TY - PAT TI - Diffraction enhanced x-ray imaging of articular cartilage AU - Chapman, L. D. AU - Hasnah, M. O. AU - Oltulu, O. AU - Zhong, Z. AU - Mollenhauer, J. Muehleman AU - C., Kuettner AU - K., Aurich AU - M., Pisano AU - E. D., Johnston AU - R. E., Thomlinson AU - W. C., AU - Sayers, D. C2 - 2003/// DA - 2003/// PY - 2003/// ER - TY - JOUR TI - Anti-ethylene properties of monoterpenes and some other naturally occurring compound in plants AU - Grichko, V. P. AU - Sisler, E. C. AU - Serek, M. T2 - new DA - 2003/// PY - 2003/// VL - 16 SP - 20 ER - TY - PAT TI - Methods, systems, and computer program products for analyzing and presenting NMR lipoprotein-based risk assessment results AU - Otvos, J. C2 - 2003/// DA - 2003/// PY - 2003/// ER - TY - PAT TI - Methods for providing personalized lipoprotein-based risk assessments AU - Otvos, J. C2 - 2003/// DA - 2003/// PY - 2003/// ER - TY - PAT TI - Method of determining presence and concentration of lipoprotein X in blood plasma and serum AU - Otvos, J. AU - Jeyarajah, E. AU - Shalaurova, I. C2 - 2003/// DA - 2003/// PY - 2003/// ER - TY - JOUR TI - Equilibrium and kinetic folding of a alpha-helical Greek key protein domain: Caspase recruitment domain (CARD) of RICK AU - Chen, YR AU - Clark, AC T2 - BIOCHEMISTRY AB - We have characterized the equilibrium and kinetic folding of a unique protein domain, caspase recruitment domain (CARD), of the RIP-like interacting CLARP kinase (RICK) (RICK-CARD), which adopts a α-helical Greek key fold. At equilibrium, the folding of RICK-CARD is well described by a two-state mechanism representing the native and unfolded ensembles. The protein is marginally stable, with a ΔGH2O of 3.0 ± 0.15 kcal/mol and an m-value of 1.27 ± 0.06 kcal mol-1 M-1 (30 mM Tris-HCl, pH 8, 1 mM DTT, 25 °C). While the m-value is constant, the protein stability decreases in the presence of moderate salt concentrations (below 200 mM) and then increases at higher salt concentrations. The results suggest that electrostatic interactions are stabilizing in the native protein, and the favorable Coulombic interactions are reduced at low ionic strength. Above 200 mM salt, the results are consistent with Hofmeister effects. The unfolding pathway of RICK-CARD is complex and contains at least three non-native conformations. The refolding pathway of RICK-CARD also is complex, and the data suggest that the unfolded protein folds via two intermediate conformations prior to reaching the native state. Overall, the data suggest the presence of kinetically trapped, or misfolded, species that are on-pathway both in refolding and in unfolding. DA - 2003/5/27/ PY - 2003/5/27/ DO - 10.1021/bi0340752 VL - 42 IS - 20 SP - 6310-6320 SN - 0006-2960 ER - TY - JOUR TI - Deletions in the transmembrane domain of a Sindbis virus glycoprotein alter virus infectivity, stability, and host range AU - Hernandez, R AU - Sinodis, C AU - Horton, M AU - Ferreira, D AU - Yang, CN AU - Brown, DT T2 - JOURNAL OF VIROLOGY AB - The alphaviruses are composed of two icosahedral protein shells, one nested within the other. A membrane bilayer derived from the host cell is sandwiched between the protein shells. The protein shells are attached to one another by protein domains which extend one of the proteins of the outer shell through the membrane bilayer to attach to the inner shell. We have examined the interaction of the membrane-spanning domain of one of the membrane glycoproteins with the membrane bilayer and with other virus proteins in an attempt to understand the role this domain plays in virus assembly and function. Through incremental deletions, we have reduced the length of a virus membrane protein transmembrane domain from its normal 26 amino acids to 8 amino acids. We examined the effect of these deletions on the assembly and function of virus particles. We found that progressive truncations in the transmembrane domain profoundly affected production of infectious virus in a cyclic fashion. We also found that membrane composition effects protein-protein and protein-membrane interactions during virus assembly. DA - 2003/12// PY - 2003/12// DO - 10.1128/JVI.77.23.12710-12719.2003 VL - 77 IS - 23 SP - 12710-12719 SN - 0022-538X ER - TY - JOUR TI - An arabinogalactan protein associated with secondary cell wall formation in differentiating xylem of loblolly pine AU - Zhang, Y AU - Brown, G AU - Whetten, R AU - Loopstra, CA AU - Neale, D AU - Kieliszewski, MJ AU - Sederoff, RR T2 - PLANT MOLECULAR BIOLOGY DA - 2003/5// PY - 2003/5// DO - 10.1023/A:1023978210001 VL - 52 IS - 1 SP - 91-102 SN - 0167-4412 KW - arabinogalactan proteins (AGPs) KW - Pinus taeda KW - plant cell wall biosynthesis KW - xylem differentiation ER - TY - JOUR TI - 1-substituted cyclopropenes: Effective blocking agents for ethylene action in plants AU - Sisler, EC AU - Alwan, T AU - Goren, R AU - Serek, M AU - Apelbaum, A T2 - PLANT GROWTH REGULATION DA - 2003/7// PY - 2003/7// DO - 10.1023/A:1025080420990 VL - 40 IS - 3 SP - 223-228 SN - 0167-6903 KW - antagonists KW - bananas KW - cyclopropenes KW - ethylene KW - inhibitor KW - receptor ER - TY - JOUR TI - Up, down and up again is a signature global gene expression pattern at the beginning of gymnosperm embryogenesis AU - Zyl, L AU - Bozhkov, PV AU - Clapham, DH AU - Sederoff, RR AU - Arnold, S T2 - GENE EXPRESSION PATTERNS AB - Somatic embryogenesis of a gymnosperm, Picea abies, represents a sequence of specifically regulated developmental stages including proembryogenic mass (PEM), PEM-to-embryo transition, and early and late embryogeny. Here, we report cDNA array analysis of expression patterns of 373 genes in the beginning of P. abies embryo development. The analysis revealed a group of 107 genes (29% of arrayed cDNAs) which were upregulated upon PEM-to-embryo transition, then downregulated during early embryogeny and finally upregulated again at the beginning of late embryogeny. This major gene expression pattern was abrogated in a developmentally arrested cell line that is unable to pass through the PEM-to-embryo transition. Thirty-five genes (9.4% of arrayed cDNAs) were found to be differentially expressed during normal embryonic pattern formation. Among them, 22 genes (5.9% of arrayed cDNAs) were directly associated with embryo pattern formation and can be considered as marker genes for early stages of P. abies embryogenesis. The majority of the marker genes encode for proteins involved in translation and posttranslational modification. Among them, 18 genes displayed the major expression pattern. DA - 2003/3// PY - 2003/3// DO - 10.1016/S1567-133X(02)00068-6 VL - 3 IS - 1 SP - 83-91 SN - 1567-133X KW - Picea abies KW - embryogenesis KW - cDNA arrays KW - gene expression profile KW - transcriptionally repressive state KW - developmentally regulated genes KW - arrested cell line ER - TY - JOUR TI - Transcript profiles of stress-related genes in developing white spruce (Picea glauca) somatic embryos cultured with polyethylene glycol AU - Stasolla, C AU - Zyl, L AU - Egertsdotter, U AU - Craig, D AU - Liu, WB AU - Sederoff, RR T2 - PLANT SCIENCE AB - The effect of polyethylene glycol (PEG) on the transcript level of 512 stress-related genes was analyzed by cDNA microarray. Major changes in gene expression between control and PEG-treated embryos were observed during the initial stages of development, upon transfer of the embryogenic tissue on maturation medium, and during the late phases of development, culminating with the generation of cotyledonary embryos. Only small changes in gene expression were observed during the intermediate phases of embryo development. The transcript levels of several genes involved in cell aging and detoxification mechanisms, including peroxidases and chitinases, were developmentally regulated during the embryogenic process. Major differences in the expression of these genes were observed between control and PEG-treated embryos. Based on their expression profiles, four different clusters of genes involved in stress response mechanisms were identified. The first group of genes, which included several heat shock proteins, was up-regulated in PEG-treated immature embryos. An opposite tendency was observed for a second cluster of genes, which included a glutathione-S-transferase, and a cysteine protease. The third class included genes repressed by PEG in fully developed embryos, whereas a fourth group of genes, which included several heat shock proteins and ubiquitin, was induced in PEG-treated embryos at the end of the culture period. Difference in transcript levels and profiles of several genes involved in cell wall and lignin biosynthesis were also observed between control and PEG-treated embryos. DA - 2003/10// PY - 2003/10// DO - 10.1016/S0168-9452(03)00228-0 VL - 165 IS - 4 SP - 719-729 SN - 0168-9452 KW - microarray KW - polyethylene glycol KW - transcript levels KW - white spruce ER - TY - JOUR TI - The effects of Ca2+ binding on the conformation of calbindin D-28K: A nuclear magnetic resonance and microelectrospray mass spectrometry study AU - Venters, RA AU - Benson, LM AU - Craig, TA AU - Paul, KH AU - Kordys, DR AU - Thompson, R AU - Naylor, S AU - Kumar, R AU - Cavanagh, J T2 - ANALYTICAL BIOCHEMISTRY AB - Calbindin D(28K) is a six-EF-hand calcium-binding protein found in the brain, peripheral nervous system, kidney, and intestine. There is a paucity of information on the effects of calcium binding on calbindin D(28K) structure. To further examine the mechanism and structural consequences of calcium binding to calbindin D(28K) we performed detailed complementary heteronuclear NMR and microelectrospray mass spectrometry investigations of the calcium-induced conformational changes of calbindin D(28K). The combined use of these two powerful analytical techniques clearly and very rapidly demonstrates the following: (i). apo-calbindin D(28K) has an ordered structure which changes to a notably different ordered conformation upon Ca(2+) loading, (ii). calcium binding is a sequential process and not a simultaneous event, and (iii). EF-hands 1, 3, 4, and 5 take up Ca(2+), whereas EF-hands 2 and 6 do not. Our results support the opinion that calbindin D(28K) has characteristics of both a calcium sensor and a buffer. DA - 2003/6/1/ PY - 2003/6/1/ DO - 10.1016/S0003-2697(03)00084-8 VL - 317 IS - 1 SP - 59-66 SN - 0003-2697 KW - calbindin D-28K KW - EF-hand KW - conformational response to calcium binding ER - TY - JOUR TI - Production and characterization of bio-immobilized keratinase in proteolysis and keratinolysis AU - Wang, JJ AU - Swaisgood, HE AU - Shih, JCH T2 - ENZYME AND MICROBIAL TECHNOLOGY AB - Extracellular production of keratinase–streptavidin fusion protein (KER–STP) was accomplished by the cloning of Bacillus subtilis with a transforming plasmid carrying the kerA-stp fusion gene. The fusion protein was readily immobilized onto a biotinylated solid matrix by mixing in the culture medium. The properties and reaction kinetics of free and immobilized keratinase (KE) were characterized and compared. Heat stability and pH tolerance were greatly improved by immobilization, but the catalytic efficiency (kcat/Km) was reduced by eightfold. The yield of bio-immobilization using bioselective adsorption of the fusion protein was approximately 20%, as estimated from the activity of free KE. HPLC analysis of reaction products demonstrated the hydrolysis of feather keratin, casein, and bovine serum albumin (BSA) by the immobilized KE. The rates of reactions are lower than those of the free enzyme. On the other hand, the stability of the enzyme was greatly improved. DA - 2003/6/12/ PY - 2003/6/12/ DO - 10.1016/S0141-0229(03)00060-7 VL - 32 IS - 7 SP - 812-819 SN - 0141-0229 KW - keratinase KW - bio-immobililzation KW - streptavidin KW - fusion protein ER - TY - JOUR TI - Pituitary tumorigenesis targeted by the ovine follicle-stimulating hormone beta-subunit gene regulatory region in transgenic mice AU - Pernasetti, F AU - Spady, TJ AU - Hall, SB AU - Rosenberg, SB AU - Givens, ML AU - Anderson, S AU - Paulus, M AU - Miller, WL AU - Mellon, PL T2 - MOLECULAR AND CELLULAR ENDOCRINOLOGY AB - Targeted tumorigenesis in transgenic mice has been a powerful tool for the study of gene expression and oncogenesis, as well as for the production of differentiated immortal cell lines from rare cell types. Follicle-stimulating hormone (FSH) is secreted by the gonadotrope cells of the anterior pituitary gland and plays a pivotal role in mammalian reproduction. Here we have used the regulatory region of the ovine FSH beta gene to direct expression of the SV40 T antigen oncogene to gonadotrope cells in the pituitary of transgenic mice. Two of five transgenic mouse lines bearing this fusion gene rapidly developed pituitary tumors, with appearance of adenomatous foci as early as 6 weeks of age, resulting in death by 12 weeks of age in both genders. Histologic examination of tumor development over time revealed that increases in cell proliferation and dysplasia were accompanied by decreases in synthesis of pituitary hormones, indicating dedifferentiation of the pituitary cells. Histological features observed in these tumors were in agreement with this rapid transformation of cell phenotype. Tumors were multifocal in origin, and the most highly transformed cell types observed consisted of giant pale basophilic cells with enormous hyperploid nuclei associated with infiltrating neuronal-like cells, which were very abundant at later stages of tumor development. Mitotic indices were much higher in transgenic than wild-type pituitaries, as expected. Morphologic analysis of the gonads of these transgenic