha-helix formation: Discontinuous molecular dynamics on an intermediate-resolution protein model AU - Smith, AV AU - Hall, CK T2 - PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS AB - Abstract An intermediate‐resolution model of small, homogeneous peptides is introduced, and discontinuous molecular dynamics simulation is applied to study secondary structure formation. Physically, each model residue consists of a detailed three‐bead backbone and a simplified single‐bead side‐chain. Excluded volume and hydrogen bond interactions are constructed with discontinuous (i.e., hard‐sphere and square‐well) potentials. Simulation results show that the backbone motion of the model is limited to realistic regions of Φ–Ψ conformational space. Model polyalanine chains undergo a locally cooperative transition to form α‐helices that are stabilized by backbone hydrogen bonding, while model polyglycine chains tend to adopt nonhelical structures. When side‐chain size is increased beyond a critical diameter, steric interactions prevent formation of long α‐helices. These trends in helicity as a function of residue type have been well documented by experimental, theoretical, and simulation studies and demonstrate the ability of the intermediate‐resolution model developed in this work to accurately mimic realistic peptide behavior. The efficient algorithm used permits observation of the complete helix–coil transition within 15 min on a single‐processor workstation, suggesting that simulations of very long times are possible with this model. Proteins 2001;44:344–360. © 2001 Wiley‐Liss, Inc. DA - 2001/8/15/ PY - 2001/8/15/ DO - 10.1002/prot.1100 VL - 44 IS - 3 SP - 344-360 SN - 1097-0134 KW - helix-coil transition KW - computer simulation KW - four-bead protein model ER - TY - JOUR TI - Use of echocardiography for the diagnosis of heartworm disease in cats: 43 cases (1985-1997) AU - DeFrancesco, Teresa C. AU - Atkins, Clarke E. AU - Miller, Matthew W. AU - Meurs, Kathryn M. AU - Keene, Bruce W. T2 - Journal of the American Veterinary Medical Association AB - Abstract Objective —To determine the usefulness of echocardiography in the diagnosis of heartworm disease in cats and to compare this modality with other tests. Design —Retrospective study. Animals —43 cats with heartworm infection that had echocardiographic examinations at 2 veterinary teaching hospitals between 1985 and 1997. Twenty-two of these 43 cats also underwent radiography of the thorax and heartworm antibody and heartworm antigen testing. Procedure —Cats were determined to be infected with Dirofilaria immitis infection on the basis of 1 or more of the following findings: positive modified Knott or antigen test result, echocardiographic evidence of heartworm disease, or confirmation of the disease on postmortem examination. The percentage of echocardiographs in which heartworms were evident was compared with the percentage of radiographs in which pulmonary artery enlargement was evident and results of antigen or antibody tests in cats in which all tests were performed. Results —Overall, heartworms were detectable by use of echocardiography in 17 of 43 cats, most often in the pulmonary arteries. In the 22 cats in which all tests were performed, antibody test results were positive in 18, antigen test results were positive in 12, and pulmonary artery enlargement was evident radiographically and heartworms were identifiable echocardiographically in 14. Heartworm infection was diagnosed exclusively by use of echocardiography in 5 cats in which the antigen test result was negative. Conclusions and Clinical Relevance —Although echocardiography was less sensitive than antigen testing, it was a useful adjunctive test in cats that had negative antigen test results in which there was a suspicion of heartworm disease. The pulmonary arteries should be evaluated carefully to increase the likelihood of detection of heartworms echocardiographically. ( J Am Vet Med Assoc2001;218:66–69) DA - 2001/1// PY - 2001/1// DO - 10.2460/javma.2001.218.66 VL - 218 IS - 1 SP - 66-69 J2 - Journal of the American Veterinary Medical Association LA - en OP - SN - 0003-1488 UR - http://dx.doi.org/10.2460/javma.2001.218.66 DB - Crossref ER - TY - JOUR TI - The ACVD task force on canine atopic dermatitis (XXII): nonsteroidal anti-inflammatory pharmacotherapy AU - Marsella, R AU - Olivry, T T2 - VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY AB - The pharmacotherapy of canine atopic dermatitis has relied primarily on the use of glucocorticoids and anti-histamines. During the last decade, other anti-inflammatory drugs have been investigated in clinical trials. This paper will review the studies using misoprostol, cyclosporine, tacrolimus, phosphodiesterase inhibitors, capsaicin, leukotriene inhibitors and serotonin-reuptake inhibitors for treatment of dogs with atopic dermatitis. For each drug the mechanism of action, the rationale for use in atopic dermatitis, the clinical efficacy, reported adverse effects and strength of recommendation for treatment of canine atopic dermatitis are described. At the time of this writing, there is fair evidence to support the recommendation for using cyclosporine, misoprostol and pentoxifylline for treatment of canine atopic dermatitis. This recommendation can be strengthened by the performance of additional blinded randomized controlled trials with larger number of dogs. In contrast, there is insufficient evidence to recommend for or against treatment with tacrolimus, leukotriene inhibitors, serotonin-reuptake antagonists and capsaicin. DA - 2001/9/20/ PY - 2001/9/20/ DO - 10.1016/S0165-2427(01)00315-4 VL - 81 IS - 3-4 SP - 331-345 SN - 1873-2534 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035922086&partnerID=MN8TOARS KW - atopic dermatitis KW - arofylline KW - capsaicin cyclosporine KW - dog KW - fluoxetine KW - misoprostol KW - pentoxifylline KW - tacrolimus KW - zileuton ER - TY - JOUR TI - The ACVD task force on canine atopic dermatitis (XX): glucocorticoid pharmacotherapy AU - Olivry, T AU - Sousa, CA T2 - VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY AB - Glucocorticoids (GCs) have been the most commonly prescribed drugs for treatment of canine atopic dermatitis (AD) during the last decades. In spite of this widespread usage, there are a few studies documenting their efficacy. Fortunately, recently completed clinical trials were designed with oral GCs used as "standard of care" for treatment of canine AD. These studies provided high quality evidence in favor of the strong efficacy of oral low-dose glucocorticoid formulations to control skin lesions and pruritus in dogs with AD. Consequently, there is good evidence to support the recommendation of use of oral glucocorticoids for treatment of canine AD. DA - 2001/9/20/ PY - 2001/9/20/ DO - 10.1016/S0165-2427(01)00314-2 VL - 81 IS - 3-4 SP - 317-322 SN - 1873-2534 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035922085&partnerID=MN8TOARS KW - atopy KW - atopic dermatitis KW - corticosteroids KW - dogs KW - glucocorticoids KW - prednisone ER - TY - JOUR TI - The ACVD task force on canine atopic dermatitis (XVIII): histopathology of skin lesions AU - Olivry, T AU - Hill, PB T2 - VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY AB - For years, the histopathology of skin lesions of canine atopic dermatitis was deemed non-specific for this diagnosis. However, more recent studies have established that canine atopic skin lesions exhibit an inflammatory pattern characterized as a chronic, hyperplastic and spongiotic, mixed perivascular dermatitis. The nature of epidermal and dermal inflammatory cell infiltrates has now been characterized using modern immunological techniques. Epitheliotropic cells include Langerhans' cells, T-lymphocytes and rare eosinophils. Dermal cells are composed of mast cells, dermal antigen-presenting cells, T-lymphocytes and occasional intact and degranulated eosinophils. This paper provides an historical review of the landmark papers that have elucidated the pathology of canine atopic dermatitis. DA - 2001/9/20/ PY - 2001/9/20/ DO - 10.1016/S0165-2427(01)00305-1 VL - 81 IS - 3-4 SP - 305-309 SN - 0165-2427 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035922096&partnerID=MN8TOARS KW - allergic skin disease KW - atopy KW - dermatopathology KW - dog ER - TY - JOUR TI - The ACVD task force on canine atopic dermatitis (XIX): general principles of therapy AU - Olivry, T AU - Sousa, CA T2 - VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY AB - The treatment of canine atopic dermatitis is multifaceted and consists of a combination of actions that include the use of allergen avoidance, anti-inflammatory agents, allergen-specific immunotherapy and antimicrobial drugs. The importance and order of these treatment steps vary from patient to patient. General recommendations for each of the therapeutic steps are highlighted in this paper. Specific details are covered in other papers of this issue. DA - 2001/9/20/ PY - 2001/9/20/ DO - 10.1016/S0165-2427(01)00347-6 VL - 81 IS - 3-4 SP - 311-316 SN - 0165-2427 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035922084&partnerID=MN8TOARS KW - allergen avoidance KW - antibiotics KW - antifungal drugs KW - atopy KW - dog KW - immunotherapy KW - pharmacotherapy ER - TY - JOUR TI - The ACVD task force on canine atopic dermatitis (IX): the controversy surrounding the route of allergen challenge in canine atopic dermatitis AU - Olivry, Thierry AU - Hill, Peter B T2 - Veterinary Immunology and Immunopathology AB - For decades, the dogma that environmental allergens trigger cutaneous inflammation led to the denomination of canine atopic dermatitis as "allergic inhalant dermatitis". Definitive proof for a respiratory route of allergen challenge is lacking, however. Recent observations suggest, in fact, that skin inflammation could occur because of epidermal allergenic contact. The aim of this paper is to review the evidence published in favor and against the two suspected routes of allergen provocation. DA - 2001/9// PY - 2001/9// DO - 10.1016/S0165-2427(01)00311-7 VL - 81 IS - 3-4 SP - 219-225 J2 - Veterinary Immunology and Immunopathology LA - en OP - SN - 0165-2427 UR - http://dx.doi.org/10.1016/s0165-2427(01)00311-7 DB - Crossref KW - allergen KW - allergic skin disease KW - atopy KW - dog ER - TY - JOUR TI - Testing equality of survival functions of quality-adjusted lifetime AU - Zhao, HW AU - Tsiatis, AA T2 - BIOMETRICS AB - We present a method for comparing the survival functions of quality-adjusted lifetime from two treatments. This test statistic becomes the ordinary log-rank test when quality-adjusted lifetime is the same as the survival time. Simulation experiments are conducted to examine the behavior of our proposed test statistic under both null and alternative hypotheses. In addition, we apply our method to a breast cancer trial for comparing the distribution of quality-adjusted lifetime between two treatment regimes. DA - 2001/9// PY - 2001/9// DO - 10.1111/j.0006-341X.2001.00861.x VL - 57 IS - 3 SP - 861-867 SN - 0006-341X KW - counting processes KW - hypothesis