TY - BOOK TI - NOM and MIB, who wins in the competition for activated carbon adsorption sites? AU - Hepplewhite, C. AU - Newcombe, G. AU - Knappe, D.R.U. DA - 2004/// PY - 2004/// VL - 49 SE - 257-265 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-3042814758&partnerID=MN8TOARS ER - TY - ER - TY - CHAP TI - Introduction to Biochemical and Molecular Methods in Toxicology AU - Hodgson, Ernest AU - Leblanc, Gerald A. AU - Meyer, Sharon A. AU - Smart, Robert C. T2 - A Textbook of Modern Toxicology A2 - Hodgson, E. AB - A brief introduction to recently developed molecular and cellular methods currently in use in toxicology, including cell culture techniques (suspension cell culture, monolayer cell culture, indicators of toxicity in cultured cells), molecular cloning techniques (molecular cloning, cDNA and genomic libraries, Northern and Southern blot analysis, PCR, evaluation of gene expression, regulation and function) as well as immunochemical methods. PY - 2004/3/8/ DO - 10.1002/0471646776.ch2 SP - 13-22 PB - John Wiley & Sons, Inc. UR - http://dx.doi.org/10.1002/0471646776.ch2 ER - TY - CHAP TI - Chemical Carcinogenesis AU - Smart, Robert C. T2 - A Textbook of Modern Toxicology A2 - Hodgson, E. AB - General aspects of cancer biology as well as human cancer causes, incidence and mortality rates are discussed. The identification and classification of potential human carcinogens is discussed. The mechanisms through which chemical carcinogens induce cancer are described as is the involvement of oncogenes and tumor suppressor genes in this process. Finally the usefulness and limitation of mutagenicity assays for the identification of carcinogens is discussed. PY - 2004/3/8/ DO - 10.1002/0471646776.ch12 SP - 225-250 PB - John Wiley & Sons, Inc. UR - http://dx.doi.org/10.1002/0471646776.ch12 ER - TY - RPRT TI - Policies for Strengthening Markets for Recyclables: A Worldwide Perspective AU - Loughlin, D.L. AU - Barlaz, M.A. A3 - Environmental Research and Education Foundation DA - 2004/// PY - 2004/// PB - Environmental Research and Education Foundation ER - TY - RPRT TI - State-of-The Practice Review of Bioreactor Landills AU - Benson, C.H. AU - Barlaz, M.A. AU - Lane, D.T. AU - Rawe, J.M. A3 - U.S. Environmental Protection Agency DA - 2004/8// PY - 2004/8// PB - U.S. Environmental Protection Agency ER - TY - RPRT TI - Critical Review of Forest Products Decomposition in Municipal Solid Waste Landfills AU - Barlaz, M.A. A3 - National Council for Air and Stream Improvement DA - 2004/// PY - 2004/// M1 - 872 M3 - Technical Bulletin PB - National Council for Air and Stream Improvement SN - 872 ER - TY - SOUND TI - Research Needs for Sustainable Solid Waste Management: A Perspective from North America AU - Barlaz, M.A. DA - 2004/// PY - 2004/// M3 - Invited lecture ER - TY - SOUND TI - The Application of Life-Cycle Analysis to Integrated Solid Waste Management Planning AU - Barlaz, M.A. DA - 2004/// PY - 2004/// M3 - Invited lecture ER - TY - SOUND TI - The Application of a Life-Cycle Analysis Decision Support Tool to Integrated Waste Management Planning in the United States AU - Barlaz, M.A. DA - 2004/// PY - 2004/// M3 - Invited lecture ER - TY - SOUND TI - Bioreactor Landfills for Refuse Decomposition AU - Barlaz, M.A. DA - 2004/// PY - 2004/// M3 - Invited lecture ER - TY - CONF TI - The Effect of Aging on the Bioavailability of Toluene Sorbed to Municipal Solid Waste Components AU - Chen, Y. AU - Knappe, D.R.U. AU - Barlaz, M.A. T2 - 3rd Intercontinental Landfill Research Symposium C2 - 2004/// CY - Lake Toya, Japan DA - 2004/// PY - 2004/11/30/ ER - TY - CONF TI - Measurement of Decomposition Rates in Landfills AU - Barlaz, M.A. T2 - 3rd Intercontinental Landfill Research Symposium C2 - 2004/// CY - Lake Toya, Japan DA - 2004/// PY - 2004/11/30/ ER - TY - CONF TI - Methanogen community structure during residential food waste decomposition in a simulated landfill AU - Staley, B.F. AU - Barlaz, M.A. AU - de los Reyes, F.L. T2 - Anaerobic Digestion 10th World Conference C2 - 2004/// CY - Montreal, CA DA - 2004/// PY - 2004/8/29/ ER - TY - CONF TI - Trace Organic Compounds in Landfill Gas Produced During the Decomposition of Refuse and Individual Waste Components AU - Barlaz, M.A. AU - Staley, B.F. AU - Cowie, S.J. AU - Hater, G.R. T2 - Anaerobic Digestion 10th World Conference C2 - 2004/// CY - Montreal, CA DA - 2004/// PY - 2004/8/29/ ER - TY - CONF TI - Production of Non-Methane Organic Compounds (NMOCs) During the Decomposition of Refuse and Individual Waste Components AU - Barlaz, M.A. AU - Staley, B.F. AU - Cowie, S.J. AU - Hater, G.R. T2 - Solid Waste Association of North America (SWANA) Landfill Symposium C2 - 2004/// CY - Monterey, CA DA - 2004/// PY - 2004/6/21/ ER - TY - CONF TI - Evaluation of a Biologically Active Cover for Mitigation of Landfill Gas Emissions AU - Barlaz, M.A. AU - Green, R AU - Chanton, J.P. AU - Goldsmith, C.D. AU - Hater, G.R. T2 - WasteTech C2 - 2004/// CY - Dallas, TX DA - 2004/// PY - 2004/5/17/ ER - TY - CONF TI - Accurate and Expedited Diagnosis of Amyloidotic Transthyretin Neuropathy: Proteomic and Genomic Approach AU - Klein, C.J. AU - Kim, C.H. AU - Dyck, P.J. AU - Zeldenrust, S.R. AU - Bergen, H.R. AU - O'Brien, J.F. AU - Nepomuceno, A.I. AU - Butz, M.L. AU - Thibodeau, S.I. AU - Muddiman, D.C. AU - Dyck, P.J. T2 - American Neurological Association, 129th Annual Meeting C2 - 2004/// C3 - Annals of Neurology CY - Toronto, Ontario, Canada DA - 2004/// PY - 2004/10/3/ VL - 56 SP - 29 M1 - S8 ER - TY - JOUR TI - Detection of Genetic Variants of Transthyretin by Liquid Chromatography–Dual Electrospray Ionization Fourier-Transform Ion-Cyclotron-Resonance Mass Spectrometry AU - Nepomuceno, Angelito I AU - Mason, Christopher J AU - Muddiman, David C AU - Bergen, H Robert, III AU - Zeldenrust, Steven R T2 - Clinical Chemistry AB - One of the numerous proteins causing amyloidosis is transthyretin (TTR), a protein usually responsible for the transport of thyroxine and retinol-binding protein. Variants within TTR cause it to aggregate and form insoluble fibers that accumulate in tissue, leading to organ dysfunction.TTR was immunoprecipitated from serum by use of a polyclonal antibody and subsequently reduced with tris(2-carboxyethyl)phosphine. The purified TTR was then analyzed by fast-gradient liquid chromatography-dual-electrospray ionization Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometry. DNA sequencing was performed on all samples used in this study.Because of the inherent limitations in achieving high mass measurement accuracy based on the most abundant isotopic mass, we applied a fitting procedure that allowed determination of monoisotopic mass. Wild-type TTR (mean molecular mass, 13,761 Da) and its associated variant forms could be distinguished because of the high molecular mass accuracy afforded by FT-ICR (< or = 3 ppm) except for instances involving isobaric species or when isotopic distributions overlapped significantly. The [M + 11 H+]11+ charge state for all samples was used to determine the mass accuracies for both wild-type and variant forms of the protein. We correctly assigned seven of seven TTR variants. Moreover, using a combination of proteomic and genomic technologies, we discovered and characterized a previously unreported cis double mutation with a mass only 2 Da different from wild-type TTR. Furthermore, DNA sequencing of the TTR gene for all individuals in this study completely agreed with the intact protein measurements.FT-ICR mass spectrometry has sufficient mass accuracy to identify genetic variants of immunoaffinity-purified TTR. We believe that 91% of known TTR variants could be detected by this technique. DA - 2004/9/1/ PY - 2004/9/1/ DO - 10.1373/clinchem.2004.033274 VL - 50 IS - 9 SP - 1535-1543 LA - en OP - SN - 0009-9147 1530-8561 UR - http://dx.doi.org/10.1373/clinchem.2004.033274 DB - Crossref ER - TY - JOUR TI - Identification of Transthyretin Variants by Sequential Proteomic and Genomic Analysis AU - Bergen, H Robert, III AU - Zeldenrust, Steven R AU - Butz, Malinda L AU - Snow, Denise S AU - Dyck, Peter J AU - Dyck, P James B AU - Klein, Christopher J AU - O’Brien, John F AU - Thibodeau, Stephen N AU - Muddiman, David C T2 - Clinical Chemistry AB - Abstract Background: Transthyretin-associated hereditary amyloidosis (ATTR) is an inherited disease in which variants in the primary structure of transthyretin (TTR; prealbumin) lead to the extracellular polymerization of insoluble protein fibrils, causing organ failure and ultimately death when major organs are involved. We have developed an integrated approach to molecular diagnosis with initial analysis of intact plasma TTR by electrospray ionization mass spectrometry (MS) and referral of positive samples for DNA sequence analysis and real-time PCR to confirm the common Gly6Ser polymorphism. Methods: Samples from 6 patients previously diagnosed with ATTR and from 25 controls with (n = 15) or without (n = 10) polyneuropathy were analyzed in a blinded fashion for the presence of variant TTR. TTR protein was extracted with an immunoaffinity resin from 20 μL of archived plasma samples. The purified TTR was reduced with tris(2-carboxyethyl)phosphine and analyzed by MS. The appearance of two peaks (or a single peak shifted in mass indicative of a homozygous variant), including the wild-type mass of 13 761 Da, was indicative of the presence of a variant, and the individual was referred for DNA sequence analysis. Results: MS analysis of intact reduced TTR correctly identified each of six samples known to contain variant TTR. These results were corroborated by subsequent DNA sequence analysis. Additionally, all Gly6Ser polymorphisms were correctly called based on the +30 mass shift and an equal relative abundance of the +30 polymorphism relative to wild-type TTR. No false-positive results were seen. Conclusions: This referral method eliminates the necessity of sequencing most samples and allows screening for the familial forms of amyloidosis in a broad patient population in a timely fashion. This method correctly identified all previously known variants and also identified a novel variant, Val94Ala. DA - 2004/9/1/ PY - 2004/9/1/ DO - 10.1373/clinchem.2004.033266 VL - 50 IS - 9 SP - 1544-1552 LA - en OP - SN - 0009-9147 1530-8561 UR - http://dx.doi.org/10.1373/clinchem.2004.033266 DB - Crossref ER - TY - JOUR TI - Discovery of Ovarian Cancer Biomarkers in Serum Using NanoLC Electrospray Ionization TOF and FT-ICR Mass Spectrometry AU - Bergen, H. Robert AU - Vasmatzis, George AU - Cliby, William A. AU - Johnson, Kenneth L. AU - Oberg, Ann L. AU - Muddiman, David C. T2 - Disease Markers AB - Treatment of cancer patients is greatly facilitated by detection of the cancer prior to metastasis. One of the obstacles to early cancer detection is the lack of availability of biomarkers with sufficient specificity. With modern differential proteomic techniques, the potential exists to identify high specificity cancer biomarkers. We have delineated a set of protocols for the isolation and identification of serum biomarkers for ovarian cancer that exist in the low molecular weight serum fraction. After isolation of the low molecular weight fraction by ultrafiltration, the potential biomarkers are separated by reversed phase nano liquid chromatography. Detection via TOF or FT-ICR yields a data set for each sample. We compared stage III/IV ovarian cancer serum with postmenopausal age-matched controls. Using bioinformatics tools developed at Mayo, we normalized each sample for intensity and chromatographic alignment. Normalized data sets are subsequently compared and potential biomarkers identified. Several candidate biomarkers were found. One of these contains the sequence of fibrinopeptide-A known to be elevated in many types of cancer including ovarian cancer. The protocols utilized will be examined and would be applicable to a wide variety of cancers or disease states. DA - 2004/// PY - 2004/// DO - 10.1155/2004/797204 VL - 19 IS - 4-5 SP - 239-249 J2 - Disease Markers LA - en OP - SN - 0278-0240 1875-8630 UR - http://dx.doi.org/10.1155/2004/797204 DB - Crossref ER - TY - JOUR TI - Identification and Characterization of Novel, Naturally Processed Measles Virus Class II HLA-DRB1 Peptides AU - Ovsyannikova, Inna G. AU - Johnson, Kenneth L. AU - Muddiman, David C. AU - Vierkant, Robert A. AU - Poland, Gregory A. T2 - Journal of Virology AB - ABSTRACT Previously, we identified a naturally processed and presented measles virus (MV) 19-amino-acid peptide, ASDVETAEGGEIHELLRLQ (MV-P), derived from the phosphoprotein and eluted from the human leukocyte antigen (HLA) class II molecule by using mass spectrometry. We report here the identification of a 14-amino-acid peptide, SAGKVSSTLASELG, derived from the MV nucleoprotein (MV-N) bound to HLA-DRB1*0301. Peripheral blood mononuclear cells (PBMC) from 281 previously vaccinated measles-mumps-rubella II (MMR-II) subjects (HLA discordant) were studied for peptide recognition by T cells. Significant gamma interferon (IFN-γ) responses to MV-P and MV-N peptides were observed in 55.9 and 15.3% of subjects, respectively. MV-P- and MV-N-specific interleukin-4 (IL-4) responses were detected in 19.2 and 23.1%, respectively, of PBMC samples. Peptide-specific cytokine responses and HLA-DRB1 allele associations revealed that, for the MV-P peptide, the allele with the strongest association with both IFN-γ ( P = 0.02) and IL-4 ( P = 0.03) secretion was DRB1*0301. For MV-N, the allele with the strongest association with IFN-γ secretion was DRB1*1501 ( P = 0.04), and the alleles with the strongest associations with IL-4 secretion were DRB1*1103 and DRB1*1303 ( P = 0.01). These results indicate that HLA class II MV proteins can be processed, presented, and identified, and the ability to generate cell-mediated immune responses can be demonstrated. This information is promising for new vaccine design strategies with peptide-based vaccines. DA - 2004/1/1/ PY - 2004/1/1/ DO - 10.1128/jvi.78.1.42-51.2004 VL - 78 IS - 1 SP - 42-51 J2 - JVI LA - en OP - SN - 0022-538X 1098-5514 UR - http://dx.doi.org/10.1128/jvi.78.1.42-51.2004 DB - Crossref ER - TY - JOUR TI - “In Silico” Design of New Uranyl Extractants Based on Phosphoryl-Containing Podands:  QSPR Studies, Generation and Screening of Virtual Combinatorial Library, and Experimental Tests AU - Varnek, A. AU - Fourches, D. AU - Solov'e, V. P. AU - Baulin, V. E. AU - Turanov, A. N. AU - Karandashev, V. K. AU - Fara, D. AU - Katritzky, A. R. T2 - Journal of Chemical Information and Computer Sciences AB - This paper is devoted to computer-aided design of new extractants of the uranyl cation involving three main steps: (i) a QSPR study, (ii) generation and screening of a virtual combinatorial library, and (iii) synthesis of several predicted compounds and their experimental extraction studies. First, we performed a QSPR modeling of the distribution coefficient (logD) of uranyl extracted by phosphoryl-containing podands from water to 1,2-dichloroethane. Two different approaches were used: one based on classical structural and physicochemical descriptors (implemented in the CODESSA PRO program) and another one based on fragment descriptors (implemented in the TRAIL program). Three statistically significant models obtained with TRAIL involve as descriptors either sequences of atoms and bonds or atoms with their close environment (augmented atoms). The best models of CODESSA PRO include its own molecular descriptors as well as fragment descriptors obtained with TRAIL. At the second step, a virtual combinatorial library of 2024 podands has been generated with the CombiLib program, followed by the assessment of logD values using developed QSPR models. At the third step, eight of these hypothetical compounds were synthesized and tested experimentally. Comparison with experiment shows that developed QSPR models successfully predict logD values for 7 of 8 compounds from that “blind test” set. DA - 2004/7// PY - 2004/7// DO - 10.1021/ci049976b VL - 44 IS - 4 SP - 1365-1382 J2 - J. Chem. Inf. Comput. Sci. LA - en OP - SN - 0095-2338 UR - http://dx.doi.org/10.1021/ci049976b DB - Crossref ER - TY - JOUR TI - Factor analysis of pesticide use patterns among pesticide applicators in the Agricultural Health Study AU - Samanic, Claudine AU - Hoppin, Jane A AU - Lubin, Jay H AU - Blair, Aaron AU - Alavanja, Michael C R T2 - Journal of Exposure Science & Environmental Epidemiology AB - Exposure to certain pesticides has been linked with both acute and chronic adverse health outcomes such as neurotoxicity and risk for certain cancers. Univariate analyses of pesticide exposures may not capture the complexity of these exposures since use of various pesticides often occurs simultaneously, and because specific uses have changed over time. Using data from the Agricultural Health Study, a cohort study of 89,658 licensed pesticide applicators and their spouses in Iowa and North Carolina, we employed factor analysis to order to characterize underlying patterns of self-reported exposures to 50 different pesticides. Factor analysis is a statistical method used to explain the relationships between several correlated variables by reducing them to a smaller number of conceptually meaningful, composite variables, known as factors. Three factors emerged for farmer applicators (N=45,074): (1) Iowa agriculture and herbicide use, (2) North Carolina agriculture and use of insecticides, fumigants and fungicides, and (3) older age and use of chlorinated pesticides. The patterns observed for spouses of farmers (N=17,488) were similar to those observed for the farmers themselves, whereas five factors emerged for commercial pesticide applicators (N=4,384): (1) herbicide use, (2) older age and use of chlorinated pesticides, (3) use of fungicides and residential pest treatments, (4) use of animal insecticides, and (5) use of fumigants. Pesticide exposures did not correlate with lifestyle characteristics such as race, smoking status or education. This heterogeneity in exposure patterns may be used to guide etiologic studies of health effects of farmers and other groups exposed to pesticides. DA - 2004/7/28/ PY - 2004/7/28/ DO - 10.1038/sj.jea.7500396 VL - 15 IS - 3 SP - 225-233 J2 - J Expo Sci Environ Epidemiol LA - en OP - SN - 1559-0631 1559-064X UR - http://dx.doi.org/10.1038/sj.jea.7500396 DB - Crossref KW - pesticides KW - farmers KW - custom applicators KW - factor analysis KW - herbicides KW - insecticides KW - fungicides KW - fumigants KW - Iowa KW - North Carolina ER - TY - JOUR TI - Roles of uptake, biotransformation, and target site sensitivity in determining the differential toxicity of chlorpyrifos to second to fourth instar Chironomous riparius (Meigen) AU - Buchwalter, D.B. AU - Sandahl, J.F. AU - Jenkins, J.J. AU - Curtis, L.R. T2 - Aquatic Toxicology AB - Early life stages of aquatic organisms tend to be more sensitive to various chemical contaminants than later life stages. This research attempted to identify the key biological factors that determined sensitivity differences among life stages of the aquatic insect Chironomous riparius. Specifically, second to fourth instar larvae were exposed in vivo to both low and high waterborne concentrations of chlorpyrifos to examine differences in accumulation rates, chlorpyrifos biotransformation, and overall sensitivity among instars. In vitro acetylcholinesterase (AChE) assays were performed with chlorpyrifos and the metabolite, chlorpyrifos-oxon, to investigate potential target site sensitivity differences among instars. Earlier instars accumulated chlorpyrifos more rapidly than later instars. There were no major differences among instars in the biotransformation rates of chlorpyrifos to the more polar metabolites, chlorpyrifos-oxon, and chlorpyridinol (TCP). Homogenate AChE activities from second to fourth instar larvae were refractory to chlorpyrifos, even at high concentrations. In contrast, homogenate AChE activities were responsive in a dose-dependent manner to chlorpyrifos-oxon. In general, it appeared that chlorpyrifos sensitivity differences among second to fourth instar C. riparius were largely determined by differences in uptake rates. In terms of AChE depression, fourth instar homogenates were more sensitive to chlorpyrifos and chlorpyrifos-oxon than earlier instars. However, basal AChE activity in fourth instar larvae was significantly higher than basal AChE activity in second to third instar larvae, which could potentially offset the apparent increased sensitivity to the oxon. DA - 2004/2// PY - 2004/2// DO - 10.1016/j.aquatox.2003.08.004 VL - 66 IS - 2 SP - 149-157 J2 - Aquatic Toxicology LA - en OP - SN - 0166-445X UR - http://dx.doi.org/10.1016/j.aquatox.2003.08.004 DB - Crossref KW - chlorpyrifos KW - acetylcholinesterase KW - biotransformation KW - life stages KW - Chironomous ER - TY - JOUR TI - Estrogen receptor expression in a human primitive neuroectodermal tumor cell line from the cerebral cortex: estrogen stimulates rapid ERK1/2 activation and receptor-dependent cell migration AU - Kirby, Michelle AU - Zsarnovszky, Attila AU - Belcher, Scott M T2 - Biochemical and Biophysical Research Communications AB - Primitive neuroectodermal tumors (PNETs) are the most common form of pediatric brain tumor. Most often these malignant childhood brain tumors arise from neuroepithelial precursor cells in the cerebellum, and less frequently in the cerebral cortex. Because the normal PNET precursor cells from the cerebrum and cerebellum transiently express high levels of estrogen receptors (ERs), we hypothesized that the PNET cells of the cerebrocortical-derived cell line PFSK1 may also express ERs and would be responsive to estrogen. Results of immunoblot studies using ER-specific antiserum indicate that both ERα and ERβ are expressed in PFSK1 cells. The ability of estrogen to rapidly activate MAPK signaling was tested; low physiological concentrations of E2 stimulated ERK1/2 phosphorylation and nuclear translocation within 15 min of exposure. Exogenously added 17β-estradiol (E2) could not stimulate PFSK1 growth, however E2 significantly increased PFSK1 cell migration, suggesting that rapid actions of E2 and ER-mediated processes might contribute to the metastatic phenotype of some PNETs. DA - 2004/7// PY - 2004/7// DO - 10.1016/j.bbrc.2004.05.049 VL - 319 IS - 3 SP - 753-758 J2 - Biochemical and Biophysical Research Communications LA - en OP - SN - 0006-291X UR - http://dx.doi.org/10.1016/j.bbrc.2004.05.049 DB - Crossref KW - ER alpha KW - ER beta KW - ERK1/2 KW - estrogen KW - estrogen receptor KW - growth KW - hormone responsive KW - MAPK KW - migration KW - non-genomic KW - PNET KW - signal transduction ER - TY - JOUR TI - Detection of Purkinje cell loss following drug exposures to developing rat pups using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for calbindin-D28k mRNA expression AU - Ge, Yun AU - Belcher, Scott M. AU - Pierce, Dwight R. AU - Light, Kim E. T2 - Toxicology Letters AB - A technique is described that allows for the identification and quantification of Purkinje cell loss in cerebellum subsequent to developmental toxic exposures. This technique relies upon the extensively validated findings that the Purkinje cell is the only site of expression in the cerebellum of the calcium binding protein calbindin-D28k. Thus, analysis of mRNA expression specific to this protein by comparison to matched controls provides a reliable means of determining whether cell loss has occurred. Purkinje cell loss was induced in rat pups by ethanol exposure on postnatal day (PN) 4 or valproic acid administration to pregnant dams on gestational day 13. Analysis was conducted on PN5 or PN10 and the results compared to parallel groups of pups where the Purkinje cells were counted by traditional means. When compared to matched control rat pups the decrease in calbindin-D28k mRNA expression indicates Purkinje cell loss regardless of whether the cell loss was induced by prenatal valproic acid or postnatal ethanol exposure. The availability of a biochemical alternative to histological cell counting allows for more detailed analyses of the mechanisms of Purkinje cell death induced by these two toxicants, including analyses of the early alterations in signal transduction proteins. DA - 2004/5// PY - 2004/5// DO - 10.1016/j.toxlet.2004.02.003 VL - 150 IS - 3 SP - 325-334 J2 - Toxicology Letters LA - en OP - SN - 0378-4274 UR - http://dx.doi.org/10.1016/j.toxlet.2004.02.003 DB - Crossref ER - TY - JOUR TI - Analytical Performance of a Venturi Device Integrated into an Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometer for Analysis of Nucleic Acids AU - Hawkridge, Adam M. AU - Zhou, Li AU - Lee, Milton L. AU - Muddiman, David C. T2 - Analytical Chemistry AB - A voltage-assisted venturi device modeled after an industrial air amplifier was used to improve the ion transmission efficiency of a 16.2 kDa oligonucleotide and a 53-mer PCR product in the high-pressure region between an electrospray ionization (ESI) emitter and the sampling orifices of two Fourier transform ion cyclotron resonance mass spectrometers (FT-ICR-MS). The venturi device increased the total ion abundance of the oligonucleotide and the PCR product by more than 6-fold relative to the best achievable signal without the device. Furthermore, the average charge states of the oligonucleotide and PCR product shifted from 12.5- to 14.5- and 10.9- to 12.6-, respectively, with the addition of the venturi device. Specific to FT-ICR mass spectrometry, this increase in the charge state directly translates to an increase in theoretical mass resolving power (>10000 full width half-maximum for the results presented here at 7 T). Adduction was still observed while using the device, suggesting that it is "soft" relative to other high-pressure ion focusing methods. DA - 2004/7// PY - 2004/7// DO - 10.1021/ac049677l VL - 76 IS - 14 SP - 4118-4122 J2 - Anal. Chem. LA - en OP - SN - 0003-2700 1520-6882 UR - http://dx.doi.org/10.1021/ac049677l DB - Crossref ER - TY - JOUR TI - Utility of accurate monoisotopic mass measurements to confidently identify lambda exonuclease generated single-stranded amplicons containing 7-deaza analogs by electrospray ionization FT-ICR mass spectrometry AU - Frahm, Jennifer L AU - Mason, Christopher J AU - Muddiman, David C T2 - International Journal of Mass Spectrometry AB - A 53-base pair region on the long arm of chromosome 22 was amplified using PCR with 7-deaza-modified deoxynucleotides. Increased amplification efficiency was achieved by doubling the concentration of the modified deoxynucleotide triphosphates. Incorporation of 7-deaza purines has been previously shown to selectively eliminate fragmentation pathways during gas-phase sequencing of nucleic acids by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and infrared multiphoton dissociation. However, 7-deaza analogs result in significant duplex stability precluding interrogation of the single-stranded species by tandem mass spectrometry. Herein, we demonstrate the use of lambda exonuclease to successfully overcome this problem and are able to obtain single-stranded PCR products containing 7-deaza adenine and guanine nucleobases. Mass accuracy was used as our metric to determine complete incorporation of 7-deaza residues in PCR products>15 kDa; ≤ 3 ppm neutral monoisotopic mass measurement accuracies were routinely achieved. High mass measurement accuracy was obtained using a dual electrospray source and subsequently, using an isotopic fitting algorithm, the best fit between the theoretical and experimental isotopic distributions was determined using a chi-square value. Theoretical isotopic distributions were generated using an average nucleotide (averatide) chemical formula developed herein which was based on the relative frequencies of AT and GC base pairs in the human genome. Single-stranded PCR products were fragmented using SORI-CID and as expected, cleavage at the 7-deaza modified sites was not observed. Collectively, this integrated approach can facilitate top–down sequencing of PCR products by a variety of tandem mass spectrometry methods. DA - 2004/5// PY - 2004/5// DO - 10.1016/j.ijms.2004.02.004 VL - 234 IS - 1-3 SP - 79-87 J2 - International Journal of Mass Spectrometry LA - en OP - SN - 1387-3806 UR - http://dx.doi.org/10.1016/j.ijms.2004.02.004 DB - Crossref ER - TY - JOUR TI - Pendimethalin exposure and cancer risk among pesticide applicators: a report from the U.S.-based agricultural health study AU - Hou, Lifang AU - Lee, Won Jin AU - Rusiecki, Jennifer AU - Hoppin, Jane A. AU - Blair, Aaron AU - Bonner, Matt AU - Lubin, Jay H. AU - Samanic, Claudine AU - Sandler, Dale P. AU - Dosemeci, Mustafa AU - Alavanja, Michael C.R. T2 - Annals of Epidemiology AB - We evaluated the incidence of cancer in relation to pendimethalin exposure among pesticide applicators in the Agricultural Health Study, a prospective cohort study of licensed pesticide applicators in Iowa and North Carolina. Pendimethalin is a widely used herbicide, intended for the control of most annual grasses and certain broadleaf weeds in most crops. This study includes 9089 pendimethalin-exposed and 26,836 nonexposed pesticide applicators. At the time of enrollment (1993–1997), detailed information on pesticide exposure and potential confounders was obtained from a self-administered questionnaire. Poisson regression analysis was used to evaluate the effect of pendimethalin exposure, categorized into tertiles, on cancer incidence, controlling for the effects of potential confounding factors. Significant increased relative risks (RRs) for rectum cancer among subjects with the highest life-time pendimethalin exposure days were observed compared with both non-pendimethalin-exposed (RR = 3.5, 95% CI = 1.3–9.2) and low-pendimethalin-exposed (RR = 9.2, 95% CI = 1.1–81.1) applicators. We also observed an increased RR for rectum cancer when we used intensity-weighted lifetime pendimethalin exposure days as the exposure metric, using non-pendimethalin-exposed applicators as the referent group (RR = 3.5, 95% CI = 1.3–9.2, Ptrend = 0.04). Relative risk for lung cancer was also increased among subjects in the upper half of the highest tertile of life-time pendimethalin exposure days (RR = 5.2, 95% CI = 1.6–17.3, Ptrend = 0.03), compared with low-pendimethalin-exposed applicators. The association was more pronounced for adenocarcinoma of both rectum and lung. Our findings suggest a possible association between pendimethalin exposure and incidence of rectum and lung cancers among pesticide applicators in the U.S.