mice showed no major developmental differences, as compared to wild-type littermates, however the length of the seminiferous tubules in transgenic males was greater than age-matched wild-type animals. Despite this phenotype difference, both genders were fertile, with normal sperm development observed in males and normal estrous cycle stages in females. Moreover, while 8 -- 10-week-old transgenic males had much lower blood levels of FSH than littermates, transgenic female FSH levels were the same as those of wild-type females. These animals offer a unique and potentially useful model of organ-specific tumorigenesis, where a multistage pathway of tumor development is evident, both histologically and temporally. Study of such models will advance our knowledge on the physiological and molecular mechanisms involved in gene expression as well as tumor formation. DA - 2003/5/30/ PY - 2003/5/30/ DO - 10.1016/S0303-7207(02)00430-6 VL - 203 IS - 1-2 SP - 169-183 SN - 0303-7207 KW - follicle-stimulating hormone KW - gonadotrope KW - T antigen KW - tumors KW - transgenic mice ER - TY - PAT TI - Methods and computer program products for determining risk of developing type 2 diabetes and other insulin resistance related disorders AU - Otvos, J. D. AU - Bennett, D. W. C2 - 2003/// DA - 2003/// PY - 2003/// ER - TY - JOUR TI - Apparent homology of expressed genes from wood-forming tissues of loblolly pine (Pinus taeda L.) with Arabidopsis thaliana AU - Kirst, M AU - Johnson, AF AU - Baucom, C AU - Ulrich, E AU - Hubbard, K AU - Staggs, R AU - Paule, C AU - Retzel, E AU - Whetten, R AU - Sederoff, R T2 - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AB - Pinus taeda L. (loblolly pine) and Arabidopsis thaliana differ greatly in form, ecological niche, evolutionary history, and genome size. Arabidopsis is a small, herbaceous, annual dicotyledon, whereas pines are large, long-lived, coniferous forest trees. Such diverse plants might be expected to differ in a large number of functional genes. We have obtained and analyzed 59,797 expressed sequence tags (ESTs) from wood-forming tissues of loblolly pine and compared them to the gene sequences inferred from the complete sequence of the Arabidopsis genome. Approximately 50% of pine ESTs have no apparent homologs in Arabidopsis or any other angiosperm in public databases. When evaluated by using contigs containing long, high-quality sequences, we find a higher level of apparent homology between the inferred genes of these two species. For those contigs 1,100 bp or longer, ≈90% have an apparent Arabidopsis homolog ( E value < 10 - 10 ). Pines and Arabidopsis last shared a common ancestor ≈300 million years ago. Few genes would be expected to retain high sequence similarity for this time if they did not have essential functions. These observations suggest substantial conservation of gene sequence in seed plants. DA - 2003/6/10/ PY - 2003/6/10/ DO - 10.1073/pnas.1132171100 VL - 100 IS - 12 SP - 7383-7388 SN - 0027-8424 ER - TY - JOUR TI - Analysis of lignin produced by cinnamyl alcohol dehydrogenase-deficient Pinus taeda cultured cells AU - Stasolla, C AU - Scott, J AU - Egertsdotter, U AU - Kadla, J AU - D O'Malley, AU - Sederoff, R AU - Zyl, L T2 - PLANT PHYSIOLOGY AND BIOCHEMISTRY AB - Comparative studies were conducted on composition of lignin produced both in vivo and in vitro by cinnamyl alcohol dehydrogenase (CAD)-deficient mutant loblolly pine (Pinus taeda L.). In vivo studies were performed using differentiating xylem obtained from two genotypes of heterozygous (CAD/cad) and two genotypes of homozygous (cad/cad) CAD-deficient mutant trees. In vitro studies were performed using a culture system in which cells, generated from the same genotypes, were induced to produce lignin in culture. Steady state RNA levels and enzyme activity of CAD were dramatically reduced in both xylem and cultured cells obtained from homozygous mutant trees, compared to their heterozygous counterparts. Light microscopic studies showed pronounced differences during the lignin formation between homozygous and heterozygous cells. Phenolic compounds in the heterozygous (CAD/cad) cells were deposited around the cell wall, accumulated preferentially in vacuoles of the homozygous (cad/cad) cells. Differences in lignin composition as revealed by thioacidolysis were also observed. Lignin of both xylem tissue and cultured cells obtained from CAD-deficient homozygotes showed lower levels of coniferyl alcohols and significant enrichments in dihydroconiferyl alcohol (DHCA) and coniferyl aldehyde, compared to their heterozygous counterparts. The striking similarities in lignin composition observed both in vivo and in vitro, open new possibilities for the use of culture systems aimed at revealing the mechanisms controlling lignin biosynthesis, and the formation of DHCA subunits. DA - 2003/5// PY - 2003/5// DO - 10.1016/S0981-9428(03)00051-2 VL - 41 IS - 5 SP - 439-445 SN - 0981-9428 KW - cinnamyl alcohol dehydrogenase KW - cultured cells KW - dihydroconiferyl alcohol KW - lignin KW - Pinus taeda KW - xylem ER - TY - JOUR TI - Reaction mechanisms in delignification of pine Kraft-AQ pulp with hydrogen peroxide using Mn(IV)-Me4DTNE as catalyst AU - Chen, CL AU - Capanema, EA AU - Gracz, HS T2 - JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY AB - Pine Kraft-AQ pulp was bleached with hydrogen peroxide catalyzed by [LMn(IV)2 (μ-O)3](ClO4)2 at 80 °C for 120 min under optimum reaction conditions. The resulting bleached pulp was hydrolyzed with cellulase to obtain insoluble and soluble residual lignins. The alkaline effluent from the bleaching was acidified to precipitate alkaline soluble lignin. These lignin preparations were purified, and then analyzed by 2D HMQC NMR spectroscopic techniques. The results showed that biphenyl (5−5) and stilbene structures are preferentially degraded in the bleaching process, while β-O-4, β-5, and β−β structures undergo degradation only to a lesser extent. This implies that hydrogen peroxide bleaching using the catalyst is more effective in delignification of softwood pulps than hardwood pulps. The possible reaction mechanisms for the delignification of residual lignin in the pine Kraft-AQ pulp in the bleaching process are discussed on the basis of the 2D HMQC NMR spectroscopic data and the model compound experiments. Keywords: Pine Kraft-AQ pulp; binucleus Mn(IV) complex; hydrogen peroxide bleaching; catalysis; residual lignins; alkaline soluble lignin; 2D HMQC NMR spectroscopic technique; epoxidation of conjugated double bonds DA - 2003/3/26/ PY - 2003/3/26/ DO - 10.1021/jf020992n VL - 51 IS - 7 SP - 1932-1941 SN - 0021-8561 KW - pine Kraft-AQ pulp KW - binucleus Mn(IV) complex KW - hydrogen peroxide bleaching KW - catalysis KW - residual lignins KW - alkaline soluble lignin KW - 2D HMQC NMR spectroscopic technique KW - epoxidation of conjugated double bonds ER - TY - JOUR TI - Potato virus X: a model system for virus replication, movement and gene expression AU - Batten, JS AU - Yoshinari, S AU - Hemenway, C T2 - MOLECULAR PLANT PATHOLOGY AB - SUMMARY Considerable research has focused on the cis ‐ and trans ‐acting components required for various aspects of the potato virus X (PVX) infection process. In addition, the development of PVX‐based vectors has facilitated analyses of the PVX infection process and provided diverse technological applications. As a result, the PVX system will continue to serve as a model for analyses of processes such as virus movement, RNA replication, and gene silencing, and as a tool for protein expression. DA - 2003/3// PY - 2003/3// DO - 10.1046/j.1364-3703.2003.00156.x VL - 4 IS - 2 SP - 125-131 SN - 1364-3703 ER - TY - JOUR TI - Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures AU - Qian, WJ AU - Gosche, MB AU - Camp, DG AU - Yu, LR AU - Tang, KQ AU - Smith, RD T2 - ANALYTICAL CHEMISTRY AB - Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line. DA - 2003/10/15/ PY - 2003/10/15/ DO - 10.1021/ac0342774 VL - 75 IS - 20 SP - 5441-5450 SN - 1520-6882 ER - TY - JOUR TI - NMR analysis of lignins in CAD-deficient plants. Part 1. Incorporation of hydroxycinnamaldehydes and hydroxybenzaldehydes into lignins AU - Kim, H AU - Ralph, J AU - Lu, FC AU - Ralph, SA AU - Boudet, AM AU - MacKay, JJ AU - Sederoff, RR AU - Ito, T AU - Kawai, S AU - Ohashi, H AU - Higuchi, T T2 - ORGANIC & BIOMOLECULAR CHEMISTRY AB - Peroxidase/H2O2-mediated radical coupling of 4-hydroxycinnamaldehydes produces 8–O–4-, 8–5-, and 8–8-coupled dehydrodimers as has been documented earlier, as well as the 5–5-coupled dehydrodimer. The 8–5-dehydrodimer is however produced kinetically in its cyclic phenylcoumaran form at neutral pH. Synthetic polymers produced from mixtures of hydroxycinnamaldehydes and normal monolignols provide the next level of complexity. Spectral data from dimers, oligomers, and synthetic polymers have allowed a more substantive assignment of aldehyde components in lignins isolated from a CAD-deficient pine mutant and an antisense-CAD-downregulated transgenic tobacco. CAD-deficient pine lignin shows enhanced levels of the typical benzaldehyde and cinnamaldehyde end-groups, along with evidence for two types of 8–O–4-coupled coniferaldehyde units. The CAD-downregulated tobacco also has higher levels of hydroxycinnamaldehyde and hydroxybenzaldehyde (mainly syringaldehyde) incorporation, but the analogous two types of 8–O–4-coupled products are the dominant features. 8–8-Coupled units are also clearly evident. There is clear evidence for coupling of hydroxycinnamaldehydes to each other and then incorporation into the lignin, as well as for the incorporation of hydroxycinnamaldehyde monomers into the growing lignin polymer. Coniferaldehyde and sinapaldehyde (as well as vanillin and syringaldehyde) co-polymerize with the traditional monolignols into lignins and do so at enhanced levels when CAD-deficiency has an impact on the normal monolignol production. The implication is that, particularly in angiosperms, the aldehydes behave like the traditional monolignols and should probably be regarded as authentic lignin monomers in normal and CAD-deficient plants. DA - 2003/1/21/ PY - 2003/1/21/ DO - 10.1039/b209686b VL - 1 IS - 2 SP - 268-281 SN - 1477-0539 ER - TY - JOUR TI - Morphological variants of Sindbis virus produced by a mutation in the capsid protein AU - Ferreira, D AU - Hernandez, R AU - Horton, M AU - Brown, DT T2 - VIROLOGY AB - Sindbis virus is a complex aggregate of RNA, protein and lipid. The virus is organized as two nested T = 4 icosahedral protein shells between which is sandwiched a lipid bilayer. The virus RNA resides within the inner protein shell. The inner protein shell is attached to the outer protein shell through contacts to proteins in the outer shell, which penetrate the lipid bilayer. The data presented in the following manuscript show that mutations in the capsid protein can result in the assembly of the virus structural proteins into icosahedra of different triangulation numbers. The triangulation numbers calculated, for these morphological variants, follow the sequence T = 4, 9, 16, 25 and 36. All fall into the class P = 1 of icosadeltahedra as was predicted by Caspar and Klug (1962). The data support their hypothesis that families of icosahedra would be developed by altering the distance between the points of insertion of the five-fold axis. This capsid protein defect also results in the incorporation of much of the capsid protein, into large cytoplasmic aggregates of protein and RNA. These observations support models suggesting that the geometry of a pre-formed nucleocapsid organizes the assembly of the virus membrane proteins into a structure of identical configuration and argues against models suggesting that assembly of the membrane glycoproteins directs the assembly of the nucleocapsid. DA - 2003/3/1/ PY - 2003/3/1/ DO - 10.1016/S0042-6822(02)00034-X VL - 307 IS - 1 SP - 54-66 SN - 0042-6822 ER - TY - JOUR TI - Investigating the possibility of monitoring lectin levels in commercial soybean meals intended for poultry feeding using steam-heated soybean meal as a model AU - Fasina, YO AU - Classen, HL AU - Garlich, JD AU - Swaisgood, HE AU - Clare, DA T2 - POULTRY SCIENCE AB - Native soybean lectins (SBL) could potentially have deleterious effects on young animals. The objectives of this study were to determine the optimum processing temperature and time at which SBL is inactivated and to investigate the possibility of using urease activity (UA) to predict residual lectin levels in soybean meal (SBM). Raw defatted SBM was steam-heated at incremental temperatures between 90 and 120 degrees C for 5 to 20 min in an autoclave. The processed meals were subjected to native-PAGE and measurement of total carbohydrate-binding lectin (TCBL), agglutinating lectin (AL), UA, and trypsin inhibitor (TI). Processing severity was evaluated by determining protein solubility in 0.2% potassium hydroxide. Results indicated that levels of all antinutrients (TCBL, AL, UA, and TI) decreased with increasing processing temperature (P < 0.05). The intensity of the lectin band on the electrophoresis gel was considerably reduced when meal was heated at 100 degrees C for 5 min. This result implied that lectin inactivation occurred at 100 degrees C. More than 90% of all the original antinutrient levels in the raw meal were destroyed when meals were heated at 100 degrees C for 5 min. Meals processed at 100 degrees C for 5 to 20 min had protein solubility values (80 to 85%) indicative of adequate processing. The denaturation pattern of UA was highly correlated with that of SBL (r > or = 0.73), indicating