test KW - martingale processes KW - quality of life KW - survival analysis ER - TY - JOUR TI - Synthesis and excited-state photodynamics of perylene-porphyrindyads. Part 3, Effects of perylene, linker, and connectivity on ultrafast energy transfer AU - Yang, S. I. AU - Lammi, R. K. AU - Prathapan, S. AU - Miller, M. A. AU - Seth, J. AU - Diers, J. R. AU - Bocian, D. F. AU - Lindsey, Jonathan AU - Holten, D. T2 - Journal of Materials Chemistry AB - New perylene–porphyrin dyads have been designed that exhibit superior light-harvesting and energy-utilization activity compared with earlier generations of structurally related dyads. The new dyads consist of a perylene mono(imide) dye (PMI) connected to a porphyrin (Por) via an ethynylphenyl (ep) linker. The PMI–ep–Por arrays were prepared with the porphyrin as either a zinc or magnesium complex (Por = Zn or Mg) or a free-base form (Por = Fb). The absorption properties of the perylene complement those of the porphyrin. Following excitation of the perylene (forming PMI*) in toluene, each array exhibits ultrafast (kENT ≥ (0.5 ps)−1) and essentially quantitative energy transfer from PMI* to the ground-state porphyrin (forming Por*). In each of the arrays, the properties of the excited porphyrin (lifetime, fluorescence yield, etc.) are basically unperturbed from those of the isolated pigment. Thus, following energy transfer, the excited porphyrin is not quenched by deleterious reactions involving the perylene accessory unit that would otherwise limit the ability of Por* to emit light or transfer energy to another stage in a molecular photonic device. Collectively, the PMI–ep–Por dyads represent the successful result of a molecular design strategy to produce arrays with excellent properties for use as light-input and energy-transduction elements for applications in molecular optoelectronics. DA - 2001/// PY - 2001/// DO - 10.1039/b102741g VL - 11 IS - 10 SP - 2420–2430 ER - TY - JOUR TI - Signaling mechanism for equine neutrophil activation by immune complexes AU - Jones, SL AU - Sharief, Y AU - Chilcoat, CD T2 - VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY AB - Neutrophils (PMN) are critical host defense cells that have a role in the pathophysiology of a variety of inflammatory diseases, particularly those diseases associated with antigen-antibody immune complexes (IC) deposited in tissues. Activation of PMN by IC is most efficient if the IC are presented immobilized on a surface. Adhesion to the immobilized IC is important for subsequent activation of PMN effector functions, such as generation of reactive oxygen metabolites. Adhesion of human PMN to immobilized IC requires the expression and activation of adhesion receptors called integrins. Of the integrins expressed on PMN, the beta 2 family has been found to be of particular importance for PMN function. The mechanism of beta 2 integrin activation during adhesion to IC has been studied in human PMN, but not in equine PMN. We show here that adhesion of equine PMN to immobilized IC requires beta 2 integrins. Like adhesion, IC-induced respiratory burst activity is dependent on beta 2 integrins. Furthermore, the signaling pathway triggering beta 2 integrin-dependent adhesion of equine PMN to IC and subsequent generation of respiratory burst activity is inhibited by the specific phosphatidylinositol 3-kinase (PI3K) antagonists wortmannin and LY294002 with IC(50) (concentration at which 50% inhibition is achieved) similar to the published values for inhibition of PI3K enzymatic activity. In contrast, PMA-induced activation of beta 2 integrin-dependent adhesion and respiratory burst activity are wortmannin and LY294002 insensitive. These data demonstrate that like in human PMN, IC-induced activation of beta 2 integrins and beta 2 integrin-dependent functions in equine PMN is dependent on PI3K activity. DA - 2001/9/28/ PY - 2001/9/28/ DO - 10.1016/S0165-2427(01)00350-6 VL - 82 IS - 1-2 SP - 87-100 SN - 0165-2427 UR - http://europepmc.org/abstract/med/11557296 KW - neutrophil KW - adhesion KW - integrin KW - phosphatidylinositol 3-kinase KW - immune complex KW - Fe receptor KW - equine ER - TY - JOUR TI - Polarized ketone inhibition of 1-naphthyl acetate esterase in azinphosmethyl-resistant and -susceptible tufted apple bud moths, Platynota idaeusalis (Walker): Novel insecticide synergists AU - Devorshak, C AU - Roe, RM T2 - PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY AB - Aliphatic and aromatic polarized trifluoromethylketones were synthesized as inhibitors of 1-naphthyl acetate (NA) esterase and potential insecticide synergists for azinphosmethyl-resistant tufted apple bud moths. A 1-NA resistance-associated esterase (RAE) was isolated from whole body homogenates of azinphosmethyl-resistant adult females. A second susceptible-associated 1-NA esterase (SE) was purified from azinphosmethyl-susceptible adult females of the tufted apple bud moth. In structure-activity studies, octylthio-1,1,1-trifluoromethyl-2-propanone (OTFP) was the most potent inhibitor of whole body 1-NA esterase from resistant and susceptible adult females. The I50s for inhibition of purified RAE and SE were 1 × 10−8.5 and 1 × 10−8.6 M, respectively. Longer and shorter n-alkyl groups or a S-phenyl substitution reduced inhibitory activity. These inhibitors were also found to compete with 1-NA substrate for the esteratic active site. OTFP alone had minimal toxicity toward third and fifth instars of the tufted apple bud moth but synergized azinphosmethyl toxicity in resistant insects. Although novel esterases were found in resistant bud moth larvae and adults, there was no evidence that the resistance-associated esterases had a unique function different from that found in susceptible insects. DA - 2001/1// PY - 2001/1// DO - 10.1006/pest.2000.2526 VL - 69 IS - 1 SP - 48-62 SN - 1095-9939 ER - TY - PAT TI - Method of treating alopecia AU - Smart, R. C. AU - Oh, H.-S. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - Linear mixed models with flexible distributions of random effects for longitudinal data AU - Zhang, DW AU - Davidian, M T2 - BIOMETRICS AB - Normality of random effects is a routine assumption for the linear mixed model, but it may be unrealistic, obscuring important features of among-individual variation. We relax this assumption by approximating the random effects density by the seminonparameteric (SNP) representation of Gallant and Nychka (1987, Econometrics 55, 363-390), which includes normality as a special case and provides flexibility in capturing a broad range of nonnormal behavior, controlled by a user-chosen tuning parameter. An advantage is that the marginal likelihood may be expressed in closed form, so inference may be carried out using standard optimization techniques. We demonstrate that standard information criteria may be used to choose the tuning parameter and detect departures from normality, and we illustrate the approach via simulation and using longitudinal data from the Framingham study. DA - 2001/9// PY - 2001/9// DO - 10.1111/j.0006-341X.2001.00795.x VL - 57 IS - 3 SP - 795-802 SN - 0006-341X KW - longitudinal data KW - multimodality KW - random effects KW - seminonparametric density KW - semiparametric mixed effects model KW - skewness ER - TY - JOUR TI - Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis AU - Zhu, Y AU - Yamamoto, T AU - Cullen, J AU - Saputelli, J AU - Aldrich, CE AU - Miller, DS AU - Litwin, S AU - Furman, PA AU - Jilbert, AR AU - Mason, WS T2 - JOURNAL OF VIROLOGY AB - ABSTRACT Hepadnaviruses replicate by reverse transcription, which takes place in the cytoplasm of the infected hepatocyte. Viral RNAs, including the pregenome, are transcribed from a covalently closed circular (ccc) viral DNA that is found in the nucleus. Inhibitors of the viral reverse transcriptase can block new DNA synthesis but have no direct effect on the up to 50 or more copies of cccDNA that maintain the infected state. Thus, during antiviral therapy, the rates of loss of cccDNA, infected hepatocytes (1 or more molecules of cccDNA), and replicating DNAs may be quite different. In the present study, we asked how these losses compared when woodchucks chronically infected with woodchuck hepatitis virus were treated with L-FMAU [1-(2-fluoro-5-methyl-β- l -arabinofuranosyl) uracil], an inhibitor of viral DNA synthesis. Viremia was suppressed for at least 8 months, after which drug-resistant virus began replicating to high titers. In addition, replicating viral DNAs were virtually absent from the liver after 6 weeks of treatment. In contrast, cccDNA declined more slowly, consistent with a half-life of ∼33 to 50 days. The loss of cccDNA was comparable to that expected from the estimated death rate of hepatocytes in these woodchucks, suggesting that death of infected cells was one of the major routes for elimination of cccDNA. However, the decline in the actual number of infected hepatocytes lagged behind the decline in cccDNA, so that the average cccDNA copy number in infected cells dropped during the early phase of therapy. This observation was consistent with the possibility that some fraction of cccDNA was distributed to daughter cells in those infected hepatocytes that passed through mitosis. DA - 2001/1// PY - 2001/1// DO - 10.1128/JVI.75.1.311-322.2001 VL - 75 IS - 1 SP - 311-322 SN - 0022-538X ER - TY - JOUR TI - Infection of Fetal Feline Brain Cells in Culture with Bartonella henselae AU - Munana, K. R. AU - Vitek, S. M. AU - Hegarty, B. C. AU - Kordick, D. L. AU - Breitschwerdt, E. B. T2 - Infection and Immunity AB - ABSTRACT Bartonella henselae is known to cause central nervous system (CNS) disease in humans, and neurological signs have been observed in experimentally infected cats. However, the pathogenesis of CNS disease remains unclear. This study was undertaken to determine whether B. henselae infects feline fetal brain cells in vitro. Microglial-cell- and astrocyte-enriched cultures were inoculated with B. henselae . Giménez staining identified bacterial organisms within microglial cells by day 7 postinoculation. The viability of the intracellular bacteria was demonstrated by incubating cultures with gentamicin and plating cell lysate on agar. Electron microscopy identified intracellular organisms with characteristic Bartonella morphology but identified no ultrastructural abnormalities within infected microglial cells. No evidence of infection was seen in Bartonella -inoculated astrocyte cultures. These findings suggest a role for microglia in the pathogenesis of B. henselae -associated neurological disease. DA - 2001/1/1/ PY - 2001/1/1/ DO - 10.1128/IAI.69.1.564-569.2001 VL - 69 IS - 1 SP - 564-569 J2 - Infection and Immunity LA - en OP - SN - 0019-9567 UR - http://dx.doi.org/10.1128/IAI.69.1.564-569.2001 DB - Crossref ER - TY - JOUR TI - Identification of arachnoid cysts in the quadrigeminal cistern using ultrasonography AU - Saito, M AU - Olby, NJ AU - Spaulding, K T2 - VETERINARY RADIOLOGY & ULTRASOUND AB - The ultrasonographic findings in three small breed dogs with intracranial arachnoid cysts are described. Ultrasound images were obtained via the foramen magnum, temporal window and persistent bregmatic fontanelle (when possible). In transverse, dorsal and sagittal transcranial ultrasound images there was marked dilation of the lateral ventricles and a well-defined, oval to triangular-shaped anechoic area between the caudal aspect of the occipital lobes, dorsal to the midbrain, and rostral to the cerebellum. These findings were consistent with a diagnosis of concurrent hydrocephalus and an arachnoid cyst within the quadrigeminal cistern. DA - 2001/// PY - 2001/// DO - 10.1111/j.1740-8261.2001.tb00966.x VL - 42 IS - 5 SP - 435-439 SN - 1058-8183 ER - TY - PAT TI - High-density non-volatile memory devices incorporating sandwich coordination compounds AU - Lindsey, J. S. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - JOUR TI - High spatial resolution multi-site EPR oximetry - The use of a convolution-based fitting method AU - Grinberg, OY AU - Smirnov, AI AU - Swartz, HM T2 - JOURNAL OF MAGNETIC RESONANCE AB - We describe a new method to enhance the spatial resolution of multi-site electron paramagnetic resonance (EPR) oximetry. The method is suitable for any shape (density distribution function) of a solid paramagnetic material implanted in tissue. It corrects distortions of lineshapes caused by the gradient and thus overcomes limitations of previous multi-site EPR oximetry methods that restricted the ratio of the particle size to the distance between sites. The new method is based on consecutive applications of magnetic field gradients with the same direction but with a different magnitude and uses a convolution-based fitting algorithm to derive Lorentzian EPR linewidths of each individual peak of the EPR spectrum. The method is applicable for any particulate EPR oxygen sensitive materials whose EPR spectra can be approximated by a Lorentzian function or a superposition of Lorentzian functions. By incorporating this model of the lineshape in the data processing, we are able to decrease significantly the number of parameters needed for the calculations and to recover the oxygen concentration, even from quite noisy spectra. We (i) describe our method and the data-processing algorithm, (ii) demonstrate our approach in model and in vivo experiments, and (iii) discuss the limitations. DA - 2001/10// PY - 2001/10// DO - 10.1006/jmre.2001.2408 VL - 152 IS - 2 SP - 247-258 SN - 1096-0856 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035743038&partnerID=MN8TOARS KW - EPR oximetry KW - multi-site EPR oximetry KW - tissue pO(2) KW - convolution-based fitting ER - TY - JOUR TI - High field electron paramagnetic resonance of Gd3+-doped glasses: Line shapes and average ion distances in silicates AU - Smirnov, AI AU - Sen, S T2 - JOURNAL OF CHEMICAL PHYSICS AB - The average distances between Gd3+ ions in multicomponent silicate glasses doped with 0.058 to up to 4.64 wt % Gd2O3 are characterized by electron paramagnetic resonance (EPR) line broadening effects at high-field (95 GHz, W band). Increase in the EPR resonant field/frequency above that of the conventional X band (9.5 GHz) simplifies the Gd3+ EPR spectra and increases the sensitivity of the EPR linewidth to concentration induced broadening effects. Least squares line shape simulations show that the broadening can be described by a Lorentzian function with a width proportional to the average Gd3+ concentration which is in good agreement with the existing theories. Analyses of the concentration dependence of Lorentzian line broadening indicate an onset of clustering of Gd3+ ions at &gt; 1wt % doping levels. DA - 2001/10/22/ PY - 2001/10/22/ DO - 10.1063/1.1403436 VL - 115 IS - 16 SP - 7650-7656 SN - 1089-7690 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035935353&partnerID=MN8TOARS ER - TY - JOUR TI - Galactomannanases man2 and man5 from Thermotoga species: Growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes AU - Parker, KN AU - Chhabra, , SR AU - Lam, D AU - Callen, W AU - Duffaud, GD AU - Snead, MA AU - Short, JM AU - Mathur, EJ AU - Kelly, RM T2 - BIOTECHNOLOGY AND BIOENGINEERING AB - Abstract The enzymatic hydrolysis of mannan‐based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding β‐mannosidase ( man2 ) and β‐mannanase ( man5 ) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M r of approximately 92 kDa. The translated nucleotide sequences for the β‐mannanase genes ( man5 ) encoded polypeptides with an M r of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90°C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that β‐mannanase and β‐mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83°C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan‐degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 322–333, 2001. DA - 2001/11/5/ PY - 2001/11/5/ DO - 10.1002/bit.10020 VL - 75 IS - 3 SP - 322-333 SN - 0006-3592 KW - Thermotoga KW - hyperthermophile KW - hemicellulase KW - glycosyl hydrolase KW - galactomannan KW - beta-mannanase KW - beta-mannosidase ER - TY - JOUR TI - Formation of antibiotic, biodegradable polymers by processing with Irgasan DP300R (Triclosan) and its inclusion compound with beta-cyclodextrin AU - Lu, J. AU - Hill, M. A. AU - Hood, M. AU - Greeson, D. F. AU - Horton, J. R. AU - Orndorff, P. E. AU - Herndon, A. S. AU - Tonelli, A. E. T2 - Journal of Applied Polymer Science AB - The inclusion compound (IC) between the FDA-approved antibacterial Irgasan DP300 (Trichlosan), and β-cyclodextrin (CD) has been formed. When the Irgasan–β-CD–IC is embedded in biodegradeable/bioabsorbable films of poly(ϵ-caprolactone) (PCL) at low levels (a few wt %), they are rendered resistant to the growth of E. coli bacteria. When these same PCL films embedded with Irgasan–β-CD–IC are used as the adhesive for laminating cotton fabrics, we observe the resulting cotton laminates to also be resistant to the growth of E. coli bacteria. These results hold promise for the fabrication of bacteria-resistant polymer films and fibers, as well as antibacterial fabrics, by means of simple melt processing with Irgasan–β-CD–IC. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 300–309, 2001 DA - 2001/// PY - 2001/// DO - 10.1002/app.1852.abs VL - 82 IS - 2 SP - 300-309 ER - TY - JOUR TI - Formation of and coalescence from the inclusion complex of a biodegradable block copolymer and alpha-cyclodextrin: A novel means to modify the phase structure of biodegradable block copolymers AU - Shuai, XT AU - Porbeni, FE AU - Wei, M AU - Shin, ID AU - Tonelli, AE T2 - MACROMOLECULES AB - A well-defined biodegradable block copolymer (PCL-b-PLLA, Mn = 1.72 × 104, Mw/Mn = 1.37) of poly(ε-caprolactone) (PCL) and poly(l-lactide) (PLLA) was synthesized by a two-step ring-opening polymerization of ε-caprolactone and l-lactide. Furthermore, we found that α-cyclodextrin (α-CD) molecules may simultaneously thread onto both PLLA and PCL blocks of PCL-b-PLLA to form an inclusion complex (IC). Washing the copolymer−α-CD IC with hot water removed the α-CD, and the copolymer chains were coalesced. Very interestingly, the coalesced copolymer sample shows a great suppression in microphase separation, compared with the as-synthesized copolymer. In contrast to the significant decrease in crystallinity of ca. 50% and up to 79% for PCL and PLLA blocks, respectively, the melting points (Tm's) and the cold crystallization temperatures (Tcc's) of both PCL and PLLA blocks of the coalesced sample increased in DSC measurements. These results may imply that only small amounts of more extended crystals, with less chain folding, were produced during the process of copolymer coalescence. Fourier transform infrared (FTIR) spectra, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and wide-angle X-ray diffraction (WAXD) measurements were employed to demonstrate formation of the block copolymer−α-CD IC as well as to gauge the suppression of the microphase separation in the coalesced sample. DA - 2001/10/9/ PY - 2001/10/9/ DO - 10.1021/ma0109626 VL - 34 IS - 21 SP - 7355-7361 SN - 1520-5835 ER - TY - JOUR TI - Calpain inhibition protects against virus-induced apoptotic myocardial injury AU - DeBiasi, RL AU - Edelstein, CL AU - Sherry, B AU - Tyler, KL T2 - JOURNAL OF VIROLOGY AB - Viral myocarditis is an important cause of human morbidity and mortality for which reliable and effective therapy is lacking. Using reovirus strain 8B infection of neonatal mice, a well-characterized experimental model of direct virus-induced myocarditis, we now demonstrate that myocardial injury results from apoptosis. Proteases play a critical role as effectors of apoptosis. The activity of the cysteine protease calpain increases in reovirus-infected myocardiocytes and can be inhibited by the dipeptide alpha-ketoamide calpain inhibitor Z-Leu-aminobutyric acid-CONH(CH(2))3-morpholine (CX295). Treatment of reovirus-infected neonatal mice with CX295 protects them against reovirus myocarditis as documented by (i) a dramatic reduction in histopathologic evidence of myocardial injury, (ii) complete inhibition of apoptotic myocardial cell death as identified by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, (iii) a reduction in serum creatine phosphokinase, and (iv) improved weight gain. These findings are the first evidence for the importance of a calpain-associated pathway of apoptotic cell death in viral disease. Inhibition of apoptotic signaling pathways may be an effective strategy for the treatment of viral disease in general and viral myocarditis in particular. DA - 2001/1// PY - 2001/1// DO - 10.1128/JVI.75.1.351-361.2001 VL - 75 IS - 1 SP - 351-361 SN - 1098-5514 ER - TY - JOUR TI - Assembly of a tetrameric α-Helical bundle: Computer simulations on an intermediate-resolution protein model AU - Marchut, A.J. AU - Smith, A.V. AU - Hall, C.K. T2 - Proteins: Structure, Function, and Genetics AB - Abstract Discontinuous molecular dynamics (DMD) simulation on an intermediate‐resolution protein model is used to study the folding of an isolated, small model peptide to an amphipathic α‐helix and the assembly of four of these model peptides into a four‐helix bundle. A total of 129 simulations were performed on the isolated peptide, and 50 simulations were performed on the four‐peptide system. Simulations efficiently sample conformational space allowing complete folding trajectories from random initial configurations to be observed within 15 min for the one‐peptide system and within 15 h for the four‐peptide system on a 500‐MHz workstation. The native structures of both the α‐helix and the four‐helix bundle are consistent with experimental characterization studies and with results from previous simulations on these model peptides. In both the one‐ and four‐peptide systems, the native state is achieved during simulations within an optimal temperature range, a phenomenon also observed experimentally. The ease with which our simulations yield reasonable estimates of folded structures demonstrates the power of the intermediate‐resolution model developed for this work and the DMD algorithm and suggests that simulations of very long times and of multiprotein systems may be possible with this model. Proteins 2001;44:376–391. © 