-based Agricultural Health Study. DA - 2004/9// PY - 2004/9// DO - 10.1016/j.annepidem.2004.07.046 VL - 14 IS - 8 SP - 608 J2 - Annals of Epidemiology LA - en OP - SN - 1047-2797 UR - http://dx.doi.org/10.1016/j.annepidem.2004.07.046 DB - Crossref ER - TY - JOUR TI - Phthalate exposure and pulmonary function. AU - Hoppin, Jane A AU - Ulmer, Ross AU - London, Stephanie J T2 - Environmental Health Perspectives AB - Exposure to phthalates is widespread because of their use in plastics, cosmetics, and other consumer products. Phthalate exposure has been associated with adverse respiratory outcomes in children. With urinary phthalate measures, we assessed the association between phthalate exposure and four pulmonary function parameters [forced vital capacity (FVC), forced expiratory volume at 1 sec (FEV1), peak expiratory flow (PEF), and maximum mid-expiratory flow] among the 240 adult Third National Health and Nutrition Examination Survey (NHANES III) participants with urinary phthalate data. Linear regression models controlled for race, age, age squared, standing height, body mass index, cumulative smoking, and current smoking. Monobutyl phthalate (MBP) was significantly associated with decrements in three measures of pulmonary function (FVC, FEV1, PEF) in males but not in females. For a change from the 25th to the 75th percentile in MBP level among men, FEV1 decreased 112 mL (SE = 51, p = 0.03). Monoethyl phthalate (MEP) was associated with lower FVC and FEV1 values in men. Monoethylhexyl phthalate (MEHP), the metabolite of the plasticizer commonly used in medical tubing, was not adversely associated with any of the pulmonary function parameters evaluated. Our results suggest that MBP and MEP, but not MEHP, may influence pulmonary function among adult males. DA - 2004/4// PY - 2004/4// DO - 10.1289/ehp.6564 VL - 112 IS - 5 SP - 571-574 J2 - Environmental Health Perspectives LA - en OP - SN - 0091-6765 1552-9924 UR - http://dx.doi.org/10.1289/ehp.6564 DB - Crossref KW - monobutyl phthalate KW - pulmonary function KW - urine samples ER - TY - JOUR TI - Cancer risk and parental pesticide application in children of Agricultural Health Study participants. AU - Flower, Kori B AU - Hoppin, Jane A AU - Lynch, Charles F AU - Blair, Aaron AU - Knott, Charles AU - Shore, David L AU - Sandler, Dale P T2 - Environmental Health Perspectives AB - Parental exposure to pesticides may contribute to childhood cancer risk. Through the Agricultural Health Study, a prospective study of pesticide applicators in Iowa and North Carolina, we examined childhood cancer risk and associations with parental pesticide application. Identifying information for 17,357 children of Iowa pesticide applicators was provided by parents via questionnaires (1993-1997) and matched against the Iowa Cancer Registry. Fifty incident childhood cancers were identified (1975-1998). Risk of all childhood cancers combined was increased [standardized incidence ratio (SIR) = 1.36; 95% confidence interval (CI), 1.03-1.79]. Risk of all lymphomas combined was also increased (SIR = 2.18; 95% CI, 1.13-4.19), as was risk of Hodgkin's lymphoma (SIR = 2.56; 95% CI, 1.06-6.14). We used logistic regression to explore associations between self-reported parental pesticide application practices and childhood cancer risk. No association was detected between frequency of parental pesticide application and childhood cancer risk. An increased risk of cancer was detected among children whose fathers did not use chemically resistant gloves [odds ratio (OR) = 1.98; 95% CI, 1.05-3.76] compared with children whose fathers used gloves. Of 16 specific pesticides used by fathers prenatally, ORs were increased for aldrin (OR = 2.66), dichlorvos (OR = 2.06), and ethyl dipropylthiocarbamate (OR = 1.91). However, these results were based on small numbers and not supported by prior biologic evidence. Identification of excess lymphoma risk suggests that farm exposures including pesticides may play a role in the etiology of childhood lymphoma. DA - 2004/4// PY - 2004/4// DO - 10.1289/ehp.6586 VL - 112 IS - 5 SP - 631-635 J2 - Environmental Health Perspectives LA - en OP - SN - 0091-6765 1552-9924 UR - http://dx.doi.org/10.1289/ehp.6586 DB - Crossref KW - agricultural workers KW - cancer KW - children KW - occupational exposure KW - pesticides ER - TY - JOUR TI - Altered expression of Bcl2, Bad and Bax mRNA occurs in the rat cerebellum within hours after ethanol exposure on postnatal day 4 but not on postnatal day 9 AU - Ge, Yun AU - Belcher, Scott M. AU - Pierce, Dwight R. AU - Light, Kim E. T2 - Molecular Brain Research AB - Previous studies have demonstrated that ethanol exposure during the vulnerable postnatal (PN) day 4–6 period results in a dose-dependent loss of Purkinje neurons in rats by apoptosis. Although the mechanism of ethanol action and the reasons for Purkinje cell vulnerability are unknown, we hypothesize that during the PN4–6 vulnerable period Purkinje cells are dependent on active trophic factor suppression of apoptosis. Furthermore, ethanol acts to prevent the reception of this trophic signaling resulting in the execution of the apoptotic pathway that includes specific alterations of proteins in the Bcl2 gene family. Ethanol exposure that occurs after this vulnerable period (i.e. PN9) would not be expected to demonstrate alterations in these apoptotic proteins since the Purkinje cells no longer demonstrate vulnerability to ethanol. The current study was undertaken to identify the alterations in mRNA expression for members of the Bcl2-family within the initial hours following ethanol administration on PN4 or PN9. Semi-quantitative reverse transcriptase with polymerase chain reaction (PCR) techniques were used to determine the expression levels of pro-apoptotic factors Bad and Bax, and anti-apoptotic Bcl2 mRNA. Ethanol was administered at four different doses (1.5, 3.0, 4.5, and 6.0 g/kg) on PN4 and analyses of whole cerebellar mRNA was conducted at 1, 4, 6, and 8 h after treatment. Doses greater than 1.5 g/kg produced significant decreases in Bcl2 and significant increases in Bad and Bax mRNA during the 8-h period after treatment. In stark contrast, when ethanol was administered at 3.0 or 6.0 g/kg to PN9 pups, no significant alterations of these apoptotic factors were identified at either 1 or 4 h after treatment. These results are in agreement with and provide further support for our hypothesis that ethanol interrupts the active suppression of apoptosis that is a crucial feature of Purkinje cell vulnerability during this time period. DA - 2004/10// PY - 2004/10// DO - 10.1016/j.molbrainres.2004.06.034 VL - 129 IS - 1-2 SP - 124-134 J2 - Molecular Brain Research LA - en OP - SN - 0169-328X UR - http://dx.doi.org/10.1016/j.molbrainres.2004.06.034 DB - Crossref KW - apoptosis KW - Bcl2 KW - Bad KW - Bax KW - RT-PCR KW - Purkinje KW - cerebellum KW - ethanol KW - mRNA ER - TY - JOUR TI - Alterations of cerebellar mRNA specific for BDNF, p75NTR, and TrkB receptor isoforms occur within hours of ethanol administration to 4-day-old rat pups AU - Ge, Yun AU - Belcher, Scott M AU - Light, Kim E T2 - Developmental Brain Research AB - Developing cerebellar Purkinje cells of the rat are extremely sensitive to ethanol during postnatal days (PN) 4–6, but not at later times during development. Ethanol exposure during this vulnerable window induces rapid apoptotic Purkinje cell death that is hypothesized to result from ethanol inhibition in brain-derived nerve growth factor (BDNF)–TrkB neurotrophic signaling that results in loss of apoptotic suppression. In this study, the effect that different concentrations of ethanol (1.5, 3.0, 4.5 and 6.0 g/kg) have on steady-state mRNA expression of BDNF and different TrkB receptor isoforms in the cerebellum on PN4 was determined at 1, 4, 6, and 8 h after treatment. Significant decreases in mRNA specific for BDNF and TrkB isoforms were detected within 1 h after ethanol administration. No significant alterations in expression of mRNA specific to the low affinity p75NTR receptor were identified. These alterations are concurrent with the PN4 vulnerable period for Purkinje cells since equivalent treatment of PN9 rat pups does not produce significant alterations in mRNA specific to BDNF or TrkB at 4 h after exposure. These results support the hypothesis that ethanol induces a disruption of BDNF–TrkB signaling that results in loss of apoptotic suppression in vulnerable Purkinje cells by growth factor withdrawal. DA - 2004/7// PY - 2004/7// DO - 10.1016/j.devbrainres.2004.04.002 VL - 151 IS - 1-2 SP - 99-109 J2 - Developmental Brain Research LA - en OP - SN - 0165-3806 UR - http://dx.doi.org/10.1016/j.devbrainres.2004.04.002 DB - Crossref KW - Purkinje cell KW - TrkB isoform KW - ethanol exposure KW - RT-PCR KW - mRNA KW - neurotrophin KW - cerebellum KW - toxicity KW - apoptosis KW - BDNF signaling ER - TY - JOUR TI - Spatial, temporal, and cellular distribution of the activated extracellular signal regulated kinases 1 and 2 in the developing and mature rat cerebellum AU - Zsarnovszky, Attila AU - Belcher, Scott M T2 - Developmental Brain Research AB - The extracellular signal regulated kinases 1 and 2 (ERK1/2) are important members of an intracellular signaling cascade that is involved in many aspects of the cellular physiology and development of neurons and glia. ERK1/2 are expressed in many brain regions including the cerebellum; however, their role during cerebellar development is poorly understood. Immunohistochemical approaches using phosphorylation-state specific antiserum that recognizes only the activated-ERK1/2 (pERK) were used to characterize the spatial and temporal patterns of activated-ERK in the developing and adult rat cerebellum. The distribution and cell type-specificity of pERK-immunoreactivity (IR) followed an age-related pattern, with the density of pERK-IR Purkinje cells decreasing between P6 and P15 and increasing at later times. Immunopositive granule cell neurons increased from P6 to P12, became decreased during much of late postnatal cerebellar development, and absent in adults. Co-localization of pERK with glial fibrillary acidic protein or the neuronal marker β-tubulin revealed that activated ERK is present in maturing Purkinje and granule cells, and the soma of Bergmann glia on P4, P10 and P15; pERK was detected in astrocytes on P10 and P15. Associated with weaning, there was a general increase in activated-ERK in all cell types on P22. In adults, pERK-IR was confined to the Purkinje cell layer and scattered cells in the corpus medullare. In summary, a high degree of developmental plasticity was observed in the spatiotemporal distribution of cerebellar pERK-IR suggesting that the ERK-pathway plays a dynamic role in regulating neuronal and glial migration, proliferation and differentiation in the developing cerebellum. In the mature cerebellum, ERK signaling may also mediate postsynaptic information processing. DA - 2004/6// PY - 2004/6// DO - 10.1016/j.devbrainres.2004.03.012 VL - 150 IS - 2 SP - 199-209 J2 - Developmental Brain Research LA - en OP - SN - 0165-3806 UR - http://dx.doi.org/10.1016/j.devbrainres.2004.03.012 DB - Crossref KW - neurotransmitters, modulators, transporters and receptors KW - second messengers and phosphorylation KW - Bergmann glia KW - cerebellum KW - development KW - active EPK1/2 KW - glia KW - granule cell KW - neuron KW - immunohistochemistry KW - kinase KW - MAPK KW - Purkinje cell KW - signal transduction ER - TY - JOUR TI - Analysis of medaka cytochrome P450 3A homotropic and heterotropic cooperativity AU - Kullman, Seth W. AU - Kashiwada, Shosaku AU - Hinton, David E. T2 - Marine Environmental Research AB - We have previously demonstrated that medaka CYP3A is associated with metabolism of several endobiotics including steroids and bile acids. In this study, we demonstrate that medaka CYP3A catalysis exhibits unusual kinetic behaviors consistent with allosteric interaction which cannot be described by hyperbolic kinetic models. Using 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC) and nonylphenol as CYP3A substrates, we describe both homotropic and heterotropic cooperative interactions. Given the role of CYP3A in maintaining the homeostatic balance for numerous endobiotics, enzymatic activation/inhibition by endocrine disruptors (EDCs) represents a putative (non-genomic) mechanism for endocrine disruption. DA - 2004/8// PY - 2004/8// DO - 10.1016/j.marenvres.2004.03.030 VL - 58 IS - 2-5 SP - 469-473 J2 - Marine Environmental Research LA - en OP - SN - 0141-1136 UR - http://dx.doi.org/10.1016/j.marenvres.2004.03.030 DB - Crossref ER - TY - JOUR TI - Absolute Quantification of the Model Biomarker Prostate-Specific Antigen in Serum by LC−MS/MS Using Protein Cleavage and Isotope Dilution Mass Spectrometry AU - Barnidge, David R. AU - Goodmanson, Marcia K. AU - Klee, George G. AU - Muddiman, David C. T2 - Journal of Proteome Research AB - Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC−MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC−MS/MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of −0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC−MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC−MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes. Keywords: LC−MS/MS • absolute quantification • serum • biomarker • prostate-specific antigen • protein DA - 2004/6// PY - 2004/6// DO - 10.1021/pr049963d VL - 3 IS - 3 SP - 644-652 J2 - J. Proteome Res. LA - en OP - SN - 1535-3893 1535-3907 UR - http://dx.doi.org/10.1021/pr049963d DB - Crossref KW - LC-MS/MS KW - absolute quantification KW - serum KW - biomarker KW - prostate-specific antigen KW - protein ER - TY - JOUR TI - Evaluation of a Cleavable Stable Isotope Labeled Synthetic Peptide for Absolute Protein Quantification Using LC−MS/MS AU - Barnidge, David R. AU - Hall, Gregory D. AU - Stocker, Jeanne L. AU - Muddiman, David C. T2 - Journal of Proteome Research AB - Protein cleavage coupled with isotope dilution mass spectrometry (PC-IDMS) has the potential to provide the absolute concentration of a specific protein, or multiple proteins, in complex mixtures. However, PC-IDMS differs from standard IDMS since the internal standard is a different molecule than the analyte at the start of the experiment, more specifically, the internal standard is a peptide and the analyte is a protein prior to cleavage. It is not until after the cleavage process that the stable isotope labeled synthetic peptide has the same physicochemical behavior as the peptide cleaved from the protein. The work presented here evaluates the use of tryptic cleavage sites incorporated into the internal standard synthetic peptide in an attempt to create an internal standard that has cleavage characteristics more similar to the protein being quantified. Results presented here suggest that an internal standard synthetic peptide incorporating internal cleavage sites does not improve the accuracy and precision of the values obtained when performing PC-IDMS. DA - 2004/6// PY - 2004/6// DO - 10.1021/pr034124x VL - 3 IS - 3 SP - 658-661 J2 - J. Proteome Res. LA - en OP - SN - 1535-3893 1535-3907 UR - http://dx.doi.org/10.1021/pr034124x DB - Crossref KW - LC-MS/MS KW - absolute quantification KW - biomarker KW - cleavable internal standard ER - TY - JOUR TI - Analysis of the Low Molecular Weight Fraction of Serum by LC-Dual ESI-FT-ICR Mass Spectrometry:  Precision of Retention Time, Mass, and Ion Abundance AU - Johnson, Kenneth L. AU - Mason, Christopher J. AU - Muddiman, David C. AU - Eckel, Jeanette E. T2 - Analytical Chemistry AB - This study quantifies the experimental uncertainty for LC retention time, mass measurement precision, and ion abundance obtained from replicate nLC-dual ESI−FT-ICR analyses of the low molecular weight fraction of serum. We used ultrafiltration to enrich the <10-kDa fraction of components from the high-abundance proteins in a pooled serum sample derived from ovarian cancer patients. The THRASH algorithm for isotope cluster detection was applied to five replicate nLC-dual ESI-FT-ICR chromatograms. A simple two-level grouping algorithm was applied to the more than 7000 isotope clusters found in each replicate and identified 497 molecular species that appeared in at least four of the replicates. In addition, a representative set of 231 isotope clusters, corresponding to 188 unique molecular species, were manually interpreted to verify the automated algorithm and to set its tolerances. For nLC retention time reproducibility, 95% of the 497 species had a 95% confidence interval of the mean of ±0.9 min or less without the use of chromatographic alignment procedures. Furthermore, 95% of the 497 species had a mass measurement precision of ≤3.2 and ≤6.3 ppm for internally and externally calibrated spectra, respectively. Moreover, 95% of replicate ion abundance measurements, covering an ion abundance range of ∼3 orders of magnitude, had a coefficient of variation of less than 62% without using any normalization functions. The variability of ion abundance was independent of LC retention time, mass, and ion abundance quartile. These measures of analytical reproducibility establish a statistical rationale for differentiating healthy and disease patient populations for the elucidation of biomarkers in the low molecular fraction of serum. DA - 2004/9// PY - 2004/9// DO - 10.1021/ac0497003 VL - 76 IS - 17 SP - 5097-5103 J2 - Anal. Chem. LA - en OP - SN - 0003-2700 1520-6882 UR - http://dx.doi.org/10.1021/ac0497003 DB - Crossref ER - TY - JOUR TI - Androgens Negatively Regulate Forkhead Transcription Factor FKHR (FOXO1) through a Proteolytic Mechanism in Prostate Cancer Cells AU - Huang, Haojie AU - Muddiman, David C. AU - Tindall, Donald J. T2 - Journal of Biological Chemistry AB - The ability of androgens to inhibit apoptosis in both normal and malignant prostatic