that UA could be used for monitoring lectin levels in commercial meals. We concluded that UA of 0.03 to 0.09 units of pH change are indicative of adequately processed meals that contain negligible lectin levels. DA - 2003/4// PY - 2003/4// DO - 10.1093/ps/82.4.648 VL - 82 IS - 4 SP - 648-656 SN - 0032-5791 KW - soybean meal KW - lectin KW - urease activity KW - trypsin inhibitor KW - protein solubility ER - TY - JOUR TI - Heterozygosity for ABCA1 gene mutations: effects on enzymes, apolipoproteins and lipoprotein particle size AU - Kuivenhoven, JA AU - Hovingh, GK AU - Tol, A AU - Jauhiainen, M AU - Ehnholm, C AU - Fruchart, JC AU - Brinton, EA AU - Otvos, JD AU - Smelt, AHM AU - Brownlee, A AU - Zwinderman, AH AU - Hayden, MR AU - Kastelein, JJP T2 - ATHEROSCLEROSIS AB - A cohort of 13 female and 14 male heterozygotes for ATP binding cassette A1 (ABCA1) gene defects was directly compared with 13 and 14 unaffected female and male family members of almost exact same age. The activities of three proteins that play key roles in HDL metabolism were measured in addition to extensive lipid and (apo) lipoprotein subfraction analysis. Compared to controls, LCAT activity was reduced by 15% in affected subjects (P<0.001) while PLTP activity was unaffected. Interestingly, CETP activity was elevated by 50% in the heterozygote siblings of one kindred but was unaffected in heterozygotes of the three other families. With respect to lipids, the heterozygotes had normal total cholesterol (TC), and LDL-cholesterol concentrations but presented with a trend towards increased triglyceride levels (13%; P=0.08). HDL metabolism, by contrast, was severely affected as illustrated by 40% reductions in HDL-cholesterol (P<0.001) with concomitant reductions in apoAI (25%; P<0.001) levels and in lipoprotein subfraction LpAI (28%; P<0.001), LpAI:AII (24%; P=0.014), and LpCIII:nonB (34%; P<0.001) concentrations. We furthermore observed reduced average HDL particle size (5%; P=0.004; 16% in female and 3.6% in male) and reduced plasma apoCIII concentration (15%; P=0.006) while apoAII, apoAIV, apoE and apoB levels were unchanged. In conclusion, heterozygosity for ABCA1 defects was associated with reduced LCAT activity in absence of effects on PLTP activity. Of special interest was our finding that the effects of compromised ABCA1 function on HDL were more pronounced in women than in men. DA - 2003/12// PY - 2003/12// DO - 10.1016/j.atherosclerosis.2003.08.014 VL - 171 IS - 2 SP - 311-319 SN - 0021-9150 KW - HDL KW - LCAT KW - CETP KW - PLTP KW - ABCA1 ER - TY - JOUR TI - Organic solvents order the dynamic switch II in Ras crystals AU - Buhrman, G AU - Serrano, V AU - Mattos, C T2 - STRUCTURE AB - Room temperature crystal structures of crosslinked H-Ras bound to GMPPNP were solved in 50% 2,2,2-trifluoroethanol, 60% 1,6-hexanediol, and 50% isopropanol. The disordered switch II region of Ras is ordered in the presence of 2,2,2-trifluoroethanol or 1,6-hexanediol. The overall backbone conformation of switch II in these organic solvents is the same as in the Ras-GMPPNP complexes with RalGDS, PI3 kinase, and RasGAP, indicating a biologically relevant form. Key polar interactions that stabilize the ordered switch are enhanced in the presence of hydrophobic cosolvents. These results suggest that hydrophobic solvents can be used in general to order short biologically relevant segments of disordered regions in protein crystals by favoring H-bonding interactions between atoms that are highly solvated and mobile in aqueous solution. DA - 2003/7// PY - 2003/7// DO - 10.1016/s0969-2126(03)00128-x VL - 11 IS - 7 SP - 747-751 SN - 1878-4186 ER - TY - JOUR TI - Mutations in the procaspase-3 dimer interface affect the activity of the zymogen AU - Pop, C AU - Feeney, B AU - Tripathy, A AU - Clark, AC T2 - BIOCHEMISTRY AB - The interface of the procaspase-3 dimer plays a critical role in zymogen maturation. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with glutamate resulted in an increase in enzyme activity of approximately 60-fold, representing a pseudoactivation of the procaspase. In contrast, substitution of V266 with histidine abolished the activity of the procaspase-3 as well as that of the mature caspase. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active site. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate binding pocket. In some cases, the amino acids affected by the mutations are >20 A from the interface. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation. DA - 2003/10/28/ PY - 2003/10/28/ DO - 10.1021/bi034999p VL - 42 IS - 42 SP - 12311-12320 SN - 0006-2960 ER - TY - JOUR TI - Lipoproteins in the DCCT/EDIC cohort: Associations with diabetic nephropathy AU - Jenkins, AJ AU - Lyons, TJ AU - Zheng, DY AU - Otvos, JD AU - Lackland, DT AU - McGee, D AU - Garvey, WT AU - Klein, RL T2 - KIDNEY INTERNATIONAL AB - Lipoproteins may contribute to diabetic nephropathy. Nuclear magnetic resonance (NMR) can quantify subclasses and mean particle size of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL), and LDL particle concentration. The relationship between detailed lipoprotein analyses and diabetic nephropathy is of interest.In a cross-sectional study, lipoproteins from 428 women and 540 men from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) cohort were characterized by conventional lipid enzymology, NMR, apolipoprotein levels, and LDL oxidizibility. Linear regression was performed for each lipoprotein parameter versus log albumin excretion rate (AER), with and without covariates for age, diabetes duration, HbA1c, hypertension, body mass index, waist-hip ratio, and DCCT treatment group. Significance was taken at P < 0.05.By multivariate analysis, conventional profile, total triglycerides, total- and LDL cholesterol, but not HDL cholesterol, were associated with AER. NMR-determined large, medium, and small VLDL were associated with AER in both genders (except large VLDL in women), and intermediate density lipoprotein (IDL) was associated with AER (men only). LDL particle concentration and ApoB were positively associated with AER (in men and in the total cohort), and there was a borderline inverse association between LDL diameter and AER in men. Small HDL was positively associated with AER and a borderline negative association was found for large HDL. No associations were found with ApoA1, Lp(a), or LDL oxidizibility.Potentially atherogenic lipoprotein profiles are associated with renal dysfunction in type 1 diabetes and further details are gained from NMR analysis. Longitudinal studies are needed to determine if dyslipoproteinemia can predict patients at risk of nephropathy, or if lipoprotein-related interventions retard nephropathy. DA - 2003/9// PY - 2003/9// DO - 10.1046/j.1523-1755.2003.00164.x VL - 64 IS - 3 SP - 817-828 SN - 1523-1755 KW - diabetes mellitus KW - nephropathy KW - lipoproteins KW - NMR ER - TY - JOUR TI - Characterisation of PtMYB1, an R2R3-MYB from pine xylem AU - Patzlaff, A AU - Newman, LJ AU - Dubos, C AU - Whetten, R AU - Smith, C AU - McInnis, S AU - Bevan, MW AU - Sederoff, RR AU - Campbell, MM T2 - PLANT MOLECULAR BIOLOGY DA - 2003/11// PY - 2003/11// DO - 10.1023/B:PLAN.0000019066.07933.d6 VL - 53 IS - 4 SP - 597-608 SN - 1573-5028 KW - MYB KW - phenylalanine ammonia-lyase KW - phenylpropanoid KW - pine KW - xylem ER - TY - JOUR TI - Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems AU - Wang, JJ AU - Swaisgood, HE AU - Shih, JCH T2 - BIOTECHNOLOGY AND BIOENGINEERING AB - Immobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties. Conventional purification followed by chemical immobilization is a laborious and costly process. A new genetic construct was developed to produce the keratinase-streptavidin fusion protein. Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step. The method was tested in both the Bacillus subtilis and Escherichia coli systems. In B. subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium. In E. coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract. The overall efficiencies were approximately the same, 24-28%, using both systems. DA - 2003/2/20/ PY - 2003/2/20/ DO - 10.1002/bit.10485 VL - 81 IS - 4 SP - 421-429 SN - 0006-3592 KW - keratinase KW - immobilization KW - streptavidin KW - fusion protein ER - TY - JOUR TI - Sub-classification of response regulators using the surface characteristics of their receiver domains AU - Kojetin, DJ AU - Thompson, RJ AU - Cavanagh, J T2 - FEBS LETTERS AB - The omnipresent bacterial switch known as a two-component system is comprised of a response regulator and a sensor kinase with which it interacts. Sensor kinases have been classified and further sub-classified into groups based on their sequence similarity, loop lengths and domain organization. Response regulators have been classified predominantly by the identity and function of their output domains. Here, comparative based homology modeling of the receiver domains of the OmpR sub-family of response regulators in Bacillus subtilis and Escherichia coli suggests further sub-classification is possible. A color-coded scale is used to show trends in surface hydrophobicity. For the OmpR receiver domains modeled these trends allow further sub-classification. The specific surface regions used for this sub-classification procedure correlate with clusters of residues that are important for interaction with cognate four helix bundle HisKA/Hpt domains. DA - 2003/11/20/ PY - 2003/11/20/ DO - 10.1016/S0014-5793(03)01167-0 VL - 554 IS - 3 SP - 231-236 SN - 0014-5793 KW - OmpR sub-families KW - homology modeling ER - TY - JOUR TI - Naturally-occurring modification restricts the anticodon domain conformational space of tRNA(Phe) AU - Stuart, JW AU - Koshlap, KM AU - Guenther, R AU - Agris, PF T2 - JOURNAL OF MOLECULAR BIOLOGY AB - Post-transcriptional modifications contribute chemistry and structure to RNAs. Modifications of tRNA at nucleoside 37, 3'-adjacent to the anticodon, are particularly interesting because they facilitate codon recognition and negate translational frame-shifting. To assess if the functional contribution of a position 37-modified nucleoside defines a specific structure or restricts conformational flexibility, structures of the yeast tRNA(Phe) anticodon stem and loop (ASL(Phe)) with naturally occurring modified nucleosides differing only at position 37, ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)), and ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)), were determined by NMR spectroscopy and restrained molecular dynamics. The ASL structures had similarly resolved stems (RMSD approximately 0.6A) of five canonical base-pairs in standard A-form RNA. The "NOE walk" was evident on the 5' and 3' sides of the stems of both RNAs, and extended to the adjacent loop nucleosides. The NOESY cross-peaks involving U(33) H2' and characteristic of tRNA's anticodon domain U-turn were present but weak, whereas those involving the U(33) H1' proton were absent from the spectra of both ASLs. However, ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) exhibited the downfield shifted 31P resonance of U(33)pGm(34) indicative of U-turns; ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)) did not. An unusual "backwards" NOE between Gm(34) and A(35) (Gm(34)/H8 to A(35)/H1') was observed in both molecules. The RNAs exhibited a protonated A(+)(38) resulting in the final structures having C(32).A(+)(38) intra-loop base-pairs, with that of ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) being especially well defined. A single family of low-energy structures of ASL(Phe)-(Cm(32),Gm(34), m(1)G(37),m(5)C(40)) (loop RMSD 0.98A) exhibited a significantly restricted conformational space for the anticodon loop in comparison to that of ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)) (loop RMSD 2.58A). In addition, the ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) average structure had a greater degree of similarity to that of the yeast tRNA(Phe) crystal structure. A comparison of the resulting structures indicates that modification of position 37 affects the accuracy of decoding and the maintenance of the mRNA reading frame by restricting anticodon loop conformational space. DA - 2003/12/12/ PY - 2003/12/12/ DO - 10.1016/j.jmb.2003.09.058 VL - 334 IS - 5 SP - 901-918 SN - 1089-8638 KW - methylation KW - anticodon dynamics KW - tRNA position 37 KW - codon recognition KW - frameshifting ER - TY - JOUR TI - Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNA(Phe) binding AU - Noah, JW AU - Shapkina, TG AU - Nanda, K AU - Huggins, W AU - Wollenzien, P T2 - BIOCHEMISTRY AB - The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes. DA - 2003/12/16/ PY - 2003/12/16/ DO - 10.1021/bi035369q VL - 42 IS - 49 SP - 14386-14396 SN - 0006-2960 ER - TY - JOUR TI - Compounds interacting with the ethylene receptor in plants AU - Sisler, E. C. AU - Serek, M. T2 - Plant Biology (Stuttgart, Germany) DA - 2003/// PY - 2003/// VL - 5 IS - 5 SP - 473-480 ER - TY - JOUR TI - Characterisation of a pine MYB that regulates lignification AU - Patzlaff, A AU - McInnis, S AU - Courtenay, A AU - Surman, C AU - Newman, LJ AU - Smith, C AU - Bevan, MW AU - Mansfield, S AU - Whetten, RW AU - Sederoff, RR AU - Campbell, MM T2 - PLANT JOURNAL AB - Summary A member of the R2R3‐MYB family of transcription factors was cloned from a cDNA library constructed from RNA isolated from differentiating pine xylem. This MYB, Pinus taeda MYB4 ( Pt MYB4), is expressed in cells undergoing lignification, as revealed by in situ RT‐PCR. Electrophoretic mobility shift assays (EMSAs) showed that recombinant Pt MYB4 protein