2001 Wiley‐Liss, Inc. DA - 2001/// PY - 2001/// DO - 10.1002/prot.1103 VL - 44 IS - 3 SP - 376-391 SN - 0887-3585 1097-0134 UR - http://dx.doi.org/10.1002/prot.1103 KW - discontinuous molecular dynamics KW - protein folding KW - protein misfolding ER - TY - JOUR TI - Semiquantitative immunohistochemical analysis for hypoxia in human tumors AU - Raleigh, JA AU - Chou, SC AU - Bono, EL AU - Thrall, DE AU - Varia, MA T2 - INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS AB - The goal of this study was to develop a semiquantitative scoring system for measuring hypoxia in human tumors by an immunohistochemical marker approach.Eighteen patients diagnosed with squamous cell carcinoma of the uterine cervix or head and neck were infused intravenously with a solution of pimonidazole hydrochloride at a dose of 0.5 gm/m2. Twenty-four hours later, four biopsies on average from each tumor were fixed in formalin, processed into paraffin blocks, and sectioned. Tissue sections were immunostained for the presence of pimonidazole adducts. Microscopic images (x200) of immunostaining were captured and quantitated by standard image analysis. Images with known amounts of hypoxia spanning ranges of > 0% to 5%, > 5% to 15%, > 15% to 30%, and >30% were assigned scores of +1, +2, +3, and +4, respectively. Three observers then used this calibrated scoring system to analyze hypoxia in tumor sections in a blinded fashion.Excellent interobserver reproducibility was obtained with the calibrated, semiquantitative, immunohistochemical assay for hypoxia in squamous cell carcinomas.The calibrated, semiquantitative assay shows promise as an approach to simplifying the quantitation of human tumor hypoxia by immunohistochemical techniques. DA - 2001/2/1/ PY - 2001/2/1/ DO - 10.1016/s0360-3016(00)01505-4 VL - 49 IS - 2 SP - 569-574 SN - 1879-355X KW - immunohistochemical assay KW - pimonidazole KW - quantitation ER - TY - JOUR TI - Purification and characterization of a phosphoric triester hydrolase from the tufted apple bud moth, Platynota idaeusalis (Walker) AU - Devorshak, C AU - Roe, RM T2 - JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY AB - Whole body homogenates from azinphosmethyl-resistant fifth instars of the tufted apple bud moth demonstrated 11.8-fold elevated phosphoric triester hydrolase (methyl paraoxonase) activity as compared to susceptible insects of the same species. Elevated phosphoric triester hydrolase (PTEH) activity associated with resistance was also found in the Colorado potato beetle but not in the German cockroach or tobacco budworm. Phosphoric triester hydrolase activity in the tufted apple bud moth was minimal in resistant and susceptible third instars and in adult males and females and was highest in whole body homogenates and in the alimentary canal of resistant fifth instars. A microtiterplate assay was developed, which successfully diagnosed resistance in individual fifth instars based on increased phosphoric triester hydrolase (methyl paraoxonase) activity. Phosphoric triester hydrolase was purified 289-fold from fifth instars of resistant bud moths, but any additional resolution resulted in the loss of enzyme activity. Phosphoric triester hydrolase demonstrated an apparent molecular weight of 41,000 with an isoelectric point of 5.28. Methyl paraoxonase activity was increased by calcium, cobalt, manganese, and octylthio-1,1,1-trifluoro-2-propanone and decreased by mercury, phosphate ions, tin, and ethylenediaminetetraacetic acid. Iron, potassium chloride, lithium, magnesium, sodium chloride, and lead had no effect. DA - 2001/// PY - 2001/// DO - 10.1002/1099-0461(2001)15:1<55::AID-JBT7>3.0.CO;2-G VL - 15 IS - 1 SP - 55-65 SN - 1095-6670 KW - phosphoric triester hydrolase KW - methyl paraoxonase KW - tufted apple bud moth KW - Platynota idaeusalis KW - tobacco budworm KW - Heliothis virescens KW - Colorado potato beetle KW - Leptinotarsa decemlineata KW - tobacco aphid KW - Myzus nicotianae KW - German cockroach KW - Blattella germanica KW - azinphosmethyl KW - guthion KW - insecticide resistance ER - TY - JOUR TI - Potential for milk containing penicillin G or amoxicillin to cause residues in calves AU - Musser, JMB AU - Anderson, KL AU - Rushing, JE AU - Moats, WA T2 - JOURNAL OF DAIRY SCIENCE AB - The potential for antibiotic residues in calves from consuming milk containing penicillin G or amoxicillin was investigated. Six calves were fed milk replacer, 6% body weight twice daily, containing 0.293, 2.92, or 5.85 microg of penicillin/ml (ppm) G or 0.25, 1.0, or 2.0 microg of amoxicillin/ml for three consecutive feedings. Urine and blood samples were collected after each feeding. Serum and urine samples were tested with a microbial receptor assay and a microbial growth inhibition assay to indicate potential drug residues. Penicillin G and amoxicillin were detected in the serum and urine of several calves 3 h after drinking spiked milk replacer. Possible violative drug residues in the calves were detected by the microbial growth inhibition assay up to 15 h after drinking spiked milk replacer. Penicillin G, but not amoxicillin, could be detected in urine 24 h after the final feeding of spiked milk replacer. Subsequently, six calves were fed milk replacer containing 11.7 microg of penicillin G/ml (ppm) twice daily, 6% body weight per feeding. Calves were slaughtered 3 h after the final feeding. Mean (+/-SD) concentrations of penicillin G measured by high-pressure liquid chromatography in liver, kidney, muscle, and serum were 0.409 (+/-0.167) microg/g, 0.031 (+/-0.012) microg/g 0.008 (+/-0.002) microg/g, and 0.013 (+/-0.006) mg/ml, respectively. This study indicates that calves fed milk with amoxicillin or penicillin G could possibly have violative residues if slaughtered within 24 h after feeding. Violative drug residues in liver tissue were found in calves slaughtered 3 h after consuming milk replacer containing 11.7 microg of penicillin G/ml (ppm). DA - 2001/1// PY - 2001/1// DO - 10.3168/jds.S0022-0302(01)74460-8 VL - 84 IS - 1 SP - 126-133 SN - 0022-0302 KW - penicillin KW - amoxicillin KW - residues KW - calves ER - TY - JOUR TI - Polymerase chain reaction analysis for viruses in paraffin-embedded myocardium from dogs with dilated cardiomyopathy or myocarditis AU - Maxson, Tracy R. AU - Meurs, Kathryn M. AU - Lehmkuhl, Linda B. AU - Magnon, Alex L. AU - Weisbrode, Steven E. AU - Atkins, Clarke E. T2 - American Journal of Veterinary Research AB - Abstract Objective —To perform polymerase chain reaction (PCR) analysis on paraffin-embedded myocardium from dogs with dilated cardiomyopathy (DCM) and dogs with myocarditis to screen for canine parvovirus, adenovirus types 1 and 2, and herpesvirus. Sample Population —Myocardial specimens from 18 dogs with an antemortem diagnosis of DCM and 9 dogs with a histopathologic diagnosis of myocarditis were evaluated. Procedure —Paraffin-embedded myocardial specimens were screened for viral genome by PCR analysis. Positive-control specimens were developed from cell cultures as well as paraffin-embedded tissue specimens from dogs with clinical and histopathologic diagnoses of viral infection with canine parvovirus, adenovirus types 1 and 2, and herpesvirus. The histologic characteristics of all myocardial specimens were classified regarding extent, location, and type of inflammation and fibrosis. Results —Canine adenovirus type 1 was amplified from 1 specimen from a dog with DCM. Canine parvovirus, adenovirus type 2, and herpesvirus were not amplified from any myocardial specimens. Histologic analysis of specimens from dogs with DCM revealed variable amounts of fibrosis; myocardial inflammation was observed in 1 affected dog. Histopathologic analysis of specimens from dogs with myocarditis disclosed variable degrees of inflammation and fibrosis. C onclusions and Clinical Relevance —Viral agents canine parvovirus, adenovirus types 1 and 2, and herpesvirus are not commonly associated with DCM or active myocarditis in dogs. Additional studies evaluating for nucleic acid from viruses that less commonly affect dogs or different types of infectious agents may be warranted to gain insight into the cause of DCM and myocarditis in dogs. ( Am J Vet Res 2001;62: 130–135) DA - 2001/1// PY - 2001/1// DO - 10.2460/ajvr.2001.62.130 VL - 62 IS - 1 SP - 130-135 J2 - American Journal of Veterinary Research LA - en OP - SN - 0002-9645 UR - http://dx.doi.org/10.2460/ajvr.2001.62.130 DB - Crossref ER - TY - JOUR TI - Risk factors for reduced postoperative fecal output in horses: 37 cases (1997-1998) AU - Little, D AU - Redding, WR AU - Blikslager, AT T2 - JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION AB - To determine prevalence and risk factors for development of ileus of the large intestine after surgery in horses, identified by reduced postoperative fecal output (RPFO).Retrospective study.37 horses that developed RPFO after undergoing general anesthesia for reasons unrelated to the gastrointestinal tract.Fecal output was obtained from medical records as number of defecations per 24-hour period after surgery; RPFO was defined as < or = 3 defecations per 24-hour period after surgery. The reference population included 48 horses that defecated > or = 4 times during the same period. Demographic, clinical, and surgical variables were evaluated for their association with development of RPFO by use of logistic regression analysis.Ten (12%) horses, all of which had RPFO, developed signs of colic after surgery. Horses > or = 5 years old that underwent orthopedic procedures of > 60 minutes' duration and that did not receive phenylbutazone after surgery were at significant risk for developing RPFO.Results suggest that after surgery unrelated to the gastrointestinal tract in horses, there is an intermediate clinical phase characterized by reduced fecal output preceding overt signs of colic. Recognition of RPFO may reduce morbidity and mortality of such horses. DA - 2001/2/1/ PY - 2001/2/1/ DO - 10.2460/javma.2001.218.414 VL - 218 IS - 3 SP - 414-420 SN - 0003-1488 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035253634&partnerID=MN8TOARS ER - TY - JOUR TI - Pharmacokinetics and adverse effects of butorphanol administered by single intravenous injection or continuous intravenous infusion in horses AU - Sellon, DC AU - Monroe, VL AU - Roberts, MC AU - Papich, MG T2 - AMERICAN JOURNAL OF VETERINARY RESEARCH AB - To determine an infusion rate of butorphanol tartrate in horses that would maintain therapeutic plasma drug concentrations while minimizing development of adverse behavioral and gastrointestinal tract effects.10 healthy adult horses.Plasma butorphanol concentrations were determined by use of high-performance liquid chromatography following administration of butorphanol by single IV injection (0.1 to 0.13 mg/kg of body weight) or continuous IV infusion (loading dose, 17.8 microg/kg; infusion dosage, 23.7 microg/kg/h for 24 hours). Pharmacokinetic variables were calculated, and changes in physical examination data, gastrointestinal tract transit time, and behavior were determined over time.A single IV injection of butorphanol was associated