cells has been well documented. However, the underlying mechanisms are understood poorly. Here we demonstrated that forkhead transcription factor FKHR (FOXO1)-induced death of LNCaP cells was blocked by a synthetic androgen R1881. Androgen treatment also resulted in a reduction in transcriptional activity of FKHR in these cells. Moreover, treatment of LNCaP cells with R1881 led to a decrease in the intact FKHR protein (70 kDa) and an increase in a faster migrating protein band (60 kDa). Androgen-enhanced appearance of the 60-kDa protein was diminished specifically by lysosomal acidic cysteine protease inhibitors. Mass spectrometry analyses of the purified FLAG-tagged 70- and 60-kDa proteins demonstrated that the 60-kDa species is a FKHR protein product that lacks about 120 amino acid residues of the C-terminal end. Mutagenesis of the basic amino acid Arg537 in the protease cleavage region, as suggested by mass spectrometry, abrogated both the androgen-induced accumulation of the 60-kDa product and decrease in cell death induced by FKHR, suggesting that the residue Arg537 is a potential protease cleavage site. Finally, ectopic expression of the first 537 amino acids of FKHR produced an inhibitory effect on transcriptional activity of the intact protein. Together, these results suggest that androgens induce increased activity of an acidic cysteine protease, which in turn cleaves FKHR. This provides a mechanism by which androgens protect prostate cancer cells from the killing effect of FKHR. The ability of androgens to inhibit apoptosis in both normal and malignant prostatic cells has been well documented. However, the underlying mechanisms are understood poorly. Here we demonstrated that forkhead transcription factor FKHR (FOXO1)-induced death of LNCaP cells was blocked by a synthetic androgen R1881. Androgen treatment also resulted in a reduction in transcriptional activity of FKHR in these cells. Moreover, treatment of LNCaP cells with R1881 led to a decrease in the intact FKHR protein (70 kDa) and an increase in a faster migrating protein band (60 kDa). Androgen-enhanced appearance of the 60-kDa protein was diminished specifically by lysosomal acidic cysteine protease inhibitors. Mass spectrometry analyses of the purified FLAG-tagged 70- and 60-kDa proteins demonstrated that the 60-kDa species is a FKHR protein product that lacks about 120 amino acid residues of the C-terminal end. Mutagenesis of the basic amino acid Arg537 in the protease cleavage region, as suggested by mass spectrometry, abrogated both the androgen-induced accumulation of the 60-kDa product and decrease in cell death induced by FKHR, suggesting that the residue Arg537 is a potential protease cleavage site. Finally, ectopic expression of the first 537 amino acids of FKHR produced an inhibitory effect on transcriptional activity of the intact protein. Together, these results suggest that androgens induce increased activity of an acidic cysteine protease, which in turn cleaves FKHR. This provides a mechanism by which androgens protect prostate cancer cells from the killing effect of FKHR. Androgens are critical for proliferation and apoptosis in both normal and malignant prostatic epithelial cell (1Isaacs J.T. Vitam. Horm. 1994; 49: 433-502Google Scholar). Orchitectomy results in extensive apoptosis and involution of the rat ventral prostate and human prostate cancer xenografts (2Kerr J.F. Searle J. Virchows Arch. B Cell Pathol. 1973; 13: 87-102Google Scholar, 3Kyprianou N. English H.F. Isaacs J.T. Cancer Res. 1990; 50: 3748-3753Google Scholar, 4Brandstrom A. Westin P. Bergh A. Cajander S. Damber J.E. Cancer Res. 1994; 54: 3594-3601Google Scholar). Regression of prostatic tumors in patients following androgen ablation therapy is associated with apoptotic death in malignant prostatic epithelium (5Westin P. Stattin P. Damber J.E. Bergh A. Am. J. Pathol. 1995; 146: 1368-1375Google Scholar, 6Cardillo M. Berchem G. Tarkington M.A. Krajewski S. Krajewski M. Reed J.C. Tehan T. Ortega L. Lage J. Gelmann E.P. J. Urol. 1997; 158: 212-216Google Scholar). These findings suggest that androgens function as antiapoptotic factors in both normal and malignant prostatic cells. The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome ten; also known as MMAC1/TEP1) is mutated frequently in a variety of tumors including prostate, brain, and endometrium (7Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Google Scholar, 8Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Google Scholar, 9Li D.M. Sun H. Cancer Res. 1997; 57: 2124-2129Google Scholar). Although the overall prevalence of PTEN mutations in primary prostate cancer is low relative to other tumors (10Ali I.U. Schriml L.M. Dean M. J. Natl. Cancer Inst. 1999; 91: 1922-1932Google Scholar), the gene product is lost frequently in advanced prostate tumors (11McMenamin M.E. Soung P. Perera S. Kaplan I. Loda M. Sellers W.R. Cancer Res. 1999; 59: 4291-4296Google Scholar, 12Huang H. Cheville J.C. Pan Y. Roche P.C. Schmidt L.J. Tindall D.J. J. Biol. Chem. 2001; 276: 38830-38836Google Scholar). Inactivation of PTEN through different mechanisms such as deletion, methylation, or protein degradation has been implicated in progression of a number of tumors (7Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Google Scholar, 8Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Google Scholar, 13Whang Y.E. Wu X. Suzuki H. Reiter R.E. Tran C. Vessella R.L. Said J.W. Isaacs W.B. Sawyers C.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5246-5250Google Scholar, 14Lee J.O. Yang H. Georgescu M.M. Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Google Scholar, 15Vazquez F. Ramaswamy S. Nakamura N. Sellers W.R. Mol. Cell. Biol. 2000; 20: 5010-5018Google Scholar, 16Georgescu M.M. Kirsch K.H. Kaloudis P. Yang H. Pavletich N.P. Hanafusa H. Cancer Res. 2000; 60: 7033-7038Google Scholar, 17Torres J. Pulido R. J. Biol. Chem. 2001; 276: 993-998Google Scholar). Heterozygous disruption results in hyperplasia of the prostate, skin, and colon. Prostate-specific homozygous deletion of PTEN alleles in mice results in metastatic prostate cancer (18Wang S. Gao J. Lei Q. Rozengurt N. Pritchard C. Jiao J. Thomas G.V. Li G. Roy-Burman P. Nelson P.S. Liu X. Wu H. Cancer Cell. 2003; 4: 209-221Google Scholar). PTEN acts as a tumor suppressor protein primarily via its phosphatidylinositol phosphatase activity, which antagonizes the phosphatidylinositol 3-kinase/Akt pathway (19Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Google Scholar, 20Cantley L.C. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4240-4245Google Scholar). Loss of PTEN in prostate cancer cells results in the constitutive activation of the oncoprotein Akt. Moreover, restoration of PTEN expression in PTEN-mutated prostate cancer cell lines abolishes the activation of Akt and subsequently induces cell death (12Huang H. Cheville J.C. Pan Y. Roche P.C. Schmidt L.J. Tindall D.J. J. Biol. Chem. 2001; 276: 38830-38836Google Scholar, 21Davies M.A. Koul D. Dhesi H. Berman R. McDonnell T.J. McConkey D. Yung W.K. Steck P.A. Cancer Res. 1999; 59: 2551-2556Google Scholar, 22Persad S. Attwell S. Gray V. Delcommenne M. Troussard A. Sanghera J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3207-3212Google Scholar, 23Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Google Scholar). Active Akt phosphorylates many downstream proapoptotic proteins, which include Bad, caspase-9, and members of the FOXO subfamily of forkhead transcription factors FKHR (forkhead transcription factors in rhabdomyosarcoma) (FOXO1), FKHRL1 (FOXO3a), and AFX (FOXO4) (24Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Google Scholar, 25Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Google Scholar, 26Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Google Scholar). Phosphorylated forkhead proteins remain inactive in the cytoplasm. The forkhead proteins undergo dephosphorylation due to the inhibition of Akt activity by factors such as PTEN or phosphatidylinositol 3-kinase inhibitors. Activated forkhead proteins translocate from the cytoplasm to the nucleus and subsequently bind to promoters of their target genes (27Nakae J. Barr V. Accili D. EMBO J. 2000; 19: 989-996Google Scholar). Recent studies have suggested that FOXO forkhead transcription factors may play important roles in regulating many cellular functions including proliferation, cell survival, and DNA damage. The members of this family regulate G1 cell cycle progression by modulating expression of the cyclin-dependent kinase inhibitor p27Kip1 and D type cyclins (23Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Google Scholar, 28Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Google Scholar, 29Ramaswamy S. Nakamura N. Sansal I. Bergeron L. Sellers W.R. Cancer Cell. 2002; 2: 81-91Google Scholar). They also mediate transition from M to G1 of the cell cycle by directly regulating expression of mitotic genes such as cyclin B and polo-like kinase (plk) (30Alvarez B. Martinez A.C. Burgering B.M. Carrera A.C. Nature. 2001; 413: 744-747Google Scholar). Forkhead transcription factors affect the expression of several other genes that are involved in the cell cycle including Wip1, EXT1, and cyclin G2 (31Tran H. Brunet A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296: 530-534Google Scholar). Expression of scavenger proteins such as cytosolic catalase and superoxide dismutase and the DNA damage response gene gadd45 are regulated by FOXO forkhead transcription factors, suggesting that these proteins play a role in surveillance of DNA damage (31Tran H. Brunet A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296: 530-534Google Scholar, 32Furukawa-Hibi Y. Yoshida-Araki K. Ohta T. Ikeda K. Motoyama N. J. Biol. Chem. 2002; 277: 26729-26732Google Scholar, 33Nemoto S. Finkel T. Science. 2002; 295: 2450-2452Google Scholar). A number of proapoptotic proteins such as FasL (Fas ligand), the insulin-like growth factor-binding protein-1, Bim, NIP3, and legumain are transcriptionally regulated by members of this subfamily (31Tran H. Brunet A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296: 530-534Google Scholar, 34Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Google Scholar, 35Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Google Scholar, 36Dijkers P.F. Medema R.H. Lammers J.W. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Google Scholar, 37Gilley J. Coffer P.J. Ham J. J. Cell Biol. 2003; 162: 613-622Google Scholar). FOXO1 (FKHR) regulates cell survival in hepatic cells through modulation of gluconeo-genesis by interacting with PGC-1 (38Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Nature. 2003; 423: 550-555Google Scholar). Foxo3a (FKHRL1) knockout female mice exhibit a distinctive ovarian phenotype of global follicular activation leading to oocyte death (39Castrillon D.H. Miao L. Kollipara R. Horner J.W. DePinho R.A. Science. 2003; 301: 215-218Google Scholar). Expression of active FKHR induces death in different types of mammalian cell lines including LNCaP prostate cancer cells (23Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Google Scholar, 35Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Google Scholar). Thus, regulation of FKHR function may be a critical factor for survival of prostate cancer cells. Our laboratory and others have shown previously that androgens act as survival factors by antagonizing PTEN activity in LNCaP cells (40Murillo H. Huang H. Schmidt L.J. Smith D.I. Tindall D.J. Endocrinology. 2001; 142: 4795-4805Google Scholar, 41Li P. Nicosia S.V. Bai W. J. Biol. Chem. 2001; 276: 20444-20450Google Scholar). In the present study, we demonstrate that the FKHR-induced decrease in cell viability and increase in cell death is blocked by androgen treatment, which is concomitant with inhibition of transactivation of FKHR in androgen-treated cells. We further provide evidence that the inhibitory effect of androgens on FKHR is mediated primarily through a proteolytic mechanism. Materials—N-terminal FLAG-tagged pcDNA3 expression plasmids for wild-type FKHR, FKHR(WT), a constitutively active form, FKHR(AAA), and the luciferase reporter construct 3×IRS-Luc were kindly provided by Drs. K. L. Guan and E. D. Tang (35Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Google Scholar). The substitution mutants R537G, R554G, K559G, and P538G were constructed by PCR mutagenesis (Stratagene). Two truncation mutants WT(Δ) and AAA(Δ) were constructed from FKHR(WT) and FKHR(AAA), respectively, by deleting a C-terminal fragment after the residue Arg537. These mutations were verified by sequencing. Construction of the mammalian expression vector for PTEN has been described previously (12Huang H. Cheville J.C. Pan Y. Roche P.C. Schmidt L.J. Tindall D.J. J. Biol. Chem. 2001; 276: 38830-38836Google Scholar). A 2.5-kb fragment of the human FasL promoter was kindly provided by Dr. C. V. Paya (42Holtz-Heppelmann C.J. Algeciras A. Badley A.D. Paya C.V. J. Biol. Chem. 1998; 273: 4416-4423Google Scholar). The fragment of the FasL promoter containing the FKHR response element located between nucleotides -930 and -821 (23Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Google Scholar) was amplified by PCR and subcloned between the MluI and BglII sites of the pGL3-promoter vector (Promega). A polyclonal antibody against FKHR was purchased from Cell Signaling Technology (Beverly, MA). A monoclonal antibody against PTEN (6H2.1) was purchased from Cascade BioScience (Winchester, MA). Erk2 (D-2) monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibodies against α-FLAG were purchased from Sigma. R1881 (DuPont), bicalutamide (Zeneca Pharmaceuticals), and cycloheximide (Sigma) were dissolved in ethanol. LY294002 (Calbiochem), MG132 (Calbiochem), lactacystin (Calbiochem), and ALLnL (Sigma) were prepared in Me2SO (Sigma). Chloroquine (Sigma), ammonium chloride (Sigma), leupeptin (Sigma), and EGTA (Sigma) were prepared in water. Phenylmethylsulfonyl fluoride (Sigma) was dissolved in isopropyl alcohol (Sigma). Cell Lines and Cell Culture—The prostate cancer cell line LNCaP (purchased from the American Type Culture Collection, Manassas, VA) was cultured in RPMI 1640 medium containing 10% fetal bovine serum. In the experiments where LNCaP cells were treated with the synthetic androgen R1881 and/or the anti-androgen bicalutamide, these chemicals were refreshed every 48 h. The immortalized prostatic epithelial cell line BPH-1 was kindly provided by Dr. S. W. Hayward (43Hayward S.W. Dahiya R. Cunha G.R. Bartek J. Deshpande N. Narayan P. In Vitro Cell Dev. Biol. Anim. 1995; 31: 14-24Google Scholar) and cultured in RPMI 1640 medium (Invitrogen) containing 5% fetal bovine serum. Cell Transfections—Transient transfection of LNCaP cells was performed by electroporation as described previously (12Huang H. Cheville J.C. Pan Y. Roche P.C. Schmidt L.J. Tindall D.J. J. Biol. Chem. 2001; 276: 38830-38836Google Scholar). Cells were mixed with DNA in 400 μl of RPMI 1640 medium. The DNA/cell mixture was transferred into a 4-mm cuvette (BTX Inc., San Diego, CA) and electroporated with a 305-V/10-ms pulse using a BTX T820 square wave electroporator (BTX Inc., San Diego, CA). Transfection efficiency was monitored 12 h after transfection with green fluorescent protein (GFP) 1The abbreviations used are: GFP, green fluorescent protein; RT, reverse transcriptase; PSA, prostate-specific antigen; PBS, phosphate-buffered saline; AR, androgen receptor; MS, mass spectrometry. by examining aliquots of cells under a Zeiss fluorescence microscope with a wavelength of 488 nm. Transfection efficiency was determined by the percentage of the green cells in the whole cell population. Routinely, transfection efficiency met 60–90% was used for experiments. Western Blot Analysis—Protein samples were prepared by lysing cells in radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 mm Na3VO4, and 1 mm NaF). Equal amounts of protein (30–80 μg) from cell lysates were denatured in sample buffer, subjected to 4–12% SDS-PAGE gels (Invitrogen), and transferred to nitrocellulose membranes (Bio-Rad). The filters were immunoblotted with specific primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and visualized by enhanced chemilluminescence (Amersham Biosciences). Reverse Transcriptase (RT)-PCR and Northern Blot Analysis—Total RNA was isolated from cells treated with R1881, bicalutamide, or vehicle by TRIzol (Invitrogen). The first-strand cDNAs were synthesized by SuperScript II reverse transcriptase (RT) (Invitrogen). The forward 5′-AAGAGCGTGCCCTACTTCAA-3′ and reverse primer 5′-CAGTTCCTGCTGTCAGACAATC-3′ were used for PCR. Samples of 15 μg of total RNA from each treatment were separated on 1.2% denatured formaldehyde-agarose gels and transferred to nylon membranes (Bio-Rad). Filters were analyzed for expression of FKHR, prostate-specific antigen (PSA), or glyceraldehyde-3-phosphate dehydrogenase by using an isotope-labeled cDNA fragment of the FKHR coding region or probes as described previously (40Murillo H. Huang H. Schmidt L.J. Smith D.I. Tindall D.J. Endocrinology. 