is able to bind to DNA motifs known as AC elements. AC elements are ubiquitous in the promoters of genes encoding lignin biosynthetic enzymes. Transcriptional activation assays using yeast showed that Pt MYB4 could activate transcription in an AC‐element‐dependent fashion. Overexpression of Pt MYB4 in transgenic tobacco plants altered the accumulation of transcripts corresponding to genes encoding lignin biosynthetic enzymes. Lignin deposition increased in transgenic tobacco plants that overexpressed Pt MYB4, and extended to cell types that do not normally lignify. Taken together, these findings are consistent with the hypothesis that Pt MYB4 is sufficient to induce lignification, and that it may play this role during wood formation in pine. DA - 2003/12// PY - 2003/12// DO - 10.1046/j.1365-313X.2003.01916.x VL - 36 IS - 6 SP - 743-754 SN - 1365-313X KW - lignin KW - MYB KW - transcription KW - wood KW - pine KW - phenylpropanoid ER - TY - JOUR TI - Using capillary electrophoresis to study methylation effect on RNA-peptide interaction AU - Mucha, P. AU - Szyk, A. AU - Rekowski, P. AU - Agris, P. F. T2 - Acta Biochimica Polonica DA - 2003/// PY - 2003/// VL - 50 IS - 3 SP - 857-864 ER - TY - JOUR TI - Serum lipoproteins in the diabetes control and complications trial/epidemiology of diabetes intervention and complications cohort - Associations with gender and glycemia AU - Jenkins, AJ AU - Lyons, T AU - Zheng, DY AU - Otvos, JD AU - Lackland, DT AU - McGee, D AU - Garvey, WT AU - Klein, RL T2 - DIABETES CARE AB - OBJECTIVE—To relate the nuclear magnetic resonance (NMR)-determined lipoprotein profile, conventional lipid and apolipoprotein measures, and in vitro oxidizibility of LDL with gender and glycemia in type 1 diabetes. RESEARCH DESIGN AND METHODS—In the 1997–1999 Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications (DCCT/EDIC) cohort, serum from 428 women and 540 men were characterized by conventional lipids, NMR, apolipoprotein levels, and LDL susceptibility to in vitro oxidation. Simple and partial correlation coefficients were calculated for each lipoprotein-related parameter versus gender, with and without covariates (age, diabetes duration, concurrent HbA1c, DCCT randomization, hypertension, BMI, waist-to-hip ratio, and albuminuria). For concurrent HbA1c, data were analyzed as above, exchanging gender for HbA1c. Associations were significant if P &lt; 0.05. RESULTS—Although men and women had similar total and LDL cholesterol and triglycerides, men exhibited the following significant percent differences in NMR profiles versus women: small VLDL 41; IDL −30; medium LDL 39; small LDL 21; large HDL −32; small HDL 35; LDL particle concentration 4; VLDL and HDL diameters −8 and −4, respectively. Small VLDL, small HDL, medium LDL (women only), small LDL (men only), and LDL particle concentration were positively correlated, and HDL size was inversely correlated, with concurrent HbA1c. NMR profile was unrelated to prior DCCT randomization. Susceptibility of LDL to oxidation was unrelated to gender and glycemia. CONCLUSIONS—Male gender and poor glycemia are associated with a potentially more atherogenic NMR lipoprotein profile. Neither gender nor glycemia influence LDL oxidation in vitro. DA - 2003/3// PY - 2003/3// DO - 10.2337/diacare.26.3.810 VL - 26 IS - 3 SP - 810-818 SN - 1935-5548 ER - TY - JOUR TI - RpoS-dependent stress response and exoenzyme production in Vibrio vulnificus AU - Hulsmann, A AU - Rosche, TM AU - Kong, IS AU - Hassan, HM AU - Beam, DM AU - Oliver, JD T2 - APPLIED AND ENVIRONMENTAL MICROBIOLOGY AB - ABSTRACT Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection. Like other opportunistic pathogens, V. vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels. In order to begin to understand the ability of V. vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens. An rpoS mutant of V. vulnificus strain C7184o was constructed by homologous recombination. The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions. The most striking difference was a high sensitivity of the mutant to hydrogen peroxide. Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type. Additionally, the motility of the rpoS mutant was severely diminished. Overall, these studies suggest that rpoS in V. vulnificus is important for adaptation to environmental changes and may have a role in virulence. DA - 2003/10// PY - 2003/10// DO - 10.1128/AEM.69.10.6114-6120.2003 VL - 69 IS - 10 SP - 6114-6120 SN - 1098-5336 ER - TY - JOUR TI - Molecular characterization and functional analysis of the manganese-containing superoxide dismutase gene (sodA) from Streptococcus thermophilus AO54 AU - Andrus, JM AU - Bowen, SW AU - Klaenhammer, TR AU - Hassan, HM T2 - ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS AB - This report describes the isolation, sequencing, and functional analysis of the sodA gene, encoding Mn-superoxide dismutase, from Streptococcus thermophilus AO54. The gene was found to encode a 201 amino acid polypeptide with 88 and 83% identity to SodA from Streptococcus mutans and Streptococcus agalacticae, respectively. Primer extension analysis revealed a transcriptional start site 27 nucleotides upstream of initiation codon. The gene was expressed in Escherichia coli and was able to rescue the growth of a sodAsodB mutant in a minimal-medium containing 10−6 M paraquat. A sodA mutant of S. thermophilus was constructed and found to be more sensitive to aerobic growth than its parent strain. Supplementing the medium with MnCl2 improved the growth of the mutant, only under microaerophilic conditions. The results suggest that sodA is essential for the aerobic growth of S. thermophilus. In the absence of functional SodA, manganese ions may provide partial protection against oxygen toxicity. DA - 2003/12/1/ PY - 2003/12/1/ DO - 10.1016/j.abb.2003.09.007 VL - 420 IS - 1 SP - 103-113 SN - 1096-0384 KW - Streptococcus thermophilus KW - superoxide dismutase KW - MnSOD KW - sodA sequence KW - sodA mutant KW - SOD-null mutant KW - oxygen sensitivity KW - manganese ions KW - DNA polymerase III ER - TY - JOUR TI - Efficient RNA 2 '-O-methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C '/D ' RNPs AU - Tran, EJ AU - Zhang, , XX AU - Maxwell, ES T2 - EMBO JOURNAL AB - Article1 August 2003free access Efficient RNA 2′-O-methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C′/D′ RNPs Elizabeth J. Tran Elizabeth J. Tran Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA Search for more papers by this author Xinxin Zhang Xinxin Zhang Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA Search for more papers by this author E.Stuart Maxwell Corresponding Author E.Stuart Maxwell Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA Search for more papers by this author Elizabeth J. Tran Elizabeth J. Tran Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA Search for more papers by this author Xinxin Zhang Xinxin Zhang Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA Search for more papers by this author E.Stuart Maxwell Corresponding Author E.Stuart Maxwell Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA Search for more papers by this author Author Information Elizabeth J. Tran1, Xinxin Zhang1 and E.Stuart Maxwell 1 1Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:3930-3940https://doi.org/10.1093/emboj/cdg368 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-specific 2′-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C′/D′ RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C′/D′ motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, efficient ribonucleotide 2′-O-methylation requires that both the box C/D and C′/D′ RNPs function within the full-length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C′/D′ motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles. Introduction The small nucleolar RNAs (snoRNAs) play critical roles in ribosome biogenesis, functioning in the processing and modification of preribosomal RNA (Bachellerie et al., 2002; Kiss, 2002; Terns and Terns, 2002). The primary role of the vast majority of snoRNAs is to guide the site-specific modification of rRNA nucleotides. Guide regions within the snoRNA base pair with complementary sequences in the rRNA and direct snoRNA-associated enzymes to the designate nucleotide for ribose or base modification. Recent work has also revealed guide RNAs in Archaea (Gaspin et al., 2000; Omer et al., 2000). While archaeal organisms do not possess a nucleus, they nevertheless utilize snoRNA-like RNAs (sRNAs) for nucleotide modification. The occurrence of guide RNAs in both Eukarya and Archaea indicates that the process of RNA-guided nucleotide modification is an ancient mechanism predating the divergence of Eukarya and Archaea more than 2 billion years ago. Box C/D RNAs direct the site-specific 2′-O-ribose methylation of targeted nucleotides within rRNA and other RNA substrates (Tollervey, 1996; Tycowski et al., 1998; Jady and Kiss, 2001). Members of this family are defined by the conserved boxes C and D located at the 5′ and 3′ termini, respectively (Tyc and Steitz, 1989; Caffarelli et al., 1996; Cavaille and Bachellerie, 1996; Watkins et al., 1996, 2000). These conserved sequences fold into a stem–loop–stem structure which is essential for the binding of box C/D ribonucleoproteins (RNPs) as well as the nucleotide modification reaction itself. Additional internal sequences designated C′ and D′ boxes can be identified in eukaryotic snoRNAs and archaeal sRNAs (Kiss-Laszlo et al., 1998). Although based upon boxes C and D, the C′ and D′ sequences are not as strictly conserved and are not easily identified in all eukaryotic snoRNAs. However, both RNA motifs guide 2′-O-methylation of targeted ribonucleotides using rRNA-complementary regions located immediately upstream of boxes D and D′. Antisense sequences 10–21 nucleotides in length base pair with the target RNA and direct methylation to the designate nucleotide positioned five nucleotides upstream of box D/D′ within the snoRNA:rRNA duplex (Cavaille and Bachellerie, 1998). Eukaryotic box C/D snoRNAs associate with four core proteins: the 15.5 kDa protein, nucleolar protein 56 (Nop56p), nucleolar protein 58 (Nop58p) and fibrillarin. Assembly of the terminal box C/D snoRNP complex is required for snoRNA processing and stable accumulation of the box C/D snoRNAs (Caffarelli et al., 1996; Cavaille and Bachellerie, 1996; Watkins et al., 1996). The 15.5 kDa protein binds the terminal box C/D core motif in the absence of the other core proteins and initiates snoRNP assembly (Watkins et al., 2000). Nop56p and Nop58p are highly related snoRNP core proteins also required for ribosome biogenesis (Lafontaine and Tollervey, 1999, 2000; Newman et al., 2000). Fibrillarin is the fourth core protein and all available biochemical and genetic evidence indicates that it is the methylase enzyme (Tollervey et al., 1993; Wang et al., 2000; Galardi et al., 2002; Omer et al., 2002). Recent work has revealed the asymmetric distribution of the box C/D snoRNP core proteins upon the terminal box C/D core and internal C′/D′ motifs (Cahill et al., 2002; Szewczak et al., 2002). Based upon nucleotide modification experiments, the 15.5 kDa core protein binds exclusively to the terminal box C/D core motif. In vivo crosslinking revealed that core proteins Nop58 and Nop56 are differentially bound to the box C/D and C′/D′ motifs, respectively. Only fibrillarin, the putative methylase, is a component of both the box C/D and C′/D′ RNP complexes. Archaeal box C/D sRNP complexes exhibit a distinctly different protein composition from that of eukaryotic box C/D snoRNPs. Only three core proteins are required for sRNP assembly. Ribosomal protein L7 functions as the archaeal homolog of 15.5 kDa protein and a single archaeal Nop56/58 protein takes the place of the eukaryotic Nop56p/Nop58p protein pair (Kuhn et al., 2002; Omer et al., 2002; Tang et al., 2002). An archaeal homolog of fibrillarin which exhibits high homology with its eukaryotic counterpart is the third archaeal sRNP core protein (Amiri, 1994), and its crystal structure has revealed an S-adenosyl methionine-binding site, consistent with its role as the methylase enzyme (Wang et al., 2000). Recently, a Sulfolobus acidocaldarius box C/D sRNP complex has been assembled in vitro using recombinant core proteins (Omer et al., 2002). This complex was shown to direct 2′-O-methylation from its terminal box C/D core motif. In the work reported here, we have reconstituted a methylation-competent Methanococcus jannaschii box C/D sRNP complex which guides methylation from both the terminal box C/D core and internal C′/D′ RNA motifs. Strikingly, we demonstrate that efficient methylation requires that the terminal box C/D and internal C′/D′ RNP complexes function within the full-length sRNA molecule. RNA:protein binding studies also revealed that the internal C′/D′ motif is distinct in structure from the terminal box C/D core motif. In contrast to the asymmetric eukaryotic snoRNP, the archaeal sRNP complex is symmetric and requires all three core proteins for RNP assembly on the box C/D core and internal C′/D′ motifs. Comparative protein binding studies demonstrated the different RNA-binding capabilities of the L7 and 15.5 kDa protein homologs on the box C/D and C′/D′ motifs. These studies now provide a biochemical rationale for the symmetric versus asymmetric RNP structure of the archaeal and eukaryotic box C/D RNPs, respectively. Results sRNP core proteins L7, Nop56/58 and fibrillarin bind both the terminal box C/D core and internal C′/D′ motif to assemble symmetric RNP complexes Methanococcus