with adverse behavioral and gastrointestinal tract effects including ataxia, decreased borborygmi, and decreased defecation. Elimination half-life of butorphanol was brief (44.37 minutes). Adverse gastrointestinal tract effects were less apparent during continuous 24-hour infusion of butorphanol at a dosage that resulted in a mean plasma concentration of 29 ng/ml, compared with effects after a single IV injection. No adverse behavioral effects were observed during or after continuous infusion.Continuous IV infusion of butorphanol for 24 hours maintained plasma butorphanol concentrations within a range associated with analgesia. Adverse behavioral and gastrointestinal tract effects were minimized during infusion, compared with a single injection of butorphanol. Continuous infusion of butorphanol may be a useful treatment to induce analgesia in horses. DA - 2001/2// PY - 2001/2// DO - 10.2460/ajvr.2001.62.183 VL - 62 IS - 2 SP - 183-189 SN - 1943-5681 ER - TY - JOUR TI - Idiopathic noncirrhotic portal hypertension in dogs: 33 cases (1982-1998) AU - Bunch, SE AU - Johnson, SE AU - Cullen, JM T2 - JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION AB - Abstract Objective —To describe clinical signs, diagnostic findings, and outcome in dogs with idiopathic intrahepatic portal hypertension. Design —Retrospective study. Animals —33 dogs. Procedure —Medical records of dogs with portal hypertension of intra-abdominal origin were reviewed. Dogs with intra-abdominal portal hypertension of vascular causes or with hepatic histopathologic changes consistent with severe diffuse hepatobiliary disease were excluded. History and results of physical examination, clinicopathologic tests, diagnostic imaging studies, histologic examination, and treatment were summarized. Outcome was determined in 26 dogs. Results —Dogs were referred most often because of ascites, intermittent vomiting or diarrhea, and polydipsia of several months' duration. Microcytosis, high serum alkaline phosphatase and alanine transaminase activities, hepatic dysfunction, urine specific gravity ≤ 1.021, and abdominal transudate were the predominant clinicopathologic features. Microhepatia, abdominal effusion, and multiple anomalous venous anastomoses were the major findings of diagnostic imaging. Hepatic histopathologic changes were consistent with idiopathic noncirrhotic portal hypertension and were indistinguishable from those of dogs with surgically created portocaval anastomosis. Outcome was determined for 19 dogs released from hospital; 13 dogs remained healthy with mostly palliative treatment for periods of 5 months to 9 years. Conclusions and Clinical Relevance —The clinical signs, clinicopathologic test results, portal pressure, and gross appearance of the liver of dogs with idiopathic noncirrhotic portal hypertension may be identical to those of dogs with cirrhosis; therefore liver biopsy is crucial. Because the prognosis for idiopathic noncirrhotic portal hypertension is generally favorable, owners of affected dogs should be discouraged from choosing euthanasia. ( J Am Vet Med Assoc 2000; 218:392–399) DA - 2001/2/1/ PY - 2001/2/1/ DO - 10.2460/javma.2001.218.392 VL - 218 IS - 3 SP - 392-399 SN - 0003-1488 ER - TY - JOUR TI - Formation of a flame retardant-cyclodextrin inclusion compound and its application as a flame retardant for poly(ethylene terephthalate) AU - Huang, L AU - Gerber, M AU - Lu, J AU - Tonelli, AE T2 - POLYMER DEGRADATION AND STABILITY AB - We report the formation of an inclusion compound (IC) between a commercial flame retardant (FR) and beta-cyclodextrin (CD). The FR-CD-IC was melt-processed into PET films which were tested for flammability. The flammabilities of pure PET films, PET films containing pure CD, and PET films containing FR applied from a bath and then oven-cured, were also observed. Flammabilities, as measured using a modified AATCC Test Method 34, demonstrated that all but the PET films embedded with FR-CD-IC were either completely or substantially consumed when ignited on a single edge with a 3.8 cm flame applied for 3 s. To our knowledge this is the first demonstration of flame retardance achieved by means of delivering a FR to a polymer in the form of its inclusion compound. The high temperature stability of the FR-CD-IC crystals make them suitable for embedding in a variety of polymers that melt below 300°C. Our results suggest that incorporation of FR-CD-IC directly into polymer films or fibers during their melt-processing may be a means to protect them from burning that is superior to post-fabrication application of FRs. In addition, liquid FRs, which are difficult to incorporate and retain in solid polymers, may be included in their high-melting ICs formed with CDs and embedded directly into polymer samples. More broadly, we would expect that a host of other polymer additives may be more effectively delivered in the form of their CD-ICs. DA - 2001/// PY - 2001/// DO - 10.1016/s0141-3910(00)00175-0 VL - 71 IS - 2 SP - 279-284 SN - 0141-3910 KW - flame retardant (FR) KW - cyclodextrin (CD) KW - FR-CD-inclusion compound (IC) KW - FR delivery via FR-CD-IC KW - PET ER - TY - JOUR TI - Determination of carnitine renal threshold and effect of medium-chain triglycerides on carnitine profiles in newborn pigs AU - Heo, KN AU - Odle, J AU - Lin, X AU - Kempen, TATG AU - Han, IK T2 - ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES DA - 2001/1// PY - 2001/1// DO - 10.5713/ajas.2001.237 VL - 14 IS - 2 SP - 237-242 SN - 1011-2367 KW - carnitine KW - renal threshold KW - medium-chain triglycerides KW - newborn pigs KW - acyl intoxication ER - TY - JOUR TI - Influence of in vitro systems on embryo survival and fetal development in cattle AU - Farin, PW AU - Crosier, AE AU - Farin, CE T2 - THERIOGENOLOGY AB - In vitro systems are commonly used for the production of bovine embryos. Comparisons between in vivo and in vitro produced embryos illustrate that the morphology of preimplantation-stage embryos differ significantly, the survival of embryos and fetuses is decreased, the size distributions of the populations of conceptuses and fetuses are altered throughout gestation, and placental development is significantly changed. Taken together these findings indicate that exposure to some in vitro environments during the first 7 days of life can profoundly influence fetal and placental development in cattle. An understanding of how in vitro oocyte maturation, in vitro fertilization, and embryo culture systems influence both fetal and placental development should result in systems that consistently produce normal embryos, fetuses, and calves. DA - 2001/1/1/ PY - 2001/1/1/ DO - 10.1016/S0093-691X(00)00452-0 VL - 55 IS - 1 SP - 151-170 SN - 1879-3231 KW - bovine KW - embryo KW - fetus KW - in vitro production KW - gene expression KW - large offspring syndrome ER - TY - JOUR TI - Immunodiagnosis of Ehrlichia canis Infection with Recombinant Proteins AU - McBride, J. W. AU - Corstvet, R. E. AU - Breitschwerdt, E. B. AU - Walker, D. H. T2 - Journal of Clinical Microbiology AB - Ehrlichia canis causes a potentially fatal rickettsial disease of dogs that requires rapid and accurate diagnosis in order to initiate appropriate therapy leading to a favorable prognosis. We recently reported the cloning of two immunoreactive E. canis proteins, P28 and P140, that were applicable for serodiagnosis of the disease. In the present study we cloned a new immunoreactive E. canis surface protein gene of 1,170 bp, which encodes a protein with a predicted molecular mass of 42.6 kDa (P43). The P43 gene was not detected in E. chaffeensis DNA by Southern blot, and antisera against recombinant P43 (rP43) did not react with E. chaffeensis as detected by indirect fluorescent antibody (IFA) assay. Forty-two dogs exhibiting signs and/or hematologic abnormalities associated with canine ehrlichiosis were tested by IFA assay and by recombinant Western immunoblot. Among the 22 samples that were IFA positive for E. canis, 100% reacted with rP43, 96% reacted with rP28, and 96% reacted with rP140. The specificity of the recombinant proteins compared to the IFAs was 96% for rP28, 88% for P43 and 63% for P140. The results of this study demonstrate that the rP43 and rP28 are sensitive and reliable serodiagnostic antigens for E. canis infections. DA - 2001/1/1/ PY - 2001/1/1/ DO - 10.1128/JCM.39.1.315-322.2001 VL - 39 IS - 1 SP - 315-322 J2 - Journal of Clinical Microbiology LA - en OP - SN - 0095-1137 UR - http://dx.doi.org/10.1128/JCM.39.1.315-322.2001 DB - Crossref ER - TY - JOUR TI - Coinfection with Three Ehrlichia Species in Dogs from Thailand and Venezuela with Emphasis on Consideration of 16S Ribosomal DNA Secondary Structure AU - Suksawat, J. AU - Pitulle, C. AU - Arraga-Alvarado, C. AU - Madrigal, K. AU - Hancock, S. I. AU - Breitschwerdt, E. B. T2 - Journal of Clinical Microbiology AB - As part of a larger study to investigate tick-borne infections in dogs from Thailand and Venezuela, documentation of coinfection with three Ehrlichia species in two dogs, one from each country, became the focus of the present study. Although neither dog had clinical signs attributable to ehrlichiosis, both dogs were anemic and neutropenic and the Thai dog was thrombocytopenic. Genus- and species-specific PCR targeting the 16S rRNA genes indicated that both dogs were coinfected with Ehrlichia canis, E. platys, and E. equi. To our knowledge, these results provide the first molecular documentation for the presence of E. equi in dogs from these countries. Using universal bacterial PCR primers, one nearly full-length 16S rRNA gene could be amplified from each dog. The sequences were identical to each other and almost identical to that of E. platys (AF156784), providing the first E. platys 16S ribosomal DNA (rDNA) sequences reported from these two geographically divergent countries. To determine whether these sequence differences allow differentiation between these two strains and other published 16S rDNA E. platys sequences, we performed a phylogenetic analysis of the rRNA, incorporating the consideration of secondary structure. DA - 2001/1/1/ PY - 2001/1/1/ DO - 10.1128/JCM.39.1.90-93.2001 VL - 39 IS - 1 SP - 90-93 J2 - Journal of Clinical Microbiology LA - en OP - SN - 0095-1137 UR - http://dx.doi.org/10.1128/JCM.39.1.90-93.2001 DB - Crossref ER - TY - JOUR TI - Canine Rocky Mountain spotted fever: A retrospective study of 30 cases AU - Gasser, AM AU - Birkenheuer, AJ AU - Breitschwerdt, EB T2 - JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION AB - Rocky Mountain spotted fever (RMSF) was diagnosed in 30 dogs examined at North Carolina State University, Veterinary Teaching Hospital between 1984 and 1997. Historical, physical examination, and laboratory abnormalities were reviewed. Diagnostic criteria included a four-fold rise in antibody titer to Rickettsia rickettsii (R. rickettsii) (n=15) or a single R. rickettsii antibody titer of 1:1,024 or greater (n=15; when this initial titer was determined one week or more after the onset of clinical signs). Fifteen (50%) dogs were greater than seven years of age, and 13 (43%) dogs were