2001; 142: 4795-4805Google Scholar, 44Huang H. Reed C.P. Zhang J.S. Shridhar V. Wang L. Smith D.I. Cancer Res. 1999; 59: 2981-2988Google Scholar). Luciferase Reporter Assay—LNCaP cells were harvested after transfections, and cell lysates were prepared by adding lysis buffer directly to the cells on ice. Firefly luciferase and Renilla luciferase activities in cell lysates were determined using a dual luciferase kit (Promega, Madison, WI). Renilla luciferase activities of cells were used as internal controls. Immunofluorescence Chemistry and Confocal Microscopy—For cell viability assays, cells were replated into 6-well plates after transfection. After 12 h, cells were treated with 1 nm R1881 or ethanol for an additional 36 h. Cells were examined under a Zeiss LSM-510 confocal laser microscope. Cells were photographed with a wavelength of 488 nm for GFP. For immunofluorescence chemistry, cells on coverslips (Eppendorf Scientific, Inc., Hamburg, Germany) were washed briefly in 1× PBS and fixed for 20 min in 2% paraformaldehyde (Toussins) in 1× PBS. Cells were permeabilized by incubating with 0.3% Triton X-100 for 15 min. Cells were washed three times in 1× PBS and incubated in blocking buffer (5% goat serum in 1× PBS) for 1 h at room temperature. Cells were incubated with a rabbit anti-AR polyclonal antibody (1:500) and a mouse anti-FLAG monoclonal antibody (1:1000) for 2 h at room temperature. After washing with 1× PBS three times for 5 min each, cells were incubated for 1 h at room temperature with the following secondary antibodies: Alexa Fluor 594 goat anti-rabbit IgG conjugate (Molecular Probes, Inc., Eugene, OR) (1:1000) and Alexa Fluor 488 goat anti-mouse IgG conjugate (Molecular Probes) (1:1000) prepared in blocking buffer for 1 h at room temperature. Coverslips were washed with three changes of 1× PBS for 5 min each and mounted in ProLong (Molecular Probes). Cells were analyzed with a laser-scanning microscope LSM510. Argon ion and HeNe lasers were used to excite fluorescein isothiocyante and Texas Red fluorescence, respectively, and UV laser was used to excite 4′,6-diamidino-2-phenylindole. Cell Death Assay—Nuclear fragment and chromatin condensation were measured as described previously (40Murillo H. Huang H. Schmidt L.J. Smith D.I. Tindall D.J. Endocrinology. 2001; 142: 4795-4805Google Scholar). Briefly, cells treated for 48 h after transfections were collected at 4 °C. Supernatant medium was aspirated, and cells were treated with fixative solution (4% formaldehyde in 1× PBS). Bis-benzimide was added at a final concentration of 1 μg/ml and incubated for 10 min at room temperature. Cell aliquots were placed on slides and viewed under UV and a wavelength of 488 nm (Carl Zeiss Axiophot). Green cells with signs of chromatin condensation and/or nuclear fragmentation (apoptotic) were scored as dead. Purification of FLAG-tagged FKHR Proteins—LNCaP cells grown in 50 150-mm dishes were transfected with FLAG-tagged FKHR by electroporation. All subsequent steps were performed at 4 °C. Cells were washed once with ice-cold 1× PBS and lysed in Triton lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100) plus 1% of protease inhibitor mixture (Sigma). Cells were lysated at 4 °C for 30 min. Whole cell lysates were cleared at 12,000 × g for 20 min. Protein samples were incubated with anti-FLAG M2 affinity resins (Sigma) in protein binding buffer (190 mm NaCl, 50 mm Tris-HCl, pH 7.4, 6 mm EDTA, 2.5% Triton X-100) for 2 h. The beads were washed three times with washing buffer (150 mm NaCl, 10 mm Tris-HCl, pH 9.0, 5 mm EDTA, 0.1% Triton X-100) and once with Tris-buffered saline (10 mm Tris-HCl, pH 7.4, 150 mm NaCl). Proteins were eluted with SDS-PAGE sample buffer (Invitrogen), analyzed by SDS-PAGE, and stained with GelCode blue (Pierce) and Western blotting using anti-FKHR antibody (Cell Signaling). In-gel Trypsin Digestion of FLAG-tagged FKHR—The GelCode blue-stained gel bands were destained with 50% acetonitrile, 50 mm Tris, pH 8.1, for 30 min at room temperature and then reduced with 20 mm dithiothreitol, 50 mm Tris, pH 8.1, for 20 min at 55 °C, followed by alkylation with 40 mm iodoacetamide, 50 mm Tris, pH 8.1, for 20 min at room temperature. Overnight digestion was performed with trypsin (Promega Corp., Madison WI) in 25 mm Tris, pH 8.1, at 37 °C. Peptides were extracted from the gel bands, first with 2% formic acid followed by 70% acetonitrile, 30% water, 0.1% formic acid. Mass Spectrometry—Liquid chromatography-MS/MS analysis of the peptides was performed on a ThermoFinnigan LCQ Deca ion trap mass spectrometer (ThermoFinnigan, San Jose, CA). The LCQ Deca ion trap was coupled with an Applied Biosystems 140D pump, with a 1:100 split into a New Objective ProteoPep C18 PicoFrit column (75 μm × 5.0 cm) mounted on the New Objective PicoView source (New Objective, Woburn, MA). Peptides were loaded onto a 100-μm × 2.0-cm C18 trap and then eluted and chromatographed with a gradient of 4% acetonitrile, 0.1% formic acid to 40% acetonitrile, 0.1% formic acid in 50 min. The LCQ was set to run in data-dependant triple play mode consisting of full scan (400–1900 atomic mass units), zoom scan on most abundant ion, followed by MS/MS mode on that ion. Once a precursor ion was fragmented, it was placed on an exclusion list for 5 min, to avoid repeating tandem MS analysis of the same precursor ion. The MS/MS raw data were converted to DTA files using Bioworks 3.0 and correlated to theoretical fragmentation patterns using the SEQUEST search algorithm with tryptic peptide sequences from the NCBI data base downloaded in February of 2003. Statistics—Statistical analyses were performed by Student's t test. Values of p < 0.05 were considered significant and are presented under “Results.” Androgens Protect LNCaP Cells from Apoptosis Induced by FKHR—Ectopic expression of PTEN in LNCaP cells induces apoptosis, which can be inhibited by androgen treatment (40Murillo H. Huang H. Schmidt L.J. Smith D.I. Tindall D.J. Endocrinology. 2001; 142: 4795-4805Google Scholar, 41Li P. Nicosia S.V. Bai W. J. Biol. Chem. 2001; 276: 20444-20450Google Scholar). Downstream of PTEN, members of the FOXO subfamily of forkhead transcription factors are critical effectors of cell death, since overexpression of active FKHR mediates death in LNCaP cells (23Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Google Scholar). To examine whether androgens affect FKHR-induced death in these cells, we conducted cell viability analyses. In cells without androgen treatment, fewer cells remained viable after being transfected with a constitutively active form of FKHR, FKHR(AAA), in comparison with cells transfected with the empty vector (Fig. 1, A and B, left panel). However, the killing effect of FKHR(AAA) was inhibited by the treatment of cells with 1 nm of the synthetic androgen R1881 (Fig. 1B, right panel). There was only a slight decease in cell viability when cells were transfected with wild-type FKHR, FKHR(WT) (Fig. 1C, left panel). This confirms a previous report that wild-type FKHR is only partially active because of its phosphorylation state (23Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Google Scholar). This slight difference was attenuated following androgen treatment (Fig. 1C, right panel). Co-transfection of FKHR(WT) along with PTEN resulted in a marked decrease in cell viability (Fig. 1D, left panel), which is consistent with the decreased phosphorylation of FKHR and thereby activation of FKHR in cells in the presence of PTEN (23Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Google Scholar). This effect of PTEN was inhibited by androgen treatment (Fig. 1D, right panel). As shown in Fig. 1E, PTEN-induced loss of cell viability was also inhibited markedly by androgens. Next we measured apoptosis by a nuclear condensation and fragmentation assay (40Murillo H. Huang H. Schmidt L.J. Smith D.I. Tindall D.J. Endocrinology. 2001; 142: 4795-4805Google Scholar). The constitutively active FKHR induced more than 15% apoptosis in LNCaP cells, and this was largely inhibited by androgens (Fig. 2). The death of LNCaP cells induced by FKHR(WT) alone or plus PTEN was markedly abrogated by androgen treatment. Androgens also inhibited PTEN-induced cell death (Fig. 2). Importantly, the androgen-mediated effects were reversed completely by co-treatment of cells with an androgen antagonist, bicalutamide, indicating that this is an androgen receptor-dependent event. Therefore, androgens inhibit cell death that is either induced by activated FKHR or potentiated by PTEN. Moreover, the androgen receptor appears to mediate these effects. Transactivation of FKHR Is Diminished by Androgen Action—Akt remains constitutively activated in PTEN-mutated LNCaP cells (7Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Google Scholar, 21Davies M.A. Koul D. Dhesi H. Berman R. McDonnell T.J. McConkey D. Yung W.K. Steck P.A. Cancer Res. 1999; 59: 2551-2556Google Scholar, 22Persad S. Attwell S. Gray V. Delcommenne M. Troussard A. Sanghera J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3207-3212Google Scholar, 45Wu X. Senechal K. Neshat M.S. Whang Y.E. Sawyers C.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15587-15591Google Scholar). This results in FKHR phosphorylation and disruption of its transactivation ability. However, the entire process can be reversed in LNCaP cells by forced expression of PTEN (23Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Google Scholar). Therefore, we were interested to determine whether PTEN-stimulated transactivation of FKHR is regulated by androgens. LNCaP cells were co-transfected transiently with PTEN and a luciferase reporter 3× IRS, which contains three copies of an FKHR response element from the promoter of the insulin-like growth factor-binding protein-1 gene. Ectopic expression of PTEN resulted in an ∼8-fold increase in the transcriptional activity of endogenous FKHR protein in cells without R1881 treatment (Fig. 3A, white column 2 versus 1). However, this effect of PTEN was diminished by androgen treatment (Fig. 3A, black column 2 versus white column 2). Co-expression of PTEN and FKHR in LNCaP cells resulted in a robust increase in activity of the reporter gene (Fig. 3A, white columns 3 and 4) in comparison with the level activated by endogenous FKHR. However, this increase was also inhibited by the treatment of cells with 1 nm R1881 (Fig. 3A, black columns 3 and 4). These results suggest that androgens inhibit transcriptional activities of both the endogenous and transfected FKHR protein. A similar inhibitory effect of androgens on transactivation of FKHR was observed i DA - 2004/1/15/ PY - 2004/1/15/ DO - 10.1074/jbc.M314143200 VL - 279 IS - 14 SP - 13866-13877 J2 - J. Biol. Chem. LA - en OP - SN - 0021-9258 1083-351X UR - http://dx.doi.org/10.1074/jbc.M314143200 DB - Crossref ER - TY - JOUR TI - A method for calculating 16o/18o peptide ion ratios for the relative quantification of proteomes AU - Johnson, Kenneth L. AU - Muddiman, David C. T2 - Journal of the American Society for Mass Spectrometry AB - A method is described for the identification and relative quantification of proteomes using accurate mass tags (AMT) generated by nLC-dual ESI-FT-ICR-MS on a 7T instrument in conjunction with stable isotope labeling using 16O/18O ratios. AMTs were used for putative peptide identification, followed by confirmation of peptide identity by tandem mass spectrometry. For a combined set of 58 tryptic peptides from bovine serum albumin (BSA) and human transferrin, a mean mass measurement accuracy of 1.9 ppm +/-0.94 ppm (CIM99%) was obtained. This subset of tryptic peptides was used to measure 16O/18O ratios of 0.36 +/- 0.09 (CIM99%) for BSA (micro = 0.33) and 1.48 +/- 0.47 (CIM99%) for transferrin (micro = 1.0) using a method for calculating 16O/18O ratios from overlapping isotopic multiplets arising from mixtures of 16O, 18O1, and 18O2 labeled C-termini. The model amino acid averagine was used to calculate a representative molecular formula for estimating and subtracting the contributions of naturally occurring isotopes solely as a function of peptide molecular weight. The method was tested against simulated composite 16O/18O spectra where peptide molecular weight, 16O/18O ratio, 18O1/18O2 ratios, and number of sulfur atoms were varied. Relative errors of 20% or less were incurred when the 16O/18O ratios were less than three, even for peptides where the number of sulfur atoms was over- or under-estimated. These data demonstrate that for biomarker discovery, it is advantageous to label the proteome representing the disease state with 18O; and the method is not sensitive to variations in 18O1/18O2 ratio. This approach allows a comprehensive differentiation of expression levels and tentative identification via AMTs, followed by targeted analysis of over- and under-expressed peptides using tandem mass spectrometry, for applications such as the discovery of disease biomarkers. DA - 2004/4// PY - 2004/4// DO - 10.1016/j.jasms.2003.11.016 VL - 15 IS - 4 SP - 437-445 J2 - J Am Soc Mass Spectrom LA - en OP - SN - 1044-0305 1879-1123 UR - http://dx.doi.org/10.1016/j.jasms.2003.11.016 DB - Crossref ER - TY - JOUR TI - Determination of the relative energies of activation for the dissociation of aromatic versus aliphatic phosphopeptides by ESI-FTICR-MS and IRMPD AU - Flora, Jason W. AU - Muddiman, David C. T2 - Journal of the American Society for Mass Spectrometry AB - Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI-FTICR-MS) coupled with infrared multiphoton dissociation (IRMPD) is potentially a powerful method for rapid phosphopeptide mapping of complex proteolytic digests. The dissociation of deprotonated phosphopeptides by IRMPD is energetically favorable over unmodified deprotonated peptides because of a lower energy of activation and a higher internal energy under identical irradiation conditions. The energies of activation for dissociation are determined for model peptides phosphorylated on an aliphatic side chain (serine) and an aromatic side chain (tyrosine). The determination of phosphorylation location provides important biochemical information identifying the kinase involved in specific phosphorylation mechanisms. The data presented in this manuscript also support the theory that for phosphopeptides, the phosphate moiety's P-O stretch is in direct resonance with the infrared laser (10.6 microm), thus increasing the relative absorptivity of the modified species. A greater extinction coefficient affords more extensive photon absorption and subsequently a greater internal energy at the rapid exchange limit. DA - 2004/1// PY - 2004/1// DO - 10.1016/j.jasms.2003.10.004 VL - 15 IS - 1 SP - 121-127 J2 - J Am Soc Mass Spectrom LA - en OP - SN - 1044-0305 1879-1123 UR - http://dx.doi.org/10.1016/j.jasms.2003.10.004 DB - Crossref ER - TY - JOUR TI - Informed use of proteolytic inhibitors in biomarker discovery AU - Bergen, H. Robert AU - Klug, Michael G. AU - Bolander, Mark E. AU - Muddiman, David C. T2 - Rapid Communications in Mass Spectrometry AB - Rapid Communications in Mass SpectrometryVolume 18, Issue 9 p. 1001-1002 Letter to the Editor Informed use of proteolytic inhibitors in biomarker discovery H. Robert Bergen III, H. Robert Bergen III W. M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, MN 55905, USASearch for more papers by this authorMichael G. Klug, Michael G. Klug W. M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, MN 55905, USA Department of Orthopedic Surgery, Mayo Clinic College of Medicine, Rochester, MN 55905, USASearch for more papers by this authorMark E. Bolander, Mark E. Bolander Department of Orthopedic Surgery, Mayo Clinic College of Medicine, Rochester, MN 55905, USASearch for more papers by this authorDavid C. Muddiman, Corresponding Author David C. Muddiman [email protected] W. M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, MN 55905, USA Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USAMedical Science Building 3-115, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, MN 55905, USA.Search for more papers by this author H. Robert Bergen III, H. Robert Bergen III W. M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, MN 55905, USASearch for more papers by this authorMichael G. Klug, Michael G. Klug W. M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, MN 55905, USA Department of Orthopedic Surgery, Mayo Clinic College of Medicine, Rochester, MN 55905, USASearch for more papers by this authorMark E. Bolander, Mark E. Bolander Department of Orthopedic Surgery, Mayo Clinic College of Medicine, Rochester, MN 55905, USASearch for more papers by this authorDavid C. Muddiman, Corresponding Author David C. Muddiman [email protected] W. M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, MN 55905, USA Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USAMedical Science Building 3-115, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, MN 55905, USA.Search for more papers by this author First published: 01 April 2004 https://doi.org/10.1002/rcm.1439Citations: 5Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1 Bergen HR III, Zeldenrust SR, Naylor S. Amyloid 2003; 10: 190. 