jannaschii sR8 was selected as the model sRNA for in vitro sRNP assembly. Figure 1A (upper panel) shows the predicted secondary structure of sR8 with its terminal box C/D core motif and internal box C′/D′ motif. The sR8 sRNP complex was assembled in vitro by incubating radiolabeled sR8 sRNA with recombinant M.jannaschii sRNP core proteins L7, Nop56/58 and fibrillarin (Figure 1A, lower panel). sRNP assembly exhibited an ordered addition of core proteins with L7 binding first, followed by Nop56/58 and fibrillarin. RNP assembly also required elevated temperatures around 70°C (data not shown). Both the required order of protein binding and the elevated temperature for assembly are consistent with recent observations reported for in vitro assembly of an S.acidocaldarius box C/D sRNP complex (Omer et al., 2002). We suspect that the elevated temperature induces conformational changes in the core proteins and/or the sRNA that are critical for protein binding. Figure 1.Archaeal sR8 sRNP assembly requires a defined order of core protein addition and forms symmetric RNP complexes on the terminal box C/D core and internal C′/D′ motifs. (A) The folded structure of M.jannaschii sR8 sRNA with terminal box C/D core and internal C′/D′ motifs is based upon the consensus structure of the snoRNA box C/D motif implied from the crystal structure of the15.5 kDa binding site on the U4 snRNA (Vidovic et al., 2000; Watkins et al., 2000). Lower panel: sR8 sRNP complexes were assembled by incubating L7, Nop56/58 and fibrillarin core proteins, either individually or in different combinations, with 5′-radiolabeled sR8 sRNA. Assembled complexes were resolved on 4% native polyacrylamide gels and RNPs were visualized by autoradiography. Migration positions of the partially assembled (RNP I and RNP II) and complete (RNP III) RNP complexes are indicated. (B and C) The terminal box C/D core and internal C′/D′ RNA half-molecules are derived from the wild-type sR8 full-length sRNA. Lower panels: terminal box C/D core and internal C′/D′ RNPs were assembled by incubating 5′-radiolabeled RNA with the indicated sRNP core proteins. Migration positions of the partial (RNP I and RNP II) and fully assembled RNPs (RNP III) are indicated. The slower migrating complex in lane 2 of (C) is non-specific L7 binding to the RNA at elevated L7 concentrations. Download figure Download PowerPoint Addition of all three core proteins assembled several large complexes (designated RNP III, lane 8). We do not know the distinct composition of each larger RNP but believe that incomplete assembly upon one or both RNA motifs or alternative conformations of the assembled sR8 RNP generates the multiple complexes. Addition of Nop56/58 to the L7:sR8 RNP complex resulted in the formation of only modest amounts of higher-order RNP II (lane 5). In contrast, addition of both Nop56/58 and fibrillarin resulted in assembly of RNP III complexes in greater amounts. We believe that Nop56/58 and fibrillarin bind the L7:sR8 RNP as a dimer despite the fact that Nop56/58 alone exhibits some binding activity. The highly charged nature of Nop56/58 when it is not complexed with fibrillarin causes significant aggregation of the L7:sR8 RNP (our unpublished results) and the resultant loss of soluble RNP complex (lane 5). To study the structure and function of the box C/D and C′/D′ RNPs independently of each other, sR8 ‘halfmers’ were constructed (Figure 1B and C, upper panels). The C/D RNA contains the 5′/3′ terminal stem, boxes C and D, and the guide sequence upstream of box D. The C′/D′ RNA possesses internal boxes C′ and D′ as well as both guide sequences. The second, box D-associated guide sequence and terminal stem were added to the C′/D′ halfmer to facilitate RNP assembly. Smaller RNAs lacking these added elements assembled little RNP complex, suggesting a perturbed C′/D′ structure (data not shown). RNP assembly for each RNA motif exhibited the same order of protein binding as the complete sR8 sRNA, and the fully assembled RNP III complex contained all three sRNP core proteins (Figure 1B and C, lower panels). Analysis of L7 binding to sR8, C/D and C′/D′ RNAs to form RNP I complexes revealed a predominantly slower migrating complex for full-length sR8 compared with the faster migrating complexes for the C/D and C′/D′ halfmers (compare Figure 1A, B and C, lanes 2 of lower panels). This is consistent with L7 binding both C/D and C′/D′ motifs on the full-length sR8 versus a single L7 protein on each halfmer. Nuclease mapping of L7 binding upon sR8 has demonstrated L7 binding to both motifs (data not shown). Interestingly, subsequent binding of the Nop56/58 and fibrillarin proteins to the C/D and C′/D′ halfmers to assemble RNP complexes II and III is less efficient than that observed for the full-length sR8 RNA, particularly for the terminal box C/D core motif. We suspect that the smaller size of the box C/D RNA with a constrained loop structure likely affects the interaction of Nop56/58 and fibrillarin with the RNA, thus assembling less RNP II and III complexes. These results demonstrate that both archaeal RNP complexes are symmetric with respect to core protein composition and require all three core proteins for RNP assembly. Nop56/58 and fibrillarin associate through protein–protein interactions and can bind the C′/D′ motif in the absence of L7 The limited binding of Nop56/58 and fibrillarin to sR8 in the absence of L7 (Figure 1A, lane 7) suggested that these two core proteins working together as a complex can bind box C/D sRNAs. Therefore, Nop56/58 and fibrillarin were incubated with the sR8 half-molecules to assess their binding to the box C/D and C′/D′ motifs (Figure 2A). Despite very limited protein binding, these two core proteins interacted with the internal C′/D′ motif but not the terminal box C/D core motif. Competition studies demonstrated the specificity of this interaction, indicating that each motif presents a unique folded structure for protein binding. Although considerably weaker in binding affinity than when assembled in the complete RNP complex, these results clearly demonstrated the ability of Nop56/58 complexed with fibrillarin to bind the C′/D′ motif independently of L7. This observation has implications for the evolution of RNA-binding capabilities of these core proteins and the contrasting organization of the archaeal and eukaryotic box C/D RNPs (see Discussion). Figure 2.Nop56/58 and fibrillarin interact via protein–protein interactions and can bind the internal C′/D′ motif in the absence of core protein L7. (A) Nop56/58 and fibrillarin core proteins bind specifically to the C′/D′ motif in the absence of L7 core protein. Nop56/58 and fibrillarin were incubated with radiolabeled box C/D or C′/D′ RNA. Assembled RNP complexes were resolved on a native polyacrylamide gel and visualized by autoradiography. Competition experiments included a 1000-fold molar excess of non-radiolabeled box C/D or C′/D′ RNA. (B) Nop56/58 interacts with fibrillarin. sRNP core proteins were incubated in equimolar concentrations. Twenty percent of the protein mixture was removed as the applied sample (A) and the remainder applied to affinity resin. Bound proteins were eluted (E), resolved on 14% SDS–polyacrylamide gels and visualized by Coomassie Blue staining. Incubated protein combinations are indicated above each gel lane. (C and D) Pulldown analysis demonstrates the presence of L7 in the C′/D′ RNP complex. His-tagged fibrillarin, Nop56/58 and L7 were incubated with the C/D and C′/D′ RNAs in various combinations as indicated and fibrillarin was affinity-selected via the His tag. Co-isolated sRNP core proteins (upper panels of C and D) and RNAs (lower panels of C and D) were resolved on polyacrylamide gels and visualized by Coomassie Blue and EtBr staining, respectively. Download figure Download PowerPoint Protein–protein interactions between the sRNP core proteins were explored using in vitro ‘pulldown’ experiments. sRNP core proteins were incubated in pairs, with one of the proteins possessing a His tag, and coprecipitation of the untagged core protein with its His-tagged partner was assessed by SDS–PAGE (Figure 2B). No interaction between L7 and Nop56/58 or between L7 and fibrillarin was noted. However, interaction between fibrillarin and Nop56/58 was observed, consistent with our belief that these two proteins likely bind the sRNA as a complex. The possibility that Nop56/58 and fibrillarin were interacting via contaminating RNA in the recombinant protein preparations was ruled out by pretreating the proteins with RNase (data not shown). The recently reported cocrystal structure of the Archaeoglobus fulgidus Nop56/58–fibrillarin complex (Aittaleb et al., 2003) is consistent with our pulldown experiments and supports the idea that these proteins function in vivo as a dimer. The ability of the Nop56/58–fibrillarin complex to bind the C′/D′ motif in the absence of L7 raised the question as to whether L7 is ultimately displaced upon Nop56/58–fibrillarin binding. This possibility was assessed in RNP ‘pulldown’ experiments using His-tagged fibrillarin for RNP assembly on the RNA halfmers (Figure 2C and D). Co-isolation of the box C/D or C′/D′ RNA using His-tagged fibrillarin required the presence of all three core proteins for efficient RNP assembly. Identical results were obtained for both RNAs, thus demonstrating the symmetry of each RNP complex with respect to core protein content. Of particular note in the control experiments (Figure 2D, lane 8) was the small amount of Nop56/58 co-isolated with His-tagged fibrillarin when L7 was absent. The highly charged character of Nop56/58 in the presence of RNA results in non-specific aggregation and loss of soluble material for gel analysis. This aggregation is not a problem when Nop56/58 and fibrillarin are incubated together in the absence of RNA (Figure 2B). The same aggregation was noted when these two proteins were incubated with RNA for gel shift analysis (Figure 1A). However, the small amounts of radiolabeled RNA used in these experiments minimized these solubility problems. Collectively, these experiments demonstrate the importance of L7 for both box C/D and C′/D′ RNP assembly and the symmetry of the assembled RNP complexes. The terminal box C/D RNP is the minimal methylation complex but efficient methylation requires that the box C/D and C′/D′ RNPs are juxtaposed in the full-length RNA The ability of the in vitro assembled sR8 sRNP to guide the methylation of RNA substrates was assessed by monitoring the incorporation of [3H]CH3 donated from S-adenosyl-L-methionine (SAM) into substrate RNAs. Target substrates of 21 and 16 nucleotides contained sequences complementary to the terminal box C/D RNP guide sequence (D target) and the internal C′/D′ guide sequence (D′ target), respectively (Figure 3A). Determination of TCA-precipitable counts into the target RNAs revealed that both the terminal box C/D core and internal C′/D′ motifs of the assembled sR8 RNP guided methylation of their target RNAs (Figure 3B). Similar to in vitro methylation guided by the S.acidocaldarius box C/D sRNP (Omer et al., 2002), an elevated temperature of 68°C was required for 2′-O-methylation activity (data not shown). Target RNAs possessing a methyl group at the 2′-O-ribose position of the designate nucleotide prior to incubation with the assembled sRNP (D-CH3 and D′-CH3) (Figure 3A) showed no incorporation of [3H]CH3. Incubation of both target RNAs in the same methylation reaction correspondingly increased the incorporation of CH3 into RNA, and electrophoretic analysis of these substrates demonstrated that CH3 incorporation was blocked when either substrate was previously methylated at the designate nucleotide (Figure 3C). At present, we do not know whether both RNAs can be methylated by the sRNP simultaneously or if the binding of one substrate precludes binding of the second. Figure 3.In vitro assembled sR8 sRNP guides site-specific 2′-O-methylation from both terminal box C/D and internal C′/D′ RNPs. Efficient methylation requires juxtapositioning of both RNP complexes in the full-length sRNA. (A) Schematic presentation of M.jannaschii sR8 base paired with D and D′ target RNAs. D-CH3 and D′-CH3 target RNAs possessing a previously 2′-O-methylated sugar at the designate nucleotide are illustrated above. (B) The assembled sR8 sRNP complex guides site-specific methylation of both the D and D′ target RNAs. Assembled sR8 sRNP was incubated at 70°C with the indicated target RNAs (in parentheses) and [3H]SAM. At various times, aliquots of the reaction were collected and TCA precipitated, and [3H]methyl incorporation was measured by scintillation counting. (C) Methylation of D and D′ RNA substrates demonstrates site-specific 2′-O-methylation at designate nucleotides of the target RNAs. Target RNAs in various combinations indicated above the gel were resolved by electrophoresis and 2′-O-methylated RNAs revealed by radiography. (D) Efficient methylation requires juxtapositioned RNPs. RNP complexes assembled upon the box C/D and C′/D′ halfmer RNAs were incubated with their respective target RNAs and assayed for methylation. sR8 (D) target RNA (solid square) is the control level of methylation for the box C/D core RNP when positioned in the full-length sR8 sRNP. Download figure Download PowerPoint The ability of the terminal box C/D core and internal C′/D′ RNP complexes to guide methylation independently was subsequently examined. Strikingly, guided methylation from each assembled half-molecule RNP was adversely affected (Figure 3D). The level of 2′-O- methylation for the terminal box C/D RNP complex was reduced to