between two and seven years of age. There was no sex predilection. Only five (17%) dogs had a history of known tick exposure. Presumably due to delayed diagnosis, dogs with antibody titers of 1:1,024 or greater at the time of presentation had a higher incidence of more severe neurological dysfunction (e.g., ataxia, hyperesthesia, vestibular disease, and seizures) and cutaneous lesions (e.g., hyperemia, edema, petechiae, ecchymoses, and necrosis). Laboratory findings included anemia, leukocytosis accompanied by toxic granulation of neutrophils, hypoalbuminemia, and coagulation abnormalities; signs were generally more severe in the 15 dogs with R. rickettsii antibody titers of 1:1,024 or greater at the time of presentation. Twelve (40%) dogs in this study were severely thrombocytopenic (less than 75 x10(3) platelets/microl; reference range, 200 to 450 x 10(3)/microl), without clinical evidence of fulminant disseminated intravascular coagulation. In this study, the survival rate following R. rickettsii infection was 100%. DA - 2001/// PY - 2001/// DO - 10.5326/15473317-37-1-41 VL - 37 IS - 1 SP - 41-48 SN - 0587-2871 ER - TY - JOUR TI - Bacteria and ornamental fish AU - Lewbart, GA T2 - SEMINARS IN AVIAN AND EXOTIC PET MEDICINE AB - Bacterial disease is the most common infectious problem of ornamental fishes, and most bacterial infections are caused by gram-negative organisms. The majority of bacterial fish pathogens are natural inhabitants of the aquatic environment, whether it be freshwater or marine. Extrinsic stressors, including shipping, crowding, poor water quality, and inadequate nutrition, may predispose an ornamental fish to bacterial disease. This article reviews the important bacterial pathogens of ornamental fish in a systematic manner, discusses the problems of over-the-counter antimicrobial compounds and antibiotic resistance, and reviews treatment and management options. DA - 2001/1// PY - 2001/1// DO - 10.1053/saep.2001.19543 VL - 10 IS - 1 SP - 48-56 SN - 1055-937X KW - bacteria KW - ornamental fish KW - pet fish KW - antibiotics ER - TY - JOUR TI - A novel serotype-specific gene cassette (gltA-gltB) is required for expression of teichoic acid-associated surface antigens in Listeria monocytogenes of serotype 4b AU - Lei, XH AU - Fiedler, F AU - Lan, Z AU - Kathariou, S T2 - JOURNAL OF BACTERIOLOGY AB - Listeria monocytogenes serotype 4b strains account for about 40% of sporadic cases and many epidemics of listeriosis. Mutations in a chromosomal locus resulted in loss of reactivity with all three monoclonal antibodies (MAbs) which were specific to serotype 4b and the closely related serotypes 4d and 4e. Here we show that this locus contains a serotype 4b-4d-4e-specific gene cassette (3,071 bp) which consists of two genes, gltA and gltB, and is flanked by palindromic sequences (51 and 44 nucleotides). Complete loss of reactivity with the three serotype-specific MAbs resulted from insertional inactivation of either gltA or gltB. The gltA and gltB mutants were characterized by loss and severe reduction, respectively, of glucose in the teichoic acid, whereas galactose, the other serotype-specific sugar substituent in the teichoic acid, was not affected. Within L. monocytogenes, only strains of serotypes 4b, 4d, and 4e harbored the gltA-gltB cassette, whereas coding sequences on either side of the cassette were conserved among all serotypes. Comparative genomic analysis of a serotype 1/2b strain showed that the 3,071-bp gltA-gltB cassette was replaced by a much shorter (528-bp) and unrelated region, flanked by inverted repeats similar to their counterparts in serotype 4b. These findings indicate that in the evolution of different serotypes of L. monocytogenes, this site in the genome has become occupied by serotype-specific sequences which, in the case of serotype 4b, are essential for expression of serotype-specific surface antigens and presence of glucose substituents in the teichoic acids in the cell wall. DA - 2001/2// PY - 2001/2// DO - 10.1128/jb.183.4.1133-1139.2001 VL - 183 IS - 4 SP - 1133-1139 SN - 0021-9193 ER - TY - PAT TI - Method of repelling insects AU - Roe, R. M. C2 - 2001/// DA - 2001/// PY - 2001/// ER - TY - PCOMM TI - Untitled - Reply AU - Gross, TL AU - Olivry, T AU - Tobin, D AB - Veterinary DermatologyVolume 12, Issue 6 p. 352-352 Reply Thelma Lee Gross, Thelma Lee Gross California Dermopathology Service and IDEXX Veterinary Services, West Sacramento, CA 95605, USASearch for more papers by this author Thierry Olivry, Thierry Olivry North Carolina State University, College of Veterinary Medicine, Raleigh, NC 27606, USASearch for more papers by this authorDesmond Tobin, Desmond Tobin University of Bradford, Bradford DB7 1DP, UKSearch for more papers by this author Thelma Lee Gross, Thelma Lee Gross California Dermopathology Service and IDEXX Veterinary Services, West Sacramento, CA 95605, USASearch for more papers by this author Thierry Olivry, Thierry Olivry North Carolina State University, College of Veterinary Medicine, Raleigh, NC 27606, USASearch for more papers by this authorDesmond Tobin, Desmond Tobin University of Bradford, Bradford DB7 1DP, UKSearch for more papers by this author First published: 30 April 2002 https://doi.org/10.1046/j.0959-4493.2001.00250.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume12, Issue6December 2001Pages 352-352 RelatedInformation DA - 2001/12// PY - 2001/12// DO - 10.1046/j.0959-4493.2001.00250.x SP - 352-352 ER - TY - JOUR TI - The future of antiinflammatory therapy AU - Jones, SL AU - Blikslager, A T2 - VETERINARY CLINICS OF NORTH AMERICA-EQUINE PRACTICE AB - The cells and mediators that make up the inflammatory response have the potential to injure tissues and contribute to the pathophysiology of many inflammatory diseases. Strategies to reduce neutrophil migration into sites of inflammation and subsequent activation by inhibiting integrin-mediated adhesion hold promise for successful treatment of a variety of inflammatory diseases. New pharmacologic agents that specifically target prostanoid mediators of inflammation by specifically inhibiting the activity of cyclooxygenase 2 are potent antiinflammatory agents with fewer gastrointestinal side effects than nonspecific cyclooxygenase inhibitors. These areas of antiinflammatory research are rapidly yielding drugs with diverse future applications in equine medicine. DA - 2001/8// PY - 2001/8// DO - 10.1016/S0749-0739(17)30060-3 VL - 17 IS - 2 SP - 245-+ SN - 0749-0739 ER - TY - JOUR TI - The ACVD task force on canine atopic dermatitis (XXIII): are essential fatty acids effective? AU - Olivry, T AU - Marsella, R AU - Hillier, A T2 - VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY AB - Essential fatty acids (EFAs) exhibit the potential to affect allergic inflammation through the modulation of prostaglandin and leukotriene production, the inhibition of cellular activation and cytokine secretion as well as the alteration of the composition and function of the epidermal lipid barrier. Because of these multi-facetted effects, EFA have been proposed for treatment of canine atopic dermatitis (AD) since 1987. To date, more than 20 trials have been performed, reporting the efficacy of either oral EFA supplements or EFA-rich diets. Unfortunately, most of these studies were found to exhibit one or more of the following deficiencies: heterogeneity of diagnoses used as inclusion criteria, short duration of supplementation, lack of randomization of treatment allocation, lack of blinding of investigators and/or owners, lack of placebo or active controls, lack of documentation of plasma or skin EFA profiles during supplementation, as well as lack of standardization of the basal diets or supplements which could have provided additional EFA. Consequently, there is presently insufficient evidence to recommend for or against the use of EFA to control clinical signs of canine AD. Evidence of efficacy must await the performance of blinded, randomized and controlled trials of at least 3 months duration in which diets are identical for all of study subjects. In these trials, clinical efficacy should be evaluated in relation to plasma and cutaneous EFA treatment-induced alterations. DA - 2001/9/20/ PY - 2001/9/20/ DO - 10.1016/S0165-2427(01)00316-6 VL - 81 IS - 3-4 SP - 347-362 SN - 1873-2534 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035922065&partnerID=MN8TOARS KW - atopic dermatitis KW - dog KW - essential fatty acids KW - epidermal lipids KW - therapy ER - TY - JOUR TI - The ACVD task force on canine atopic dermatitis (VII): mediators of cutaneous inflammation AU - Marsella, Rosanna AU - Olivry, Thierry T2 - Veterinary Immunology and Immunopathology AB - Controversy still exists on the role of various inflammatory mediators in the pathogenesis of canine atopic dermatitis. The objective of this article is to review the most recent information available on the inflammatory mediators that have been implicated in the pathogenesis of this disease. Studies on the role of histamine, serotonin, leukotrienes and various cytokines are presented in a comparative manner reviewing the experimental evidence for a role in the pathogenesis and the arguments against it. DA - 2001/9// PY - 2001/9// DO - 10.1016/S0165-2427(01)00300-2 VL - 81 IS - 3-4 SP - 205-213 J2 - Veterinary Immunology and Immunopathology LA - en OP - SN - 0165-2427 UR - http://dx.doi.org/10.1016/s0165-2427(01)00300-2 DB - Crossref KW - basophils KW - cytokines KW - histamine KW - leukotrienes KW - mast cells KW - serotonin ER - TY - JOUR TI - Semiparametric nonlinear mixed-effects models and their applications - Comment AU - Lin, X. H. AU - Zhang, D. W. T2 - Journal of the American Statistical Association DA - 2001/// PY - 2001/// VL - 96 IS - 456 SP - 1288-1291 ER - TY - JOUR TI - Mixture effects of JP-8 additives on the dermal disposition of jet fuel components AU - Baynes, RE AU - Brooks, JD AU - Budsaba, K AU - Smith, CE AU - Riviere, JE T2 - TOXICOLOGY AND APPLIED PHARMACOLOGY AB - Aliphatic and aromatic components in formulated jet fuels can cause occupational dermatitis. However, the influence of JP-8 performance additives (DIEGME, 8Q21, and Stadis450) on the dermal disposition of fuel components is not well understood. These additives are formulated with commercial Jet-A to form military JP-8 fuel. The purpose of this study is to assess the influence of these additives on the dermal disposition of marker aromatic and aliphatic components, naphthalene and dodecane, respectively. Porcine skin sections in an in vitro system were used to characterize chemical-biological interactions that modulate diffusion of jet fuel components and isolated perfused porcine skin flaps (IPPSFs) were used to evaluate diffusion in a viable skin model with an intact microvasculature. In these 5-h studies, Jet-A, Jet-A + DIEGME, Jet-A + 8Q21, and Jet-A + Stadis450, Jet-A + DIEGME + 8Q21, Jet-A + DIEGME + Stadis450, Jet-A + 8Q21 + Stadis450, and JP-8 mixtures were tested. In general, naphthalene absorption (0.76-2.39% dose) was greater than dodecane absorption (0.10-0.84% dose), while the IPPSFs alone demonstrated that