2 Riordan JF, Vallee BL. Methods Enzymol. 1972; 25: 449. 3 Brewer C, Riehm J. Anal. Biochem. 1967; 18: 248. 4 Bergen HR III, Vasmatzis G, Cliby WA, Johnson KL, Oberg AL, Muddiman DC. Dis. Markers 2004; in press. Citing Literature Volume18, Issue915 May 2004Pages 1001-1002 ReferencesRelatedInformation DA - 2004/4/23/ PY - 2004/4/23/ DO - 10.1002/rcm.1439 VL - 18 IS - 9 SP - 1001-1002 J2 - Rapid Commun. Mass Spectrom. LA - en OP - SN - 0951-4198 1097-0231 UR - http://dx.doi.org/10.1002/rcm.1439 DB - Crossref ER - TY - CONF TI - Generating Urban Watershed Management Alternatives Using Evolutionary Algorithms AU - Dorn, J. L. AU - Ranjithan, S. T2 - World Water and Environmental Resources Congress 2004 AB - The design of urban drainage networks is complicated by the need to consider a number of issues that conflict and compete with the goal of managing flood impacts. These issues primarily include environmental considerations, but may also include issues such as developable land impacts, system reliability, wetland impacts, aesthetics, etc., some of which may not be modeled explicitly. Modeling to generate alternatives (MGA) is a formal optimization-based technique to find near optimal alternatives that are maximally different from one another with respect to their decision attributes. This paper presents a new evolutionary algorithm (EA)-based technique, the Solution Set Algorithm (SSA), for performing MGA and its application to design problem involving the design a least-cost drainage network using the EPA's Storm Water Management Model (SWMM). C2 - 2004/6/25/ C3 - Critical Transitions in Water and Environmental Resources Management DA - 2004/6/25/ DO - 10.1061/40737(2004)241 PB - American Society of Civil Engineers SN - 9780784407370 UR - http://dx.doi.org/10.1061/40737(2004)241 DB - Crossref ER - TY - JOUR TI - A Procedure for Life-Cycle-Based Solid Waste Management with Consideration of Uncertainty AU - Kaplan, P. Özge AU - Barlaz, Morton A. AU - Ranjithan, S. Ranji T2 - Journal of Industrial Ecology AB - The development of integrated solid‐waste management (SWM) strategies that are efficient with respect to both cost and environmental performance is a complex task. It must incorporate the numerous interrelations among different unit operations in the solid waste system (e.g., collection, recycling, and combustion), and the large number of design parameters that affect estimates of cost and environmental emissions. Uncertainty in design and operational parameters can lead to uncertainty in the estimates of cost and emissions. This article describes an extension of the capability of the Integrated Solid Waste Management Decision Support Tool (ISWM DST) to enable consideration of the effects of uncertainty in input parameters. The uncertainty analysis capability is illustrated using a hypothetical case study of a typical municipality. Results show that increased expenditure does not necessarily result in a reduction in the expected levels of environmental emissions and that some SWM alternatives may be more robust, although deterministic estimates of their expected performances are similar. The uncertainty analysis also facilitates use of the ISWM DST by policy makers responsible for evaluation of the expected effect of SWM practices on, for example, greenhouse‐gas emissions. DA - 2004/9// PY - 2004/9// DO - 10.1162/1088198043630531 VL - 8 IS - 4 SP - 155-172 J2 - Journal of Industrial Ecology LA - en OP - SN - 1088-1980 UR - http://dx.doi.org/10.1162/1088198043630531 DB - Crossref ER - TY - CHAP TI - The ion mobility mass spectrometry method and its application to duplex formation of oligonucleotides and aggregation of proteins AU - Wyttenbach, T AU - Baker, ES AU - Bernstein, SL AU - Ferzoco, A AU - Gidden, J AU - Liu, DF AU - Bowers, MT T2 - Advances in Mass Spectrometry, Vol 16 PY - 2004/// VL - 16 SP - 189-200 PB - SE - UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000226631200011&KeyUID=WOS:000226631200011 ER - TY - JOUR TI - Sodium stabilization of dinucleotide multiplexes in the gas phase AU - Baker, E.S. AU - Gidden, J. AU - Ferzoco, A. AU - Bowers, M.T. T2 - Physical Chemistry Chemical Physics AB - The aggregation and conformations of sodiated dinucleotides were studied in the gas phase. MALDI was used to generate [M − (n − 1)H + nNa]+ ions, yielding single-strand ions having n = 1–3, duplex ions with n = 1–7 and triplex ions with n = 3–10. Collision cross-sections of each sodiated complex were measured in helium using ion mobility based methods and compared to calculated cross-sections of theoretical structures generated by molecular mechanics/dynamics calculations. Three distinct single-strand conformers were observed: one with the nucleobases stacked, one with the planes oriented perpendicular to each other and one with the bases coplanar to each other. One conformer is observed for all of the duplexes except dTG·dTG, dGT·dGT and dTT·dTT (in which two conformers are observed depending on whether the guanine or thymine bases stack). For low values of n, the Na+ ions cluster around the two deprotonated phosphates. However, as n increases, the Na+ ions become more dispersed along the duplex. One conformer is also observed for all of the triplexes. For n = 3–6, three Na+ ions and the three phosphates form a quasi-planar ring with the additional Na+ ions resting above, below and in the middle of the ring. Cytosine and thymine also coordinate to the Na+ ions but adenine and guanine prefer to stack and do not coordinate to the Na+ ions in the ring. The addition of the seventh to tenth Na+ ions breaks the sodium-phosphate ring and the Na+ ions become scattered around the triplex. Differences between experimental and theoretical cross-sections (averaged over the lowest 5 kcal mol−1 structures) of each sodiated complex fell between 1–2%. DA - 2004/// PY - 2004/// DO - 10.1039/b315727j VL - 6 IS - 10 SP - 2786-2795 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-3042559886&partnerID=MN8TOARS ER - TY - JOUR TI - Sequence Dependent Conformations of Glycidyl Methacrylate/Butyl Methacrylate Copolymers in the Gas Phase AU - Baker, E.S. AU - Gidden, J. AU - Simonsick, W.J. AU - Grady, M.C. AU - Bowers, M.T. T2 - The International Journal of Mass Spectrometry AB - Sequence dependent conformations of a series of glycidyl methacrylate/butyl methacrylate (GMA/BMA) copolymers cationized by sodium were analyzed in the gas phase using ion mobility methods. GMA and BMA have the same nominal mass but vary in exact mass by 0.036 Da (CH4 versus O). Matrix assisted laser desorption/ionization (MALDI) was used to form Na + (GMA/BMA) copolymer ions and their collision cross-sections were measured in helium using ion mobility methods. The copolymer sequences from Na + (GMA/BMA)3 to Na + (GMA/BMA)5 (i.e. for the trimer to the pentamer) were studied. Analysis by molecular mechanics/dynamics indicates that each copolymer (regardless of sequence) forms a ring around the sodium ions due to Na + /oxygen electrostatic interactions. However, the structures vary in size, since the epoxy oxygen atoms in the glycidyl groups are attracted to the sodium ions while the carbon-composed butyl groups are not. This allows copolymers with more GMA segments to fold tighter (more spherically) around the sodium ion and have smaller cross-sections than copolymers with a larger amount of BMA segments in the sequence. Due to this cross-sectional difference, the GMA/BMA sequence compositions of the trimer and tetramer could be quantified. © 2004 Elsevier B.V. All rights reserved. DA - 2004/11/15/ PY - 2004/11/15/ DO - 10.1016/j.ijms.2004.04.020 VL - 238 IS - 3 SP - 279-286 UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000225740000009&KeyUID=WOS:000225740000009 KW - glycidyl methacrylate/butyl methacrylate KW - gas phase KW - MALDI KW - ion mobility ER - TY - JOUR TI - Microstructural and Conformational Studies of Polyether Copolymers AU - Jackson, A.T. AU - Scrivens, J.H. AU - Williams, J.P. AU - Baker, E.S. AU - Gidden, J. AU - Bowers, M.T. T2 - The International Journal of Mass Spectrometry AB - A combined structural/conformational study of ethylene oxide/propylene oxide (EO/PO) copolymers has been undertaken. Electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI) methods have been utilised and ESI-tandem mass spectrometry (MS/MS) product ion spectra, including accurate mass measurements, utilised to establish fragmentation pathways. This has enabled end group and sequence information to be obtained. Ion mobility mass spectrometry experimental, along with theoretical, approaches has been used in tandem to probe the gas-phase conformation of selected cationised species from the block and random copolymers. The cross-sections established from these measurements and calculations have been shown to be dependent on molecular weight of the oligomer and radii of the cation but largely independent of the sequence of the ion in the gas-phase. The ion mobility results have been used to aid the understanding of the fragmentation of these copolymers by means of ESI-MS/MS. DA - 2004/11/15/ PY - 2004/11/15/ DO - 10.1016/j.ijms.2004.09.025 VL - 238 IS - 3 SP - 287-297 UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000225740000010&KeyUID=WOS:000225740000010 KW - oligomer KW - ion mobility KW - ESI-MS/MS ER - TY - JOUR TI - Isomeric structural characterization of polyhedral oligomeric silsesquioxanes (POSS) with styryl and epoxy phenyl capping agents AU - Baker, ES AU - Gidden, J AU - Anderson, SE AU - Haddad, TS AU - Bowers, MT T2 - Nano Letters AB - Ion mobility and molecular modeling methods were used to examine the gas-phase structures of sodiated POSS capped with styryl and epoxy phenyl substituents (Na+StyxEp8-xT8). Results were obtained for x = 5−7 and indicated that three distinct isomers with different collision cross-sections were present for each value of x. Theoretical modeling also yielded three different families of structures for each POSS system, and their calculated cross-sections agreed very well with experimental values (<1% difference). For Na+Sty7EpT8, the three families differ in the number of “paired” Sty groups. For Na+Sty6Ep2T8 and Na+Sty5Ep3T8, the three isomers correspond to the three different ways the Ep groups can be positioned on the POSS cage. DA - 2004/// PY - 2004/// DO - 10.1021/nl049957g VL - 4 IS - 5 SP - 779-785 UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000221410000004&KeyUID=WOS:000221410000004 ER - TY - JOUR TI - Duplex formation and the onset of helicity in poly d(CG)(n) oligonucleotides in a solvent-free environment AU - Gidden, J AU - Ferzoco, A AU - Baker, ES AU - Bowers, MT T2 - Journal of the American Chemical Society AB - The gas-phase conformations of a series of cytosine/guanine DNA duplexes were examined by ion mobility and molecular dynamics methods. Deprotonated duplex ions were formed by electrospray ionization, and their collision cross sections measured in helium were compared to calculated cross sections of theoretical models generated by molecular dynamics. The 4-mer (dCGCG) and 6-mer (dCGCGCG) duplexes were found to have globular conformations. Globular and helical structures were observed for the 8-mer (dCGCGCGCG) duplex, with the globular form being the more favored conformer. For the 10-mer (dCGCGCGCGCG), 14-mer (dCGCGCGCGCGCGCG), and 18-mer (dCGCGCGCGCGCGCGCGCG) duplexes, only helical structures were observed in the ion mobility measurements. Theory predicts that the helical structures are less stable than the globular forms in the gas phase and should collapse into the globular form given enough time. However, molecular dynamics simulations at 300 K indicate the helical structures are stable in aqueous solution and will retain their conformations for a limited time in the gas phase. The presence of helical structures in the ion mobility experiments indicates that the duplexes retain "solution structures" in the gas phase on the millisecond time scale. DA - 2004/// PY - 2004/// DO - 10.1021/ja046433+ VL - 126 IS - 46 SP - 15132-15140 UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000225233600035&KeyUID=WOS:000225233600035 ER - TY - JOUR TI - Duplex formation and the onset of helicity in oligonucleotides. AU - Gidden, J AU - Baker, ES AU - Ferzoco, A AU - Bowers, MT T2 - Abstracts of Papers of the American Chemical Society DA - 2004/// PY - 2004/// VL - 227 SP - U258 UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000223655701333&KeyUID=WOS:000223655701333 ER - TY - JOUR TI - Diastereomer assignment of an olefin-linked bis-paracyclophane by ion mobility mass spectrometry AU - Baker, ES AU - Hong, JW AU - Gidden, J AU - Bartholomew, GP AU - Bazan, GC AU - Bowers, MT T2 - Journal of the American Chemical Society AB - trans-1,2-Bis([2.2]paracyclophanyl)ethene (1) exists as a pair of diastereomers whose conformations, and thus effective collision cross sections, are quite different. The two forms can be obtained by different transition metal-catalyzed reactions. To assign meso and racemic structures, a novel method is reported in which experimental gas-phase ion mobility data are compared with theoretical structures obtained from molecular mechanics calculations. DA - 2004/// PY - 2004/// DO - 10.1021/ja039486k VL - 126 IS - 20 SP - 6255-6257 UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000221526800027&KeyUID=WOS:000221526800027 ER - TY - JOUR TI - Seed treatment and its implication for fungicide exposure assessment AU - White, Katrina E AU - Hoppin, Jane A T2 - Journal of Exposure Science & Environmental Epidemiology DA - 2004/5// PY - 2004/5// DO - 10.1038/sj.jea.7500312 VL - 14 IS - 3 SP - 195-203 J2 - J Expo Sci Environ Epidemiol LA - en OP - SN - 1559-0631 1559-064X UR - http://dx.doi.org/10.1038/sj.jea.7500312 DB - Crossref KW - pesticides KW - fungicides KW - seed treatment KW - exposure assessment KW - agricultural epidemiology ER - TY - JOUR TI - Reporting pesticide assessment results to farmworker families: development, implementation, and evaluation of a risk communication strategy. AU - Quandt, Sara A AU - Doran, Alicia M AU - Rao, Pamela AU - Hoppin, Jane A AU - Snively, Beverly M AU - Arcury, Thomas A T2 - Environmental Health Perspectives AB - The collection of environmental