approximately one-third of that observed for this complex in full-length sR8, while the assembled C′/D′ RNP was completely inactive. Incubation of the two RNP complexes together in trans did not restore the methylation activity of either RNP (Figure 4B). These results revealed two important catalytic features of the archaeal box C/D sRNP complex. First, the minimal RNP structure capable of directing nucleotide-specific 2′-O-methylation, albeit at reduced levels, is the terminal box C/D RNP. Secondly, maximal methylation efficiency of both the box C/D and C′/D′ RNP complexes requires that they be juxtapositioned within the full-length sRNA molecule. Figure 4.Mutation of either the terminal box C/D core or internal C′/D′ motifs in full-length sR8 affects guided 2′-O-methylation of both RNP complexes. (A) Schematic presentation of sR8 sRNA with various mutations in the box C, D, C′ and D′ sequences. For simplicity, the complete terminal helix is not shown but it is present in all mutant sRNAs. (B) Box C/D or C′/D′ mutations affect methylation activity of both RNPs. Methylation efficiencies of wild-type sR8, halfmer RNAs and the full-length mutants are reported as total picomoles of methyl incorporation into the target RNAs in 1 h. Methylation of the control D-CH3 or D′-CH3 target is subtracted as background. Numbers in parentheses indicate the percentage of methylation with respect to activity of the respective RNPs in the full-length sR8 sRNA. (C) RNP complexes are assembled on sR8 box C/D and C′/D′ RNA mutants. sR8 RNAs containing the individual mutants illustrated in (A) were radiolabeled and incubated with the sRNP core proteins as indicated above each gel lane. Assembled complexes were resolved on native polyacrylamide gels and visualized by autoradiography. Download figure Download PowerPoint Mutations in conserved box elements impair methylation from both guide regions The importance of positioning the two RNP complexes within the full-length sRNA for optimal methylation activity was further examined in sR8 mutagenesis experiments. Mutations were made in the critical GA dinucleotides of each box element (Figure 4A). Each mutant sRNA assembled with sRNP core proteins was assayed for methylation activity guided from both motifs (Figure 4B). Mutation of box C reduced methylation of the D target as expected, but also severely disrupted methylation of the D′ target. Similarly, mutation of the C′ sequence affected methylation from both mutated and non-mutated motifs. Mutations in box D and D′ sequences also affected methylation of both target RNAs, but resulted in greater inhibition of methylation guided by the mutated C/D or C′/D′ motifs. These results confirmed the critical nature of positioning box C/D and C′/D′ RNPs within the full-length sRNA for obtaining efficient methylation activity. The effect of the box C/D and C′/D′ mutations upon sRNP assembly was examined to determine whether the loss of methylation activity in the non-mutated motif was due to lack of RNP assembly (Figure 4C). Mutant C, D, C′ and D′ sR8 sRNAs were incubated with the core proteins and assembled RNP complexes resolved on polyacrylamide gels. All four mutant sRNAs bound all three sRNP core proteins and assembled higher-order RNP III complexes. Interestingly, closer inspection of RNP assembly indicated differences in complex formation when the terminal box C/D core motif was mutated as opposed to the internal C′/D′ motif. Binding of L7 to the box C or D mutants resulted in a faster migrating RNP I complex as compared with the C′ and D′ mutants (Figure 4C). The different migration of these RNP I complexes indicated that L7 is primarily binding the C′/D′ motif. However, some methylation is still guided by the box C/D core RNP, particularly with the box C mutant, suggesting limited L7 binding and RNP assembly on the C/D motif. In contrast, the C′ and D′ mutant sRNAs apparently bind two L7 proteins, as evidenced by slower migrating RNP I complexes, and this has been confirmed with L7 titration experiments (data not shown). Despite L7 binding at both RNA motifs and subsequent RNP assembly which mirrors formation of the wild-type RNP complex (Figure 1), methylation is adversely affected at both the mutated C′/D′ and non-mutated C/D motifs. Therefore, observed loss of methylation activity at the non-mutated motif is a consequence of crosstalk between the two RNP complexes and not a failure to assemble an RNP complex. Archaeal L7 binds cooperatively to the box C/D and C′/D′ motifs The importance of juxtaposed RNPs in guided methylation prompted us to examine the possible cooperative nature of box C/D and C′/D′ RNP assembly. Since L7 binding initiates sRNP assembly, the nature of its binding to the two RNA motifs was examined. Titration of sR8 with increasing concentrations of L7 revealed the formation of two RNP complexes (Figure 5A). Footprinting analysis has demonstrated L7 binding to both C/D and C′/D′ motifs (data not shown). The slowest migrating RNP in lane 2 is seen with excess concentrations of protein and results from non-specific binding of L7 to RNA. The strength and cooperative nature of L7 binding to sR8 were demonstrated in filter-binding experiments where L7 binding revealed a sigmoidal binding curve, indicative of cooperative binding (Figure 5B). Hill plot analysis confirmed the cooperative nature of L7 interaction with a determined slope of 1.8 (slopes >1 indicate positive cooperativity). Dissociation constants (Kd) of 9 and 19 nM were calculated. At this time, we are not able to correlate each Kd value with L7 recognition of a specific RNA motif. Figure 5.Core protein L7 exhibits cooperative binding to archaeal sR8 sRNA. (A) L7 protein binds the box C/D and C′/D′ motifs of sR8 sRNA. Increasing concentrations of L7 were incubated with radiolabeled sR8. Assembled RNP complexes were resolved on native polyacrylamide gels and visualized by autoradiography. The slowest migrating L7 RNP (lane 2) is observed only at excess L7 concentrations and represents non-specific L7 binding. (B) L7 association with the sR8 box C/D and C′/D′ motifs is cooperative. Increasing concentrations of L7 were incubated with radiolabeled sR8, assembled RNP complex blotted to nitrocellulose membranes and assembled RNP quantified using a PhosphorImager. The fraction of RNA bound in the RNP complex is plotted as a function of L7 concentration. L7 binding data are also presented as a Hill plot (inset). (C and D) L7 binds to both the terminal box C/D core and internal C′/D′ halfmer RNAs. Increasing concentrations of L7 were incubated with radiolabeled terminal box C/D (C) or internal C′/D′ halfmers (D). Assembled RNP complexes were resolved on native polyacrylamide gels and visualized by autoradiography. Download figure Download PowerPoint L7 bound both sR8 half-molecules (Figure 5C and D) with Kd values of 10 and 54 nM for the box C/D and C′/D′ motifs, respectively. Interestingly, the L7 binding affinities to the two motifs relative to each other were affected when the motifs were no longer positioned in full-length sR8 sRNA. A significant increase in one Kd was noted (9 and 19 nM versus 10 and 54 nM) when the two motifs bound L7 independently. This is consistent with the observed posi DA - 2003/8/1/ PY - 2003/8/1/ DO - 10.1093/emboj/cdg368 VL - 22 IS - 15 SP - 3930-3940 SN - 0261-4189 KW - Archaea KW - box C KW - D RNP KW - ribonucleotide methylation KW - snoRNA KW - sRNA ER - TY - JOUR TI - Efficacy of new inhibitors of ethylene perception in improvement of display life of kalanchoe (Kalanchoe blossfeldiana Poelln.) flowers AU - Kebenei, Z. AU - Sisler, E. C. AU - Winkelmann, T. AU - Serek, M. T2 - Postharvest Biology and Technology DA - 2003/// PY - 2003/// DO - 10.1016/S0925-5214(03000107-8 VL - 30 IS - 2 SP - 169-176 ER - TY - JOUR TI - Effect of 1-octylcyclopropene and 1-methylcyclopropene on vase life of sweet pea (Lathyrus odoratus L.) flowers AU - Kebenei, Z AU - Sisler, EC AU - Winkelmann, T AU - Serek, M T2 - JOURNAL OF HORTICULTURAL SCIENCE & BIOTECHNOLOGY AB - SummarySweet pea (Lathyrus odoratus L.) flowers have a very short postharvest life and are sensitive to exogenous ethylene. Exposure to 1 μl l–1 ethylene enhanced flower senescence and reduced the display life of flowers to less than 3 d. Flowers pre-treated with 200 nl l–1 1-octylcyclopropene (1-OCP) or 200 nl l–1 1-methylcyclopropene (1-MCP) for 6 h at 20°C were protected against ethylene and the display life was prolonged up to almost 7 d. 1-OCP (200 nl l–1) was applied for different exposure times of 0.5, 1, 2, 4, 6, 12 h and at temperatures of 0, 5, 10, 15, 20°C. The optimum conditions for protection against ethylene were ≥4 h exposure and temperature 20°C. No significant differences in display life were observed between 1-OCP and 1-MCP at their optimum treatment concentrations, but both compounds were excellent blockers of ethylene responses in sweet pea flowers. DA - 2003/7// PY - 2003/7// DO - 10.1080/14620316.2003.11511644 VL - 78 IS - 4 SP - 433-436 SN - 2380-4084 ER - TY - JOUR TI - Comparative genetic linkage maps of Eucalyptus grandis, Eucalyptus globulus and their F-1 hybrid based on a double pseudo-backcross mapping approach AU - Myburg, AA AU - Griffin, AR AU - Sederoff, RR AU - Whetten, RW T2 - THEORETICAL AND APPLIED GENETICS DA - 2003/10// PY - 2003/10// DO - 10.1007/s00122-003-1347-4 VL - 107 IS - 6 SP - 1028-1042 SN - 0040-5752 KW - comparative mapping KW - AFLP KW - Eucalyptus KW - transmission ratio distortion KW - genome synteny ER - TY - JOUR TI - Action spectra for UV-light induced RNA-RNA crosslinking in 16S ribosomal RNA in the ribosome AU - Zhirnov, OV AU - Wollenzien, P T2 - PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES AB - UV irradiation induces intramolecular crosslinks in ribosomal RNA in the ribosome. These crosslinks occur between nucleotides distant in primary sequence and they are specific, limited in number and have crosslinking efficiencies sufficient to allow their use in monitoring conformational changes. In this work, the frequency of crosslinking for eight 16S rRNA crosslinks was determined as a function of wavelength of irradiation. For six of the crosslinks, the action spectra correspond to the absorption spectra of at least one of the participating nucleotides. For a crosslink between nucleotides C967 and C1400 the maximum frequency of crosslinking occurs at wavelengths blue-shifted from the absorbance maximum of cytidine and for a crosslink between C1402 and C1501 the maximum frequency of crosslinking is red-shifted. Photoreversal of the crosslinks was also studied by deproteinizing crosslinked RNA under mild conditions and then re-irradiating it with specific wavelengths under conditions in which the crosslinks were reversed but not formed. The different crosslinks exhibit significantly different extents of photoreversal versus wavelength profiles. The differences in the crosslinking action spectra can be accounted for in the absorbance spectra of the nucleotides that are involved in the crosslink as well as by the photoreversal action spectra. DA - 2003/// PY - 2003/// DO - 10.1039/b208677h VL - 2 IS - 6 SP - 688-693 SN - 1474-9092 ER - TY - JOUR TI - The effects of polyethylene glycol on gene expression of developing white spruce somatic embryos AU - Stasolla, C AU - Zyl, L AU - Egertsdotter, U AU - Craig, D AU - Liu, WB AU - Sederoff, RR T2 - PLANT PHYSIOLOGY AB - Somatic embryogenic cultures of white spruce (Picea glauca) represent a valuable system to study molecular mechanisms regulating embryo development because many embryos of defined developmental stages can be generated. The inclusion of polyethylene glycol (PEG) in the maturation medium can improve the number and quality of embryos produced. To learn more about the mechanism of action of PEG, we analyzed transcript profiles of stage-specific embryos matured without (control) or with (PEG treated) PEG. RNA extracted from maturing spruce embryos was analyzed on DNA microarrays containing 2,178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has been documented previously (L. van Zyl, S. von Arnold, P. Bozhkov, Y. Chen, U. Egertsdotter, J. MacKay, R. Sederoff, J. Shen, L. Zelena, D. Clapham [2002] Comp Funct Genomics 3: 306-318). Several pine genes, including the apparent homologs to the Arabidopsis genes ZWILLE, FIDDLEHEAD, FUSCA, and SCARECROW, increased in expression after PEG treatments. These genes are known to be involved in the formation of the embryo body plan and in the control of the shoot and root apical meristems. The increased transcript levels of these genes in immature PEG-treated embryos suggest that PEG may improve the quality of spruce somatic embryos by promoting normal differentiation of the embryonic shoot and root. Changes in the transcript levels of many genes involved in sucrose catabolism and nitrogen assimilation and utilization were also observed between control and PEG-treated embryos. DA - 2003/1// PY - 2003/1// DO - 10.1104/pp.015214 VL - 131 IS - 1 SP - 49-60 SN - 1532-2548 ER - TY - JOUR TI - The C-terminal tail of Arabidopsis 14-3-3 omega functions as an autoinhibitor and may contain a tenth alpha-helix AU - Shen, W AU - Clark, AC AU - Huber, SC T2 - PLANT JOURNAL AB - Summary The eukaryotic regulatory protein 14‐3‐3 is involved in many important plant cellular processes including regulation of nitrate assimilation through inhibition of phosphorylated nitrate reductase (pNR) in darkened leaves. Divalent metal cations (Me 2+ ) and some polyamines interact with the loop 8 region of the 14‐3‐3 proteins and allow them to bind and inhibit pNR in vitro . The role of the highly