dodecane absorption was significantly greater in JP-8 than in Jet-A. Synergistic interactions with 8Q21 + Stadis450 appear to enhance systemic absorption of either naphthalene or dodecane, while DIEGME + Stadis450 increased naphthalene (1.88% dose) and dodecane (2.02% dose) penetration into the skin and fat tissues of IPPSFs. These findings were supported by the fact that 8Q21 + Stadis450 significantly increased dodecane flux and permeability in porcine skin sections, but 8Q21 alone reduced marker diffusion in both membrane systems. Furthermore, dodecane is more likely than naphthalene to remain in the stratum corneum and skin surface at 5 h, and DIEGME mixtures played a significant role in skin and surface retention of both markers. In summary, the data suggest that various combinations of these three performance additives in JP-8 can potentially alter the dermal disposition of aromatic and aliphatic fuel components in skin. More importantly, products of two-factor interactions were not predictable from single-factor exposures and, by extension, cannot be extrapolated to three-factor interactions. DA - 2001/9/15/ PY - 2001/9/15/ DO - 10.1006/taap.2001.9259 VL - 175 IS - 3 SP - 269-281 SN - 1096-0333 KW - jet fuels KW - mixtures KW - skin KW - interactions KW - absorption ER - TY - JOUR TI - MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro AU - Li, YH AU - Martin, LD AU - Spizz, G AU - Adler, KB T2 - JOURNAL OF BIOLOGICAL CHEMISTRY AB - Hypersecretion of airway mucin characterizes numerous respiratory diseases. Although diverse pathological stimuli can provoke exocytotic release of mucin from secretory cells of the airway epithelium, mechanisms involved remain obscure. This report describes a new paradigm for the intracellular signaling mechanism regulating airway mucin secretion. Direct evidence is provided that the myristoylated alanine-rich C kinase substrate (MARCKS) is a central regulatory molecule linking secretagogue stimulation at the cell surface to mucin granule release by differentiated normal human bronchial epithelial cells in vitro. Down-regulation of MARCKS expression or disruption of MARCKS function in these cells inhibits the secretory response to subsequent stimulation. The intracellular mechanism controlling this secretory process involves cooperative action of two separate protein kinases, protein kinase C and cGMP-dependent protein kinase. Upon stimulation, activated protein kinase C phosphorylates MARCKS, causing translocation of MARCKS from the plasma membrane to the cytoplasm, where it is then dephosphorylated by a protein phosphatase 2A that is activated by cGMP-dependent protein kinase, and associates with both actin and myosin. Dephosphorylated cytoplasmic MARCKS would also be free to interact with mucin granule membranes and thus could link granules to the contractile cytoskeleton, mediating their movement to the cell periphery and subsequent exocytosis. These findings suggest several novel intracellular targets for pharmacological intervention in disorders involving aberrant secretion of respiratory mucin and may relate to other lesions involving exocytosis of membrane-bound granules in various cells and tissues. Hypersecretion of airway mucin characterizes numerous respiratory diseases. Although diverse pathological stimuli can provoke exocytotic release of mucin from secretory cells of the airway epithelium, mechanisms involved remain obscure. This report describes a new paradigm for the intracellular signaling mechanism regulating airway mucin secretion. Direct evidence is provided that the myristoylated alanine-rich C kinase substrate (MARCKS) is a central regulatory molecule linking secretagogue stimulation at the cell surface to mucin granule release by differentiated normal human bronchial epithelial cells in vitro. Down-regulation of MARCKS expression or disruption of MARCKS function in these cells inhibits the secretory response to subsequent stimulation. The intracellular mechanism controlling this secretory process involves cooperative action of two separate protein kinases, protein kinase C and cGMP-dependent protein kinase. Upon stimulation, activated protein kinase C phosphorylates MARCKS, causing translocation of MARCKS from the plasma membrane to the cytoplasm, where it is then dephosphorylated by a protein phosphatase 2A that is activated by cGMP-dependent protein kinase, and associates with both actin and myosin. Dephosphorylated cytoplasmic MARCKS would also be free to interact with mucin granule membranes and thus could link granules to the contractile cytoskeleton, mediating their movement to the cell periphery and subsequent exocytosis. These findings suggest several novel intracellular targets for pharmacological intervention in disorders involving aberrant secretion of respiratory mucin and may relate to other lesions involving exocytosis of membrane-bound granules in various cells and tissues. Mammalian airways are lined by a thin layer of mucus produced and secreted by airway epithelial (goblet) cells and submucosal glands. In diseases such as asthma, chronic bronchitis, and cystic fibrosis, hypersecretion of mucus is a common lesion. Excess mucus can contribute to obstruction, susceptibility to infection, and even to destruction of airway walls and contiguous tissues. The major components of mucus are mucin glycoproteins synthesized by secretory cells and stored within cytoplasmic membrane-bound granules. Upon appropriate stimulation, these granules are released via an exocytotic process in which the granules translocate to the cell periphery where the granule membranes fuse with the plasma membrane, allowing for luminal secretion of the contents. Despite the obvious pathophysiological importance of this process, intracellular signaling mechanisms linking stimulation at the cell surface to mucin granule release have not been elucidated. It is known that a wide variety of agents and inflammatory/humoral mediators can provoke mucin secretion. These include cholinergic agonists, lipid mediators, oxidants, cytokines, neuropeptides, ATP and UTP, bacterial products, neutrophil elastase, and inhaled pollutants (reviewed in Refs. 1Adler K.B. Fischer B.M. Martin L.D. Voynow J.A. Res. Immunol. 1998; 149: 245-248Crossref Scopus (1) Google Scholar and 2Cohn L.A. Adler K.B. Exp. Lung Res. 1992; 18: 299-322Crossref PubMed Scopus (25) Google Scholar). Interestingly, many of these mucin secretagogues are also known to activate several protein kinases, and studies examining the regulation of excess secretion of mucin by airway epithelial cells from various species have consistently implicated involvement of either protein kinase C (PKC) 1PKC, protein kinase C; PKG, cGMP-dependent protein kinase; PKA, protein kinase A; MANS, myristoylated N-terminal sequence; PMA, phorbol 12-myristate 13-acetate; PSD, phosphorylation site domain; 8-Br-GMP, 8-bromo-cyclic GMP; RNS, random N-terminal sequence; MARCKS, myristoylated alanine-rich C kinase substrate; NHBE, normal human bronchial epithelial; PP2A, protein phosphatase 2A; ELISA, enzyme-linked immunosorbent assay. 1PKC, protein kinase C; PKG, cGMP-dependent protein kinase; PKA, protein kinase A; MANS, myristoylated N-terminal sequence; PMA, phorbol 12-myristate 13-acetate; PSD, phosphorylation site domain; 8-Br-GMP, 8-bromo-cyclic GMP; RNS, random N-terminal sequence; MARCKS, myristoylated alanine-rich C kinase substrate; NHBE, normal human bronchial epithelial; PP2A, protein phosphatase 2A; ELISA, enzyme-linked immunosorbent assay.(3Ko K.H. Jo M. McCracken K. Kim K.C. Am. J. Respir. Cell Mol. Biol. 1997; 16: 194-198Crossref PubMed Scopus (22) Google Scholar, 4Abdullah L.H. Conway J.D. Cohn J.A. Davis C.W. Am. J. Physiol. 1997; 273: L201-L210PubMed Google Scholar, 5Abdullah L.H. Davis S.W. Burch L. Yamauchi M. Randell S.H. Nettesheim P. Davis C.W. Biochem. J. 1996; 316: 943-951Crossref PubMed Scopus (61) Google Scholar, 6Larivee P. Levine S.J. Martinez A. Wu T. Logun C. Shelhamer J.H. Am. J. Respir. Cell Mol. Biol. 1994; 11: 199-205Crossref PubMed Scopus (45) Google Scholar) or cGMP-dependent protein kinase (PKG) (7Fischer B.M. Rochelle L.G. Voynow J.A. Akley N.J. Adler K.B. Am. J. Respir. Cell Mol. Biol. 1999; 20: 413-422Crossref PubMed Scopus (88) Google Scholar) in the secretory process. However, coordinated interactions or “cross-talk” between these two protein kinases in regulation of mucin secretion have not been demonstrated, nor have signaling events downstream of protein kinase activation that ultimately lead to the exocytotic release of mucin granules been elucidated. Previously, we reported (8Krunkosky T.M. Fischer B.M. Martin L.D. Jones N. Akley N.J. Adler K.B. Am. J. Respir. Cell Mol. Biol. 2000; 22: 685-692Crossref PubMed Scopus (133) Google Scholar) development of a procedure to culture normal human bronchial epithelial (NHBE) cells in an air/liquid interface system in which the cells differentiate to a heterogeneous population containing secretory (goblet), ciliated, and basal cells that mimic their in vivo appearance and function. These cell cultures provide an ideal in vitro model system to study mechanisms regulating mucin secretion from human airway epithelium. This report presents direct evidence demonstrating that the myristoylated alanine-rich C kinase substrate (MARCKS), a widely distributed PKC substrate for which a specific biological function has yet to be identified, is a key regulatory molecule mediating mucin granule release by NHBE cells. Secretion of mucin from these cells is maximized by activation of both PKC and PKG, and MARCKS serves as the point of convergence for coordinating the actions of these two protein kinases to control mucin granule release. The mechanism appears to involve PKC-dependent phosphorylation of MARCKS, which releases MARCKS from the plasma membrane into the cytoplasm, where it is in turn dephosphorylated by a protein phosphatase 2A (PP2A) that is activated by PKG. This dephosphorylation would allow MARCKS to regain its membrane-binding capability (9Seykora J.T. Myat M.M. Allen L.A.H. Ravetch J.V. Aderem A. J. Biol. Chem. 1996; 271: 18797-18802Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Swierczynski S.L. Blackshear P.J. J. Biol. Chem. 1996; 271: 23424-23430Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (616) Google Scholar), enabling its attachment to membranes of cytoplasmic mucin granules. In addition, MARCKS interacts with actin and myosin in the cytoplasm and thus could tether the granules to the cellular contractile apparatus, mediating subsequent granule movement and exocytosis. Expansion, cryopreservation, and culture of NHBE cells in the air/liquid interface were performed as described previously (8Krunkosky T.M. Fischer B.M. Martin L.D. Jones N. Akley N.J. Adler K.B. Am. J. Respir. Cell Mol. Biol. 2000; 22: 685-692Crossref PubMed Scopus (133) Google Scholar). Briefly, NHBE cells (Clonetics, San Diego, CA) were seeded in vented T75 tissue culture flasks (500 cells/cm2) and cultured until cells reached 75–80% confluence. Cells were then dissociated by trypsin/EDTA and frozen as passage-2. Air/liquid interface