variant C‐terminal regions of the 14‐3‐3 isoforms in regulation by polycations is not clear. In this study, we carried out structural analyses on the C‐terminal tail of the Arabidopsis 14‐3‐3ω isoform and evaluated its contributions to the inhibition of pNR. Nested C‐terminal truncations of the recombinant 14‐3‐3ω protein revealed that the removal of the C‐terminal tail renders the protein partially Mg 2+ ‐independent in both pNR binding and inhibition of activity, suggesting that the C‐terminus functions as an autoinhibitor. The C‐terminus of 14‐3‐3ω appears to undergo a conformational change in the presence of polycations as demonstrated by its increased trypsin cleavage at Lys‐247. C‐terminal truncation of 14‐3‐3ω at Thr‐255 increased its interaction with antibodies to the C‐terminus of 14‐3‐3ω in non‐denaturing conditions, but not in denaturing conditions, suggesting that the C‐terminal tail contains ordered structures that might be disrupted by the truncation. Circular dichroism (CD) analysis of a C‐terminal peptide, from Trp‐234 to Lys‐249, revealed that the C‐terminal tail might contain a tenth α‐helix, in agreement with the in silico predictions. The function of the putative tenth α‐helix is not clear because substituting two prolyl residues within the predicted helix (E245P/I246P mutant), which prevented the corresponding peptide from adopting a helical conformation, did not affect the inhibition of pNR activity in the presence or absence of Mg 2+ . We propose that in the absence of polycations, access of target proteins to their binding groove in the 14‐3‐3 protein is restricted by the C‐terminus, which acts as part of a gate that opens with the binding of polycations to loop 8. DA - 2003/5// PY - 2003/5// DO - 10.1046/j.1365-313X.2003.01739.x VL - 34 IS - 4 SP - 473-484 SN - 1365-313X KW - 14-3-3 proteins KW - nitrate reductase KW - autoinhibition KW - protein structure KW - protein-protein interaction KW - protein phosphorylation ER - TY - JOUR TI - Speciation of phosphorus in phosphorus-enriched agricultural soils using X-ray absorption near-edge structure spectroscopy and chemical fractionation AU - Beauchemin, S AU - Hesterberg, D AU - Chou, J AU - Beauchemin, M AU - Simard, RR AU - Sayers, DE T2 - JOURNAL OF ENVIRONMENTAL QUALITY AB - Knowledge of phosphorus (P) species in P-rich soils is useful for assessing P mobility and potential transfer to ground water and surface waters. Soil P was studied using synchrotron X-ray absorption near-edge structure (XANES) spectroscopy (a nondestructive chemical-speciation technique) and sequential chemical fractionation. The objective was to determine the chemical speciation of P in long-term-fertilized, P-rich soils differing in pH, clay, and organic matter contents. Samples of three slightly acidic (pH 5.5-6.2) and two slightly alkaline (pH 7.4-7.6) soils were collected from A or B horizons in two distinct agrosystems in the province of Québec, Canada. The soils contained between 800 and 2100 mg total P kg(-1). Distinct XANES features for Ca-phosphate mineral standards and for standards of adsorbed phosphate made it possible to differentiate these forms of P in the soil samples. The XANES results indicated that phosphate adsorbed on Fe- or Al-oxide minerals was present in all soils, with a higher proportion in acidic than in slightly alkaline samples. Calcium phosphate also occurred in all soils, regardless of pH. In agreement with chemical fractionation results, XANES data showed that Ca-phosphates were the dominant P forms in one acidic (pH 5.5) and in the two slightly alkaline (pH 7.4-7.6) soil samples. X-ray absorption near-edge structure spectroscopy directly identified certain forms of soil P, while chemical fractionation provided indirect supporting data and gave insights on additional forms of P such as organic pools that were not accounted for by the XANES analyses. DA - 2003/// PY - 2003/// DO - 10.2134/jeq2003.1809 VL - 32 IS - 5 SP - 1809-1819 SN - 0047-2425 ER - TY - JOUR TI - Polyolefin miscibility: Solid-state NMR investigation of phase behavior in saturated hydrocarbon blends AU - Wolak, J AU - Jia, X AU - Gracz, H AU - Stejskal, EO AU - White, JL AU - Wachowicz, M AU - Jurga, S T2 - MACROMOLECULES AB - Chain-level mixing in polyolefins is investigated for blends of polyisobutylene (PIB) and polyethylene-co-1-butene (PEB). Previous reports suggest that PIB exhibits unusual mixing behavior in certain saturated blends relative to other polyolefins, even though it is immiscible with most. Variable-temperature 1H, 2H, 13C, and 129Xe NMR experiments are used to characterize local PIB chain dynamics in blends with PEB in which the concentration of 1-butene comonomer units is 23 or 66 wt %. Results from 1D and 2D solid-state 13C exchange experiments, 1H relaxation measurements, and 2H line shape analysis indicate that local conformational dynamics of the PIB CH2 group in the polymer backbone increase significantly in blends with PEB copolymers containing 66 wt % butene comonomer (PEB-66). Even though the PEB-66 is a higher Tg polymer than PIB, PIB exhibits a lower effective Tg when the blend is formed relative to its pure state. Similar perturbations are not observed in the PIB/PEB-23 blend, indicating that this blend is not miscible at the chain level. These results are directly relevant to the length scale of glass transitions in polyolefins, indicating that local interchain packing plays an important role in the conformational dynamics that occur within a chain, and are suggestive of local configurational entropy contributions to mixing. Although 1H spin-diffusion experiments could not reveal quantitative length scales of mixing in the these blends due to insufficient contrast between the constituents, 129Xe NMR experiments showed that the PIB/PEB-66 blend was homogeneous on a 50 nm length scale. In agreement with the heterogeneous morphology indicated by the dynamic NMR experiments, 129Xe NMR showed two resolved peaks for the PIB/PEB-23 blend, indicative of phase separation on a 50 nm length scale. The compilation of all the data, most of which was obtained at natural abundance, indicates that the PIB/PEB-66 blend exhibits intimate chain-level mixing on a length scale much shorter than Rg (ca. 8 nm). More importantly, the data show that reduced chain packing constraints occur in the miscible blend and suggest that local entropic contributions are the driving force for miscibility. DA - 2003/7/1/ PY - 2003/7/1/ DO - 10.1021/ma0301449 VL - 36 IS - 13 SP - 4844-4850 SN - 0024-9297 ER - TY - JOUR TI - Photosynthetic acclimation is reflected in specific patterns of gene expression in drought-stressed loblolly pine AU - Watkinson, J. I. AU - Sioson, A. A. AU - Vasquez-Robinet, C. AU - Shukla, M. AU - Kumar, D. AU - Ellis, M. AU - Heath, L. S. AU - Ramakrishnan, N. AU - Chevone, B. AU - Watson, L. T. AU - Van Zyl, L. AU - Egertsdotter, U. AU - Sederoff, R. R. AU - Grene, R. T2 - Plant Physiology DA - 2003/// PY - 2003/// DO - 10.1104/pp.103026914 VL - 133 IS - 4 SP - 1702-1716 ER - TY - JOUR TI - Investigation of enhanced free volume in nanosilica-filled poly(1-trimethylsilyl-1-propyne) by Xe-129 NMR spectroscopy AU - Merkel, TC AU - Toy, LG AU - Andrady, AL AU - Gracz, H AU - Stejskal, EO T2 - MACROMOLECULES AB - The gas permeability of poly[1-(trimethylsilyl)-1-propyne] (PTMSP) containing nanoparticulate fumed silica increases with increasing filler content. This unusual phenomenon is explored using 129Xe NMR spectroscopy to examine the effect of filler on the free volume of the PTMSP host matrix. The 129Xe NMR chemical shift decreases regularly with increasing fumed silica concentration, consistent with an increase in the average size of free volume elements or cavities through which molecular transport can occur. A relationship between the chemical shift and gas permeability in the filled polymer is reported. DA - 2003/1/28/ PY - 2003/1/28/ DO - 10.1021/ma0256690 VL - 36 IS - 2 SP - 353-358 SN - 0024-9297 ER - TY - JOUR TI - Elucidation of the structures of residual and dissolved pine kraft lignins using an HMQC NMR technique AU - Balakshin, MY AU - Capanema, EA AU - Chen, CL AU - Gracz, HS T2 - JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY AB - Comparative studies on the structures of residual and dissolved lignins isolated from pine kraft pulp and pulping liquor have been undertaken using the (1)H-(13)C HMQC NMR technique, GPC, and sugar analysis to elucidate the reaction mechanisms in kraft pulping and the lignin reactivity. A modified procedure for the isolation of enzymatic residual lignins has resulted in an appreciable decrease in protein contaminants in the residual lignin preparations (N content < 0.2%). The very high dispersion of HMQC spectra allows identification of different lignin moieties, which signals appear overlapped in 1D (13)C NMR spectra. Elucidation of the role of condensation reactions indicates that an increase in the degree of lignin condensation during pulping results from accumulation of original condensed lignin moieties rather than from the formation of new alkyl-aryl structures. Among aryl-vinyl type moieties, only stilbene structures are accumulated in lignin in appreciable amounts. Benzyl ether lignin-carbohydrate bonds involving primary hydroxyl groups of carbohydrates have been detected in residual and dissolved lignin preparations. Structures of the alpha-hydroxyacid type have been postulated to be among the important lignin degradation products in kraft pulping. The effect of the isolation method on the lignin structure and differences between the residual and dissolved lignins are discussed. DA - 2003/10/8/ PY - 2003/10/8/ DO - 10.1021/jf034372d VL - 51 IS - 21 SP - 6116-6127 SN - 1520-5118 KW - residual lignin KW - kraft lignin KW - kraft pulping KW - NMR KW - 2D HMQC technique KW - lignin-carbohydrate complex KW - condensation reactions KW - lignin degradation products ER - TY - JOUR TI - Comparative studies on the delignification of pine kraft-anthraquinone pulp with hydrogen peroxide by binucleus Mn(IV) complex catalysis AU - Chen, CL AU - Capanema, EA AU - Gracz, HS T2 - JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY AB - Pine kraft-anthraquinone (kraft-AQ) pulp was bleached in alkaline solution with hydrogen peroxide catalyzed by either [L(1)Mn(IV)(micro-O)(3)Mn(IV)L(1)](PF(6))(2)] (C1) or [LMn(IV)(2)(micro-O)(3)] (ClO(4))(2) (C2) at 60 and 80 degrees C for 120 min with a catalyst charge of 10 ppm on pulp. The resulting bleached pulp was hydrolyzed with cellulase to obtain insoluble and soluble residual lignins. The alkaline bleaching effluents were acidified to precipitate alkaline-soluble lignins. These lignin preparations were then characterized by 2D heteronuclear multiple-quantum coherence (HMQC) NMR spectroscopic techniques. The results showed that biphenyl (5-5) and stilbene structures of the residual lignin in the pulp are preferentially degraded in both the C1- and C2-catalyzed bleachings, whereas beta-O-4, beta-5, and beta-beta structures undergo degradation to a lesser extent. In both cases, the degradation of the residual lignin increased with the increase in reaction temperature from 60 to 80 degrees C. Thus, the result of C1-catalyzed delignification is not in agreement with the observed decrease in the disappearance rate for substrates in the C1-catalyzed oxidation of lignin model compounds with hydrogen peroxide when the reaction temperature is increased from 60 to 80 degrees C. In addition, the resulting residual lignins in the C2-catalyzed bleaching at 80 degrees C are less degraded than the corresponding lignins in the C1-catalyzed bleaching at both 60 and 80 degrees C. Thus, C1 is more effective than C2 as catalyst in the binucleus Mn(IV) complex-catalyzed bleaching of pine kraft-AQ pulp with hydrogen peroxide. DA - 2003/10/8/ PY - 2003/10/8/ DO - 10.1021/jf034507f VL - 51 IS - 21 SP - 6223-6232 SN - 0021-8561 KW - pine kraft-AQ pulp KW - binucleus Mn(IV) complex KW - hydrogen peroxide bleaching KW - catalysis KW - residual lignins KW - alkaline-soluble lignin KW - 2D HMQC NMR KW - epoxidation KW - oxidative cleavage ER - TY - JOUR TI - Calbindin D-28K interacts with Ran-binding protein M: identification of interacting domains by NMR spectroscopy AU - Lutz, W AU - Frank, EM AU - Craig, TA AU - Thompson, R AU - Venters, RA AU - Kojetin, D AU - Cavanagh, J AU - Kumar, R T2 - BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS AB - Calbindin D28K is an EF-hand containing protein that plays a vital role in neurological function. We now show that calcium-loaded calbindin D28K interacts with Ran-binding protein M, a protein known to play a role in microtubule function. Using NMR methods, we show that a peptide, LASIKNR, derived from Ran-binding protein M, interacts with several regions of the calcium-loaded protein including the amino terminus and two other regions that exhibit conformational exchange on the NMR timescale. We suggest that the interaction between calbindin D28K and Ran-binding protein M may be important in calbindin D28K function. DA - 2003/4/18/ PY - 2003/4/18/ DO - 10.1016/S0006-291X(03)00499-6 VL - 303 IS - 4 SP - 1186-1192 SN - 0006-291X ER - TY - JOUR TI - A semi-pilot-scale procedure for isolating and purifying soybean (Glycine max) lectin AU - Fasina, YO AU - Swaisgood, HE AU - Garlich, JD AU - Classen, HL T2 - JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY AB - Availability of gram quantities of purified soybean lectin (SBL) to scientists will foster discovery of novel biomedical applications of the lectin and provide the opportunity to investigate the antinutritional effects of SBL in soybean-consuming food animals and poultry. Therefore, a semi-pilot-scale procedure for isolating and purifying SBL was designed. Defatted soyflour was extracted overnight with 0.9% NaCl at 4 °C. The extract obtained was filtered (0.45 μm membrane) and subjected to affinity chromatography using a column containing N-acetyl-d-galactosamine resin that is specific for SBL. Bound SBL was eluted off the column with 0.14 M galactose solution. The eluent was ultrafiltered (30 kDa), and the resulting solution (SBL and water) was freeze-dried. Electrophoretic analysis and hemagglutination assay revealed that the freeze-dried SBL was similar to Sigma-grade SBL in purity and activity (35 and 33 HU/mg protein, respectively). The procedure yielded 141 mg of SBL/100 g of soyflour. Keywords: Soybean lectin (SBL); Glycine max; lectin purification; scale-up; affinity chromatography; SDS- and native-PAGE DA - 2003/7/30/ PY - 2003/7/30/ DO - 10.1021/jf021125l VL - 51 IS - 16 SP - 4532-4538 SN - 1520-5118 KW - soybean lectin (SBL) KW - Glycine max KW - lectin purification KW - scale-up KW - affinity chromatography KW - SDS- and native-PAGE ER - TY - JOUR TI - Protein-metal ion interactions, stoichiometries and relative affinities determined by on-line size exclusion gel filtration mass spectrometry AU - Benson, LM AU - Kumar, R AU - Cavanagh, J AU - Naylor, S T2 - RAPID COMMUNICATIONS IN MASS SPECTROMETRY AB - Abstract The modulation of metal ions on protein function is well recognized and of paramount importance in protein biochemistry. To date, very few methods allow direct determination of protein‐metal ion interactions, as well as exact stoichiometric binding ratios. In this work we demonstrate the usefulness of two on‐line size exclusion gel filtration mass spectrometry approaches to directly detect protein‐metal ion adducts, as well as determine exact protein‐metal ion stoichiometries. We show that on‐line size exclusion column chromatography (SEC) and rapid in‐line desalting (RILED) coupled to microelectrospray mass spectrometry (μESI‐MS) can be used for such analyses. The SEC approach can be effectively used to both separate proteins in a complex mixture and exchange buffers prior to the electrospray process. While RILED does not allow for protein separation, it provides a much faster high‐throughput desalting procedure than the conventional SEC technique. Specifically, we show that SEC/μESI‐MS and RILED/MS can be used to determine calcium ion binding stoichiometries to a high‐affinity, metal ion binding protein, calbindin D 28K . Furthermore, the same approaches can also be used to determine metal ion binding stoichiometries of low‐affinity metal‐binding proteins such as Spo0F. Copyright © 2003 John Wiley & Sons, Ltd. DA - 2003/// PY - 2003/// DO - 10.1002/rcm.903 VL - 17 IS - 4 SP - 267-271 SN - 0951-4198 ER - TY - JOUR TI - Xanosporic acid, an intermediate in bacterial degradation of the fungal phototoxin cercosporin AU - Mitchell, TK AU - Alejos-Gonzalez, F AU - Gracz, HS AU - Danehower, DA AU - Daub, ME AU - Chilton, WS T2 - PHYTOCHEMISTRY AB - The red fungal perylenequinone phototoxin cercosporin is oxidized by Xanthomonas campestris pv zinniae to a non-toxic, unstable green metabolite xanosporic acid, identified via its lactone as 1,12-bis(2'R-hydroxypropyl)-4,9-dihydroxy-6,7-methylenedioxy-11-methoxy-3-oxaperylen-10H-10-one-2-carboxylic acid. Xanosporolactone was isolated in approximately 2:1 ratio of M:P atropisomers. DA - 2003/3// PY - 2003/3// DO - 10.1016/S0031-9422(02)00517-4 VL - 62 IS - 5 SP - 723-732 SN - 0031-9422 KW - Xanthomonas campestris pv. zinniae KW - bacterial detoxification KW - perylenequinone KW - phototoxin KW - xanosporolactone KW - xanosporic acid ER - TY - JOUR TI - Measurement of image contrast using diffraction enhanced imaging AU - Kiss, M. Z. AU - Sayers, D. E. AU - Zhong, Z. T2 - Physics in Medicine & Biology DA - 2003/// PY - 2003/// VL - 48 IS - 3 SP - 325-340 ER - TY - JOUR TI - Cis-acting regulatory elements in the potato virus X 3 ' non-translated region differentially affect minus-strand and plus-strand RNA accumulation AU - Pillai-Nair, N AU - Kim, KH AU - Hemenway, C T2 - JOURNAL OF MOLECULAR BIOLOGY AB - The 72 nt 3′ non-translated region (NTR) of potato virus X (PVX) RNA is identical in all sequenced PVX strains and contains sequences that are conserved among all potexviruses. Computer folding of the 3′ NTR sequence predicted three stem-loop structures (SL1, SL2, and SL3 in the 3′ to 5′ direction), which generally were supported by solution structure analyses. The importance of these sequence and/or structural elements to PVX RNA accumulation was further analyzed by inoculation of Nicotiana tabacum (NT-1) protoplasts with PVX transcripts containing mutations in the 3′ NTR. Analyses of RNA accumulation by S1 nuclease protection indicated that multiple sequence elements throughout the 3′ NTR were important for minus-strand RNA accumulation. Formation of SL3 was required for accumulation of minus-strand RNA, whereas SL1 and SL2 formation were less important. However, sequences within all of these predicted structures were required for minus-strand RNA accumulation, including a conserved hexanucleotide sequence element in the loop of SL3, and the CU nucleotide in a U-rich sequence within SL2. In contrast, 13 nucleotides that were predicted to reside in SL1 could be deleted without any significant reduction in minus or plus-strand RNA levels. Potential polyadenylation signals (near upstream elements; NUEs) in the 3′ NTR of PVX RNA were more important for plus-strand RNA accumulation than for minus-strand RNA accumulation. In addition, one of these NUEs overlapped with other sequence required for optimal minus-strand RNA levels. These data indicate that the PVX 3′ NTR contains multiple, overlapping elements that influence accumulation of both minus and plus-strand RNA. DA - 2003/2/21/ PY - 2003/2/21/ DO - 10.1016/S0022-2836(02)01369-4 VL - 326 IS - 3 SP - 701-720 SN - 0022-2836 KW - potato virus X KW - RNA replication KW - RNA structure KW - RNA virus KW - stem-loop ER - TY - JOUR TI - In-line desalting mass spectrometry for the study of noncovalent biological complexes AU - Cavanagh, J AU - Benson, LM AU - Thompson, R AU - Naylor, S T2 - ANALYTICAL CHEMISTRY AB - Electrospray ionization-mass spectrometry is becoming widely used as a high-throughput method for the study of biomolecular interactions. It allows for the analysis of complexes from heterogeneous mixtures with high sensitivity and selectivity. In many cases, biomolecules and their complexes must be stored in nonvolatile salt buffers and other solubilizing agents, such as organics or detergents, to maintain stability and integrity. To ensure an efficient electrospray process, desalting and exchanging the biomolecular solutions into a volatile buffer is imperative. Current off-line or on-line methods to accomplish this are time-consuming, frequently disrupt noncovalent interactions, and can result in considerable sample loss. Here we describe a simple, general, and highly efficient, rapid in-line desalting approach using a small gel cartridge to assist in the mass spectrometric analysis of biomolecules and their complexes. Though the method has broad applicability, we focus our analysis on proteins and demonstrate its usefulness by examining protein-metal, protein-protein, protein-DNA, and protein-RNA interactions. The method is shown to provide rapid direct analysis of analyte solutions containing salts, glycerol, organics, and involatile buffers without deleterious effects. DA - 2003/7/15/ PY - 2003/7/15/ DO - 10.1021/ac030182q VL - 75 IS - 14 SP - 3281-3286 SN - 1520-6882 ER - TY - JOUR TI - An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3 AU - Bose, K AU - Pop, C AU - Feeney, B AU - Clark, AC T2 - BIOCHEMISTRY AB - We have examined the enzymatic activity of an uncleavable procaspase-3 mutant (D9A/D28A/D175A), which contains the wild-type catalytic residues in the active site. The results are compared to those for the mature caspase-3. Although at pH 7.5 and 25 °C the Km values are similar, the catalytic efficiency (kcat) is ∼130-fold lower in the zymogen. The mature caspase-3 demonstrates a maximum activity at pH 7.4, whereas the maximum activity of procaspase-3 occurs at pH 8.3. The pKa values of both catalytic groups, H121 and C163, are shifted to higher pH for procaspase-3. We developed limited proteolysis assays using trypsin and V8 proteases, and we show that these assays allow the examination of amino acids in three of five active site loops. In addition, we examined the fluorescence emission of the two tryptophanyl residues in the active site over the pH range of 2.5−9 as well as the response to several quenching agents. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle among loops L4, L2, and L2‘. The pKa values of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate binding pocket, does not appear to be unraveled and solvent-exposed, even at lower pH. DA - 2003/10/28/ PY - 2003/10/28/ DO - 10.1021/bi034998x VL - 42 IS - 42 SP - 12298-12310 SN - 0006-2960 ER - TY - JOUR TI - Genome studies and molecular genetics - The rice genome and comparative genomics of higher plants - Editorial overview AU - Sasaki, T AU - Sederoff, RR T2 - CURRENT OPINION IN PLANT BIOLOGY AB - Rice varieties vary in their capacity for callus induction, growth, and regeneration. We identified the locus and candidate gene which conferred good callus growth and regenerative ability in the rice variety Koshihikari, a notorious poor rice line for genetic transformation. In addition, we succeeded in establishment of a new selectable marker system using the NiR gene for Agrobacterium-mediated transformation of rice, c.v. “Koshihikari.” The locus was mapped onto chromosome 1, and the nearest RFLP marker was C0178. A total of 500 segregating individuals (BC6F2 seeds) were screened for recombination by PCR-based screening and its location narrowed to a 540-kb region that had been sequenced by the International Rice Genome Sequencing Project. One ORF encoded a putative ferredoxin-nitrite reductase (NiR), which has been suggested to be required for callus induction and growth. The growth and regeneration ability of the calli initiated from Koshihikari was improved through integration of the NiR gene from Konansou. Analysis of ORFs and the promoter region of NiR indicated that the promoter region of NiR gene is responsible for growth and regeneration ability of calli in rice, c.v. Koshihikari. We established a NiR selection system for Agrobacterium-mediated transformation in rice, c.v. Koshihikari, by integration of the NiR gene isolated from rice c.v. Konansou. Transgenic rice plants regenerated from selected calli exhibited β-glucuronidase (GUS) activity. A transformation frequency of 9% was obtained. The results indicated that the NiR selection system is devoid of the disadvantages and concerns of using foreign genes (antibiotics and herbicide resistant) for selection. DA - 2003/4// PY - 2003/4// DO - 10.1016/S1369-5266(03)00018-9 VL - 6 IS - 2 SP - 97-100 SN - 1369-5266 ER - TY - JOUR TI - Effect of malic acid on the growth kinetics of Lactobacillus plantarum AU - Passos, FV AU - Fleming, HP AU - Hassan, HM AU - McFeeters, RF T2 - APPLIED MICROBIOLOGY AND BIOTECHNOLOGY DA - 2003/12// PY - 2003/12// DO - 10.1007/s00253-003-1375-7 VL - 63 IS - 2 SP - 207-211 SN - 0175-7598 ER - TY - JOUR TI - Biodefense properties of milk: The role of antimicrobial proteins and peptides AU - Clare, DA AU - Catignani, GL AU - Swaisgood, HE T2 - CURRENT PHARMACEUTICAL DESIGN AB - Mammary fluids, colostrum and milk, deliver nature's first host defense systems upon birth, and these essential liquids are critical for survival of the neonate. The identification and characterization of anti-infectious proteins were among the early scientific discoveries and this group of proteins has long been recognized for promoting health benefits in both newborns and adults. Among the more widely studied are the immunoglobulins, lactoperoxidase, lysozyme, and lactoferrin. Recently, it was shown that alpha--lactalbumin may also function in a protective capacity dependent upon its folding state. Some of these, especially lactoferrin, also display an immunomodulatory role in which case a totally separate cascade of host defense responses is initiated. It was noted that the mechanism of action for this cluster of sentry proteins does vary; thus, this protective strategy provides for a broad range of responsive reactions to infection. Presently, there is a major focus on the discovery of novel peptides that can be generated from existing milk proteins via proteolytic reactions. To date, this substrate list includes alpha--lactalbumin, beta-lactoglobulin, all casein fractions, and lactoferrin. Again, the immunoregulatory effects prompted as a result of the appearance of these peptides are currently being defined. Herein, we review the principal biological properties associated with each of these contributing milk components with a special emphasis on the role of biodefensive milk peptides. We envision future contributions emerging from this research field as an opportunity to develop effective new therapies to be used in treating infectious diseases and promoting health benefits in vivo. DA - 2003/// PY - 2003/// DO - 10.2174/1381612033454874 VL - 9 IS - 16 SP - 1239-1255 SN - 1873-4286 KW - biodefense proteins and peptides KW - antimicrobial milk proteins KW - antimicrobial milk peptides KW - immunoregulatory milk proteins KW - immunoregulatory milk peptides KW - review article ER -