culture was initiated by seeding passage-2 cells (2 × 104 cells/cm2) in Transwell® clear culture inserts (Costar, Cambridge, MA) that were thinly coated with rat tail collagen, type I (Collaborative Biomedical, Bedford, MA). Cells were cultured submerged in medium in a humidified 95% air, 5% CO2 environment for 5–7 days until nearly confluent. At that time, the air/liquid interface was created by removing the apical medium and feeding cells basalaterally. Medium was renewed daily thereafter. Cells were cultured for an additional 14 days to allow full differentiation. Before collection of “base line” and “test” mucin samples, the accumulated mucus at the apical surface of the cells was removed by washing with phosphate-buffered saline, pH 7.2. To collect the base-line secretion, cells were incubated with medium alone, and secreted mucin in the apical medium was collected and reserved. Cells were rested for 24 h and then exposed to medium containing the selected stimulatory and/or inhibitory reagents (or appropriate controls), after which secreted mucin was collected and reserved as the test sample. Incubation times for the base line and the test were the same but varied depending on the test reagent utilized. Both base line and test secretions were analyzed by ELISA using an antibody capture method described previously (12Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 570-573Google Scholar). The primary antibody for this assay was 17Q2 (Babco, Richmond, CA), a monoclonal antibody that reacts specifically with a carbohydrate epitope on human airway mucins (13Lin H. Carlson D.M. St. George J.A. Plopper C.G. Wu R. Am. J. Respir. Cell Mol. Biol. 1989; 1: 41-48Crossref PubMed Scopus (63) Google Scholar). The ratio of test/base-line mucin, similar to a “secretory index” reported previously (14Wright D.T. Fischer B.M. Li C. Rochelle L.G. Akley N.J. Adler K.B. Am. J. Physiol. 1996; 271: L854-L861PubMed Google Scholar), was used to quantify mucin secretion, allowing each culture dish to serve as its own control and thus minimizing deviation caused by variability among culture wells. Levels of mucin secretion were reported as percentage of the medium control. When labeling with [32P]phosphate, cells were preincubated for 2 h in phosphate-free Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and then labeled with 0.1 mCi/ml [32P]orthophosphate (9000 Ci/mmol, PerkinElmer Life Sciences) for 2 h. For labeling with [3H]myristic acid or 3H-amino acids, cells were incubated overnight in medium containing 50 µCi/ml [3H]myristic acid (49 Ci/mmol, PerkinElmer Life Sciences) or 0.2 mCi/ml [3H]leucine (159 Ci/mmol, PerkinElmer Life Sciences) plus 0.4 mCi/ml [3H]proline (100 Ci/mmol, PerkinElmer Life Sciences). Following labeling, cells were exposed to stimulatory reagents for 5 min. When an inhibitor was used, cells were preincubated with the inhibitor for 15 min prior to stimulation. At the end of the treatments, cells were lysed in a buffer containing 50 mmTris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 10% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin. The radiolabeling efficiency in each culture was determined by trichloroacetic acid precipitation and scintillation counting. Immunoprecipitation of MARCKS protein was carried out according to the method of Spizz and Blackshear (15Spizz G. Blackshear P.J. J. Biol. Chem. 1996; 271: 553-562Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) using cell lysates containing equal counts/min. Precipitated proteins were resolved by 8% SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Anti-human MARCKS antibody (2F12) and nonimmune control antibody (6F6) used in this assay were provided by Dr. Perry Blackshear (NIEHS, Research Triangle Park, NC). To assess MARCKS or MARCKS-associated protein complexes in different subcellular fractions, radiolabeled and treated cells were scraped into a homogenization buffer (50 mm Tris-HCl (pH 7.5), 10 mm NaCl, 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 1 mm benzamidine, 10 µg/ml pepstatin A, 10 µg/ml leupeptin) and then disrupted by nitrogen cavitation (800 pounds/square inch for 20 min at 4 °C). Cell lysates were centrifuged at 600 × g for 10 min at 4 °C to remove nuclei and unbroken cells. Post-nuclear supernatants were separated into membrane and cytosol fractions by ultracentrifugation at 400,000 × g for 30 min at 4 °C. The membrane pellet was solubilized in the lysis buffer by sonication. Immunoprecipitation was then carried out as described above. Both the myristoylated N-terminal sequence (MANS) and the random N-terminal sequence (RNS) peptides were synthesized at Genemed Synthesis, Inc. (San Francisco, CA), then purified by high pressure liquid chromatography (>95% pure), and confirmed by mass spectroscopy with each showing one single peak with an appropriate molecular mass. The MANS peptide consisted of sequence identical to the first 24 amino acids of MARCKS, i.e. the myristoylated N-terminal region that mediates MARCKS insertion into membranes (9Seykora J.T. Myat M.M. Allen L.A.H. Ravetch J.V. Aderem A. J. Biol. Chem. 1996; 271: 18797-18802Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Swierczynski S.L. Blackshear P.J. J. Biol. Chem. 1996; 271: 23424-23430Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (616) Google Scholar), MA-GAQFSKTAAKGEAAAERPGEAAVA (where MA = N-terminal myristate chain). The corresponding control peptide (RNS) contained the same amino acid composition as the MANS but arranged in random order, MA-GTAPAAEGAGAEVKRASAEAKQAF. The presence of the hydrophobic myristate moiety in these synthetic peptides enhances their permeability to the plasma membranes, enabling the peptides to be taken up readily by cells. To determine the effects of these peptides on mucin secretion, cells were preincubated with the peptides for 15 min prior to addition of secretagogues, and mucin secretion was then measured by ELISA. MARCKS antisense oligonucleotide and its corresponding control oligonucleotide were synthesized at Biognostik GmbH (Gottingen, Germany). NHBE cells were treated with 5 µm antisense or control oligonucleotide apically for 3 days (in the presence of 2 µg/ml lipofectin for the first 24 h). Cells were then incubated with secretagogues, and mucin secretion was measured by ELISA. Total RNA and protein were isolated from treated cells. MARCKS mRNA was assessed by Northern hybridization according to conventional procedures using human MARCKS cDNA (provided by Dr. Perry Blackshear, NIEHS, Research Triangle Park, NC) as a probe. MARCKS protein level was determined by Western blot using purified anti-MARCKS IgG1 (clone 2F12) as the primary detection antibody. The phosphorylation site domain (PSD) of MARCKS contains the PKC-dependent phosphorylation sites and the actin filament-binding site (16Hartwig J.H. Thelen M. Rosen A. Janmey P.A. Nairn A.C. Aderem A. Nature. 1992; 356: 618-622Crossref PubMed Scopus (618) Google Scholar). To construct a PSD-deleted MARCKS cDNA, two fragments flanking the PSD sequence (coding for 25 amino acids) were generated by polymerase chain reaction and then ligated through the XhoI site that was attached to the 5′-ends of oligonucleotide primers designed for the polymerase chain reaction. The resultant mutant cDNA and the wild-type MARCKS cDNA were each inserted into a mammalian expression vector pcDNA4/TO (Invitrogen, Carlsbad, CA). Isolated recombinant constructs were confirmed by restriction digests and DNA sequencing. HBE1 is a papilloma virus-transformed human bronchial epithelial cell line (17Yankaskas J.R. Haizlip J.E. Conrad M. Koval D. Lazarowski E. Paradiso A.M. Rinehart Jr., C.A. Sarkadi B. Schlegel R. Boucher R.C. Am. J. Physiol. 1993; 264: C1219-C1230Crossref PubMed Google Scholar) capable of mucin secretion when cultured in air/liquid interface. Transfection of HBE1 cells was carried out using the Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. Briefly, differentiated HBE1 cells grown in air/liquid interface were dissociated by trypsin/EDTA and re-seeded in 12-well culture plates at 1 × 105cells/cm2. After overnight incubation, cells were transfected with the wild-type MARCKS cDNA, the PSD-truncated MARCKS cDNA, or vector DNA. Cells were cultured for 48 h to allow gene expression and then exposed to secretagogues and mucin secretion measured by ELISA. All transfections were carried out in the presence of pcDNA4/TO/lacZ plasmid (Invitrogen) (DNA ratio 6:1, total 1 µg DNA, ratio of DNA to Effectene reagent = 1:25) to monitor variations in transfection efficiency. Results showed no significant difference in β-galactosidase activities in cell lysates isolated from the transfected cells, indicating similar transfection efficiency among different DNA constructs (data not shown). PP1 and PP2A activities were measured using a protein phosphatase assay system (Life Technologies, Inc.) as described (18Huang X.C. Sumners C. Richards E.M. Adv. Exp. Med. Biol. 1996; 396: 209-215Crossref PubMed Scopus (25) Google Scholar) with modification. Briefly, NHBE cells were treated with 8-Br-cGMP or medium alone for 5 min. Cells were then scraped into a lysis buffer (50 mm Tris-HCl (pH 7.4), 0.1% β-mecaptoethanol, 0.1 mm EDTA, 1 mmbenzamidine, 10 µg/ml pepstatin A, 10 µg/ml leupeptin) and disrupted by sonication for 20 s at 4 °C. Cell lysates were centrifuged and the supernatants saved for phosphatase activity assay. The assay was performed using 32P-labeled phosphorylase A as a substrate. Released 32Pi was counted by scintillation. The protein concentration of each sample was determined by the Bradford assay. PP2A activity was expressed as the sample total phosphatase activity minus the activity remaining in the presence of 1 nm okadaic acid. PP1 activity was expressed as the difference between the activities remaining in the presence of 1 nm and 1 µm okadaic acid, respectively. Protein phosphatase activities were reported as nmol of Pireleased per min/mg total protein. All reagents used in treating NHBE cells were examined for cytotoxicity by measuring the total release of lactate dehydrogenase from the cells. The assay was carried out using the Promega Cytotox 96 Kit according to the manufacturer's instructions. All experiments were performed with reagents at non-cytotoxic concentrations. Data were analyzed for significance using one-way analysis of variance with Bonferroni post-test corrections. Differences between treatments were considered significant at p < 0.05. To determine the potential role of PKC and/or PKG in the mucin secretory process, NHBE cells were exposed to the following two specific protein kinase activators: the phorbol ester, phorbol 12-myristate 13-acetate (PMA), for activation of PKC, and the nonhydrolyzable cGMP analogue, 8-Br-cGMP, for activation of PKG. Preliminary studies examining mucin secretion in response to PMA stimulation at various concentrations for different times (up to 1 µm for 2 h) indicated that activation of PKC alone did not induce significant mucin secretion from NHBE cells, although a moderate secretory response was repeatedly observed at PMA concentrations higher than 100 nm (0.05