samples presents a responsibility to return information to the affected participants. Explaining complex and often ambiguous scientific information to a lay audience is a challenge. As shown by environmental justice research, this audience frequently has limited formal education, increasing the challenge for researchers to explain the data collected, the risk indicated by the findings, and action the affected community should take. In this study we describe the development and implementation of a risk communication strategy for environmental pesticide samples collected in the homes of Latino/a migrant and seasonal farmworkers in a community-based participatory research project. The communication strategy was developed with community input and was based on face-to-face meetings with members of participating households. Using visual displays of data effectively conveyed information about individual household contamination and placed it in the context of community findings. The lack of national reference data and definitive standards for action necessitated a simplified risk message. We review the strengths and weaknesses of such an approach and suggest areas for future research in risk communication to communities affected by environmental health risks. DA - 2004/4// PY - 2004/4// DO - 10.1289/ehp.6754 VL - 112 IS - 5 SP - 636-642 J2 - Environmental Health Perspectives LA - en OP - SN - 0091-6765 1552-9924 UR - http://dx.doi.org/10.1289/ehp.6754 DB - Crossref KW - agriculture KW - children KW - community KW - environmental justice KW - exposure KW - health communication KW - house dust KW - Latino/a ER - TY - JOUR TI - Phthalate exposure and pulmonary function AU - Hoppin, J.A. AU - Ulmer, R. AU - London, S.J. T2 - Environmental Health Perspectives DA - 2004/// PY - 2004/// VL - 112 IS - 5 SP - 571-574 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-11844285846&partnerID=MN8TOARS ER - TY - JOUR TI - Health Effects of Chronic Pesticide Exposure: Cancer and Neurotoxicity AU - Alavanja, Michael C.R. AU - Hoppin, Jane A. AU - Kamel, Freya T2 - Annual Review of Public Health AB - Pesticides are widely used in agricultural and other settings, resulting in continuing human exposure. Epidemiologic studies indicate that, despite premarket animal testing, current exposures are associated with risks to human health. In this review, we describe the routes of pesticide exposures occurring today, and summarize and evaluate the epidemiologic studies of pesticide-related carcinogenicity and neurotoxicity in adults. Better understanding of the patterns of exposure, the underlying variability within the human population, and the links between the animal toxicology data and human health effects will improve the evaluation of the risks to human health posed by pesticides. Improving epidemiology studies and integrating this information with toxicology data will allow the human health risks of pesticide exposure to be more accurately judged by public health policy makers. DA - 2004/4// PY - 2004/4// DO - 10.1146/annurev.publhealth.25.101802.123020 VL - 25 IS - 1 SP - 155-197 J2 - Annu. Rev. Public Health LA - en OP - SN - 0163-7525 1545-2093 UR - http://dx.doi.org/10.1146/annurev.publhealth.25.101802.123020 DB - Crossref KW - pesticides KW - epidemiology KW - exposure assessment KW - chronic disease KW - adult health ER - TY - JOUR TI - Diesel Exhaust, Solvents, and Other Occupational Exposures as Risk Factors for Wheeze among Farmers AU - Hoppin, Jane A. AU - Umbach, David M. AU - London, Stephanie J. AU - Alavanja, Michael C. R. AU - Sandler, Dale P. T2 - American Journal of Respiratory and Critical Care Medicine AB - Farmers engage in activities that result in exposure to diesel exhaust, solvents, welding fumes, and other respiratory irritants. Using the Agricultural Health Study, a cohort of pesticide applicators in Iowa and North Carolina, we evaluated the odds of wheeze associated with nonpesticide occupational exposures. We used logistic regression models controlling for age, state, smoking, and history of asthma or atopy to evaluate odds of wheeze in the past year among the 20,898 farmers who provided complete information on all covariates. Driving diesel tractors was associated with elevated odds of wheeze (odds ratio = 1.31; 95% confidence interval = 1.13, 1.52); the odds ratio for driving gasoline tractors was 1.11 (95% confidence interval = 1.02, 1.21). A duration–response relationship was observed for driving diesel tractors but not for driving gasoline tractors. Activities involving solvent exposure, including painting and use of solvents for cleaning, were associated with an increased odds of wheeze in a duration-dependent fashion. The highest odds of wheeze for farm activities were for daily painting (odds ratio = 1.82; 95% confidence interval = 0.89, 3.73), an indication of daily solvent exposure. These results add to the growing body of evidence of adverse respiratory effects of diesel exposure on the lung and suggest exposure to solvents may contribute as well. DA - 2004/6/15/ PY - 2004/6/15/ DO - 10.1164/rccm.200309-1228OC VL - 169 IS - 12 SP - 1308-1313 J2 - Am J Respir Crit Care Med LA - en OP - SN - 1073-449X 1535-4970 UR - http://dx.doi.org/10.1164/rccm.200309-1228OC DB - Crossref KW - agriculture KW - diesel exhaust KW - occupational cohort KW - respiratory symptoms KW - solvents ER - TY - JOUR TI - Comparing questionnaire-based methods to assess occupational silica exposure AU - Parks, C.G. AU - Cooper, G.S. AU - Nylander-French, L.A. AU - Hoppin, J.A. AU - Sanderson, W.T. AU - Dement, J.M. T2 - Epidemiology AB - Background: Epidemiologic assessment of occupational exposure to silica is typically limited to long-term work in the dusty trades, primarily in jobs held by men. We compared alternative questionnaire-based methods to assess silica exposure in a recent case-control study of 265 patients with systemic lupus erythematosus (mostly women) and 355 controls randomly selected from state driver's license registries and frequency-matched by age and sex. Methods: In-person interviews included a job history (all jobs held at least 12 months) and checklist of silica-related jobs and tasks (work of at least 2 weeks). Three industrial hygienists reviewed job descriptions without knowing case-control status. Potential high- or moderate-intensity exposures were confirmed or revised based on follow-up telephone interviews. Results: In the full assessment including all work of at least 2 weeks, 9% of cases and 4% of controls were classified as medium or high silica exposure (odds ratio of disease = 2.9; 95% confidence interval = 1.3–6.4). In contrast, only 4% of cases and 9% of controls were identified by the standardized code groups index as having worked in silica-related industries or occupations for at least 12 months, providing a much lower risk estimate for disease (0.4; 0.2–0.9). Conclusions: Specific task-based questions must be included to assess the full potential of occupational silica exposure. These findings highlight the limitations of using standardized code groups to define exposure or to select jobs for industrial hygienist review. DA - 2004/// PY - 2004/// DO - 10.1097/01.ede.0000129515.54074.b2 VL - 15 IS - 4 SP - 433-441 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-4344581107&partnerID=MN8TOARS ER - TY - JOUR TI - Cancer risk and parental pesticide application in children's of agricultural health study participants AU - Flower, K.B. AU - Hoppin, J.A. AU - Lynch, C.F. AU - Blair, A. AU - Knott, C. AU - Shore, D.L. AU - Sandler, D.P. T2 - Environmental Health Perspectives DA - 2004/// PY - 2004/// VL - 112 IS - 5 SP - 631-635 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-16544378273&partnerID=MN8TOARS ER - TY - JOUR TI - Patterns of pesticide use and their determinants among wives of farmer pesticide applicators in the agricultural health study AU - Kirrane, E.F. AU - Hoppin, J.A. AU - Umbach, D.M. AU - Samanic, C. AU - Sandler, D.P. T2 - Journal of Occupational and Environmental Medicine AB - Pesticide exposure among farmers' wives is poorly characterized. Using questionnaire data from a cohort study of licensed pesticide applicators and their spouses, we investigated patterns of pesticide use among farmers' wives (n = 31,173). Wives reported a wide range of pesticide use: 36% never used pesticides during their lifetimes, whereas the heaviest pesticide users (10%) reported lifetime use of 3 or more agricultural pesticides plus commonly used residential pesticides. We identified 5 ordinal pesticide-use categories and studied factors associated with each category through polytomous logistic regression. Engaging in field work and household hygiene practices that could increase exposure were associated with pesticide use, and associations appeared to strengthen with increasing pesticide use category. Farm women reporting the heaviest pesticide use could exacerbate their exposure by engaging in practices that could increase pesticide contact. DA - 2004/// PY - 2004/// DO - 10.1097/01.jom.0000135521.15169.3e VL - 46 IS - 8 SP - 856-865 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-4143147448&partnerID=MN8TOARS ER - TY - JOUR TI - Cancer Incidence Among Pesticide Applicators Exposed to Atrazine in the Agricultural Health Study AU - Rusiecki, J. A. AU - De Roos, A. AU - Lee, W. J. AU - Dosemeci, M. AU - Lubin, J. H. AU - Hoppin, J. A. AU - Blair, A. AU - Alavanja, M. C. R. T2 - JNCI Journal of the National Cancer Institute AB - Atrazine is the most heavily applied agricultural pesticide for crop production in the United States. Both animal and human studies have suggested that atrazine is possibly carcinogenic, but results have been mixed. We evaluated cancer incidence in atrazine-exposed pesticide applicators among 53,943 participants in the Agricultural Health Study, a prospective cohort study of licensed pesticide applicators in Iowa and North Carolina.We obtained detailed pesticide exposure information using a self-administered questionnaire completed at the time of enrollment (1993-1997). Cancer incidence was followed through December 31, 2001. We used adjusted Poisson regression to calculate rate ratios (RRs) and 95% confidence intervals (CIs) of multiple types of cancer among atrazine exposed applicators. P(trend) values were calculated using atrazine exposure as a continuous variable, and all statistical tests were two-sided. Two exposure metrics were used: quartiles of lifetime days of exposure and quartiles of intensity-weighted lifetime days of exposure.36,513 (68%) applicators reported ever using atrazine; exposure was not associated with overall cancer incidence. Comparisons of cancer incidence in applicators with the highest atrazine exposure and those with the lowest exposure, assessed by lifetime days (RR(LD)) and intensity-weighted lifetime days (RR(IWLD)) of exposure yielded the following results: prostate cancer, RR(LD) = 0.88, 95% CI = 0.63 to 1.23, P(trend) =.26, and RR(IWLD) = 0.89, 95% CI = 0.63 to 1.25, P(trend) =.35; lung cancer, RR(LD) = 1.91, 95% CI = 0.93 to 3.94, P(trend) =.08, and RR(IWLD) = 1.37, 95% CI = 0.65 to 2.86, P(trend) =.19; bladder cancer, RR(LD) = 3.06, 95% CI = 0.86 to 10.81, P(trend) =.18, and RR(IWLD) = 0.85, 95% CI = 0.24 to 2.94, P(trend) =.71; non-Hodgkin lymphoma, RR(LD) = 1.61, 95% CI = 0.62 to 4.16, P(trend) =.35, and RR(IWLD) = 1.75, 95% CI = 0.73 to 4.20, P(trend) =.14; and multiple myeloma, RR(LD) = 1.60, 95% CI = 0.37 to 7.01, P(trend) =.41, and RR(IWLD) = 2.17, 95% CI = 0.45 to 10.32, P(trend) =.21.Our analyses did not find any clear associations between atrazine exposure and any cancer analyzed. However, further studies are warranted for tumor types in which there was a suggestion of trend (lung, bladder, non-Hodgkin lymphoma, and multiple myeloma). DA - 2004/9/14/ PY - 2004/9/14/ DO - 10.1093/jnci/djh264 VL - 96 IS - 18 SP - 1375-1382 J2 - JNCI Journal of the National Cancer Institute LA - en OP - SN - 0027-8874 1460-2105 UR - http://dx.doi.org/10.1093/jnci/djh264 DB - Crossref ER - TY - JOUR TI - Pesticides and Lung Cancer Risk in the Agricultural Health Study Cohort AU - Alavanja, M. C. R. AU - Dosemeci, M. AU - Samanic, C. AU - Lubin, J. AU - Lynch, C.F. AU - Knott, C. AU - Barker, J. AU - Hoppin, J.A. AU - Sandler, D.P. AU - Coble, J. AU - Thomas, K. AU - Blair, A. T2 - American Journal of Epidemiology AB - The authors examined the relation between 50 widely used agricultural pesticides and lung cancer incidence in the Agricultural Health Study, a prospective cohort study of 57,284 pesticide applicators and 32,333 spouses of farmer applicators with no prior history of lung cancer. Self-administered questionnaires were completed at enrollment (1993-1997). Cancer incidence was determined through population-based cancer registries from enrollment through December 31, 2001. A lung cancer standardized incidence ratio of 0.44 (95% confidence interval: 0.39, 0.49) was observed overall, due in large part to a low cigarette smoking prevalence. Two widely used herbicides, metolachlor and pendimethalin (for low-exposed groups to four higher exposure categories: odds ratio (OR) = 1.0, 1.6, 1.2, 5.0; p(trend) = 0.0002; and OR = 1.0, 1.6, 2.1, 4.4; p(trend) = 0.003, respectively), and two widely used insecticides, chlorpyrifos and diazinon (OR = 1.0, 1.1, 1.7, 1.9; p(trend) = 0.03; and OR = 1.0, 1.6, 2.7, 3.7; p(trend) = 0.04, respectively), showed some evidence of exposure response for lung cancer. These excesses could not be explained by previously identified lung cancer risk factors. The usage levels in this cohort are considerably higher than those typically experienced by the general population. An excess risk among spouses directly exposed to pesticides could not be evaluated at this time. DA - 2004/11/1/ PY - 2004/11/1/ DO - 10.1093/aje/kwh290 VL - 160 IS - 9 SP - 876–885 SN - 0002-9262 UR - http://dx.doi.org/10.1093/aje/kwh290 KW - lung neoplasms KW - pesticides ER - TY - JOUR TI - Cancer Incidence among Pesticide Applicators Exposed to Alachlor in the Agricultural Health Study AU - Lee, W. J. AU - Hoppin, J.A. AU - Blair, A. AU - Lubin, J.H. AU - Dosemeci, M. AU - Sandler, D.P. AU - Alavanja, M.C.R. T2 - American Journal of Epidemiology AB - The authors evaluated the incidence of cancer among pesticide applicators with exposure to alachlor in the Agricultural Health Study, a prospective cohort study of licensed pesticide applicators in Iowa and North Carolina. A total of 49,980 pesticide applicators are included in this analysis; 26,510 applicators (53%) reported use of alachlor on the enrollment questionnaire. Detailed pesticide exposure and other information were obtained from a self-administered questionnaire completed at the time of enrollment (1993–1997). Poisson regression analysis was used to evaluate the exposure-response relations between alachlor and cancer incidence controlled for the effects of potential confounding factors. A total of 1,466 incident malignant neoplasms were diagnosed during the study period, 1993–2000. Among alachlor-exposed applicators, the authors found a significant increasing trend for incidence of all lymphohematopoietic cancers associated with lifetime exposure-days (p for trend = 0.02) and intensity-weighted exposure-days (p for trend = 0.03) to alachlor. The risks of leukemia (rate ratio = 2.83, 95% confidence interval: 0.74, 10.9) and multiple myeloma (rate ratio = 5.66, 95% confidence interval: 0.70, 45.7) were increased among applicators in the highest alachlor exposure category. Our findings suggest a possible association between alachlor application and incidence of lymphohematopoietic cancers among applicators in the Agricultural Health Study. DA - 2004/2/15/ PY - 2004/2/15/ DO - 10.1093/aje/kwh040 VL - 159 IS - 4 SP - 373–380 SN - 0002-9262 UR - http://dx.doi.org/10.1093/aje/kwh040 KW - agriculture KW - cohort studies KW - herbicides KW - leukemia KW - multiple myeloma KW - neoplasms KW - occupational exposure KW - pesticides ER - TY - JOUR TI - Cancer Incidence Among Pesticide Applicators Exposed to Chlorpyrifos in the Agricultural Health Study AU - Lee, W. J. AU - Blair, A. AU - Hoppin, J. A. AU - Lubin, J. H. AU - Rusiecki, J. A. AU - Sandler, D. P. AU - Dosemeci, M. AU - Alavanja, M. C. R. T2 - JNCI Journal of the National Cancer Institute AB - Chlorpyrifos is one of the most widely used insecticides in the United States. We evaluated the incidence of cancer among pesticide applicators exposed to chlorpyrifos in the Agricultural Health Study, a prospective cohort study of licensed pesticide applicators in Iowa and North Carolina.A total of 54,383 pesticide applicators were included in this analysis. Detailed information on pesticide exposure and lifestyle factors was obtained from self-administered questionnaires completed at the time of enrollment (December 1993-December 1997). Poisson regression analysis was used to evaluate the association between chlorpyrifos exposure and cancer incidence after adjustment for potential confounders. All statistical tests were two-sided.A total of 2070 incident malignant neoplasms were diagnosed through 2001. The rate ratio for all cancers combined among chlorpyrifos-exposed applicators compared with nonexposed applicators was 0.97 (95% confidence interval = 0.87 to 1.08). For most cancers analyzed, there was no evidence of an exposure-response relationship. However, the incidence of lung cancer was statistically significantly associated with both chlorpyrifos lifetime exposure-days (P(trend) = .002) and chlorpyrifos intensity-weighted exposure-days (P(trend) = .036). After adjustment for other pesticide exposures and demographic factors, individuals in the highest quartile of chlorpyrifos lifetime exposure-days (>56 days) had a relative risk of lung cancer 2.18 (95% confidence interval = 1.31 to 3.64) times that of those with no chlorpyrifos exposure.Our findings suggest an association between chlorpyrifos use and incidence of lung cancer that deserves further evaluation. DA - 2004/11/30/ PY - 2004/11/30/ DO - 10.1093/jnci/djh324 VL - 96 IS - 23 SP - 1781-1789 J2 - JNCI Journal of the National Cancer Institute LA - en OP - SN - 0027-8874 1460-2105 UR - http://dx.doi.org/10.1093/jnci/djh324 DB - Crossref ER - TY - JOUR TI - Agricultural and residential pesticides in wipe samples from farmworker family residences in North Carolina and Virginia. AU - Quandt, Sara A AU - Arcury, Thomas A AU - Rao, Pamela AU - Snively, Beverly M AU - Camann, David E AU - Doran, Alicia M AU - Yau, Alice Y AU - Hoppin, Jane A AU - Jackson, David S T2 - Environmental Health Perspectives AB - Children of farmworkers can be exposed to pesticides through multiple pathways, including agricultural take-home and drift as well as residential applications. Because farmworker families often live in poor-quality housing, the exposure from residential pesticide use may be substantial. We measured eight locally reported agricultural pesticides and 13 pesticides commonly found in U.S. houses in residences of 41 farmworker families with at least one child < 7 years of age in western North Carolina and Virginia. Wipe samples were taken from floor surfaces, toys, and children's hands. We also collected interview data on possible predictors of pesticide presence, including characteristics of the household residents, cleaning practices, and characteristics of the home. All families were Spanish-speaking, primarily from Mexico. Results indicate that six agricultural and 11 residential pesticides were found in the homes, with agricultural, residential, or both present in 95% of homes sampled. In general, residential pesticides were more commonly found. Presence of both types of pesticides on the floor was positively associated with detection on toys or hands. Agricultural pesticide detection was associated with housing adjacent to agricultural fields. Residential pesticide detection was associated with houses judged difficult to clean. Although the likelihood of agricultural pesticide exposure has been considered high for farmworker families, these results indicate that residential pesticide use and exposure in this population merit further study. DA - 2004/3// PY - 2004/3// DO - 10.1289/ehp.6554 VL - 112 IS - 3 SP - 382-387 J2 - Environmental Health Perspectives LA - en OP - SN - 0091-6765 1552-9924 UR - http://dx.doi.org/10.1289/ehp.6554 DB - Crossref KW - agriculture KW - children KW - exposure KW - house dust KW - Latino ER - TY - JOUR TI - Association of Pesticide Exposure with Neurologic Dysfunction and Disease AU - Kamel, Freya AU - Hoppin, Jane A. T2 - Environmental Health Perspectives AB - Poisoning by acute high-level exposure to certain pesticides has well-known neurotoxic effects, but whether chronic exposure to moderate levels of pesticides is also neurotoxic is more controversial. Most studies of moderate pesticide exposure have found increased prevalence of neurologic symptoms and changes in neurobehavioral performance, reflecting cognitive and psychomotor dysfunction. There is less evidence that moderate exposure is related to deficits in sensory or motor function or peripheral nerve conduction, but fewer studies have considered these outcomes. It is possible that the most sensitive manifestation of pesticide neurotoxicity is a general malaise lacking in specificity and related to mild cognitive dysfunction, similar to that described for Gulf War syndrome. Most studies have focused on organophosphate insecticides, but some found neurotoxic effects from other pesticides, including fungicides, fumigants, and organochlorine and carbamate insecticides. Pesticide exposure may also be associated with increased risk of Parkinson disease; several classes of pesticides, including insecticides, herbicides, and fungicides, have been implicated. Studies of other neurodegenerative diseases are limited and inconclusive. Future studies will need to improve assessment of pesticide exposure in individuals and consider the role of genetic susceptibility. More studies of pesticides other than organophosphates are needed. Major unresolved issues include the relative importance of acute and chronic exposure, the effect of moderate exposure in the absence of poisoning, and the relationship of pesticide-related neurotoxicity to neurodegenerative disease. DA - 2004/6// PY - 2004/6// DO - 10.1289/ehp.7135 VL - 112 IS - 9 SP - 950-958 J2 - Environmental Health Perspectives LA - en OP - SN - 0091-6765 1552-9924 UR - http://dx.doi.org/10.1289/ehp.7135 DB - Crossref KW - fumigant KW - fungicide KW - insecticide KW - neurobehavioral performance KW - neurodegenerative disease KW - neurologic symptoms KW - organophosphate KW - Parkinson disease KW - pesticide ER - TY - JOUR TI - Promoting comparative molecular studies in environmental health research: an overview of the comparative toxicogenomics database (CTD) AU - Mattingly, C J AU - Colby, G T AU - Rosenstein, M C AU - Forrest, J N AU - Boyer, J L T2 - The Pharmacogenomics Journal AB - Promoting comparative molecular studies in environmental health research: an overview of the comparative toxicogenomics database (CTD) DA - 2004/1/28/ PY - 2004/1/28/ DO - 10.1038/sj.tpj.6500225 VL - 4 IS - 1 SP - 5-8 J2 - Pharmacogenomics J LA - en OP - SN - 1470-269X 1473-1150 UR - http://dx.doi.org/10.1038/sj.tpj.6500225 DB - Crossref ER - TY - JOUR TI - Cell and Molecular Biology of Marine Elasmobranchs: Squalus acanthias and Raja erinacea AU - Mattingly, Carolyn AU - Parton, Angela AU - Dowell, Lori AU - Rafferty, Jason AU - Barnes, David T2 - Zebrafish AB - Elasmobranchs are among the most primitive existing species exhibiting fundamental vertebrate characteristics, such as neural crest, jaws, teeth, and an adaptive immune system. They are also among the earliest-evolved vertebrates with a closed, pressurized circulatory system and related signaling molecules. Although many species are used experimentally, the spiny dogfish shark (Squalus acanthias) and little skate (Raja erinacea) have particular advantages and are the most commonly used elasmobranch biomedical models. These animals display powerful molecular systems for dealing with salt and water homeostasis, cell volume regulation, and environmental and internal osmotic sensing. They have become important unique models in studies of transport-related diseases such as cystic fibrosis and anion or xenobiotic transport. Much of this work has relied on physiological experiments combined with molecular approaches and the advantages of comparative genomic analyses to identify conserved regions representing functional protein domains. Recent work has seen the development of cell cultures and the beginning of expressed sequence tags (EST) and genomic libraries. Other areas in which elasmobranches have played critical roles include immunology and neurobiology. It also appears that sharks have tissue regenerative capability beyond what is commonly seen in mammals. For example, sharks and skates possess a region of renal regeneration, with new tubules being formed continually through adulthood. As comparative functional genomics comes of age, these comparative vertebrate models may play an increasing role in the larger picture of human biomedical research. There is plenty of ocean to share. DA - 2004/5// PY - 2004/5// DO - 10.1089/zeb.2004.1.111 VL - 1 IS - 2 SP - 111-120 J2 - Zebrafish LA - en OP - SN - 1545-8547 1557-8542 UR - http://dx.doi.org/10.1089/zeb.2004.1.111 DB - Crossref ER - TY - JOUR TI - Marine Organism Cell Biology and Regulatory Sequence Discoveryin Comparative Functional Genomics AU - Barnes, David W. AU - Mattingly, Carolyn J. AU - Parton, Angela AU - Dowell, Lori M. AU - Bayne, Christopher J. AU - Forrest, John N., Jr. T2 - Cytotechnology AB - The use of bioinformatics to integrate phenotypic and genomic data from mammalian models is well established as a means of understanding human biology and disease. Beyond direct biomedical applications of these approaches in predicting structure–function relationships between coding sequences and protein activities, comparative studies also promote understanding of molecular evolution and the relationship between genomic sequence and morphological and physiological specialization. Recently recognized is the potential of comparative studies to identify functionally significant regulatory regions and to generate experimentally testable hypotheses that contribute to understanding mechanisms that regulate gene expression, including transcriptional activity, alternative splicing and transcript stability. Functional tests of hypotheses generated by computational approaches require experimentally tractable in vitro systems, including cell cultures. Comparative sequence analysis strategies that use genomic sequences from a variety of evolutionarily diverse organisms are critical for identifying conserved regulatory motifs in the 5′-upstream, 3′-downstream and introns of genes. Genomic sequences and gene orthologues in the first aquatic vertebrate and protovertebrate organisms to be fully sequenced (Fugu rubripes, Ciona intestinalis, Tetraodon nigroviridis, Danio rerio) as well as in the elasmobranchs, spiny dogfish shark (Squalus acanthias) and little skate (Raja erinacea), and marine invertebrate models such as the sea urchin (Strongylocentrotus purpuratus) are valuable in the prediction of putative genomic regulatory regions. Cell cultures have been derived for these and other model species. Data and tools resulting from these kinds of studies will contribute to understanding transcriptional regulation of biomedically important genes and provide new avenues for medical therapeutics and disease prevention. DA - 2004/10// PY - 2004/10// DO - 10.1007/s10616-005-1719-5 VL - 46 IS - 2-3 SP - 123-137 J2 - Cytotechnology LA - en OP - SN - 0920-9069 1573-0778 UR - http://dx.doi.org/10.1007/s10616-005-1719-5 DB - Crossref ER - TY - JOUR TI - Integrated analysis of genetic, genomic and proteomic data AU - Reif, David M AU - White, Bill C AU - Moore, Jason H T2 - Expert Review of Proteomics AB - The rapid expansion of methods for measuring biological data ranging from DNA sequence variations to mRNA expression and protein abundance presents the opportunity to utilize multiple types of information jointly in the study of human health and disease. Organisms are complex systems that integrate inputs at myriad levels to arrive at an observable phenotype. Therefore, it is essential that questions concerning the etiology of phenotypes as complex as common human diseases take the systemic nature of biology into account, and integrate the information provided by each data type in a manner analogous to the operation of the body itself. While limited in scope, the initial forays into the joint analysis of multiple data types have yielded interesting results that would not have been reached had only one type of data been considered. These early successes, along with the aforementioned theoretical appeal of data integration, provide impetus for the development of methods for the parallel, high-throughput analysis of multiple data types. The idea that the integrated analysis of multiple data types will improve the identification of biomarkers of clinical endpoints, such as disease susceptibility, is presented as a working hypothesis. DA - 2004/6// PY - 2004/6// DO - 10.1586/14789450.1.1.67 VL - 1 IS - 1 SP - 67–75 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-12844275916&partnerID=MN8TOARS KW - complex systems KW - data reliability gene expression KW - joint evalution KW - protein expression KW - simulation KW - single nucleotide polymorphism ER - TY - JOUR TI - On the origins of adaptive immunity: Innate immune receptors join the tale AU - Van Den Berg, T.K. AU - Yoder, J.A. AU - Litman, G.W. T2 - Trends in Immunology AB - Among members of the Ig superfamily (IgSF), antigen receptors have the unique capacity to rearrange their variable domains, thereby creating an extensive repertoire for antigen recognition. It is assumed that antigen receptors evolved from a non-rearranging IgSF member by insertion of a transposable element. Although the nature of this predecessor is unknown, two multigene families of innate immune receptors that bear a close structural resemblance to antigen receptor chains have been identified in mammals and bony fish, respectively: signal-regulatory proteins (SIRPs) and novel immune-type receptors (NITRs). Members of both families encode V-set Ig domains with a typical antigen receptor-like joining (J) motif and possess the potential to signal through immunoreceptor tyrosine-based inhibition motifs (ITIMs) or immunoreceptor tyrosine-based activation motifs (ITAMs). By analogy to the T-cell receptor (TCR) and certain innate receptors [e.g. killer cell inhibitory receptors (KIRs)] that recognize MHC molecules, SIRP members regulate immune function by interaction with broadly expressed 'self' ligands. We propose the existence of an evolutionary and functional link between innate and adaptive immune receptors that sheds light on the nature of the antigen receptor predecessor(s). DA - 2004/// PY - 2004/// DO - 10.1016/j.it.2003.11.006 VL - 25 IS - 1 SP - 11-16 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0346961787&partnerID=MN8TOARS ER - TY - JOUR TI - On the origins of adaptive immunity:possible clues from the SIRP and NITR multigene families AU - Berg, T. K. AU - Yoder, J. A. AU - Litman, G. W. T2 - Trends in Immunology DA - 2004/// PY - 2004/// VL - 25 SP - 11-16 ER - TY - JOUR TI - Investigating the morphology, function and genetics of cytotoxic cells in bony fish AU - Yoder, Jeffrey A. T2 - Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology AB - Bony fish (teleosts) possess multiple cytotoxic cell lineages that recognize and destroy virally infected and transformed cells. In general, these lineages parallel their functional equivalents in mammals and include neutrophilic granulocytes, macrophages, cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. These four cell types have been morphologically identified in multiple fish species but only limited information is available about their function. In contrast, much work has gone into examining the function of a fifth cytotoxic cell lineage, termed nonspecific cytotoxic cells (NCC), that has been referred to as the bony fish equivalent of NK cells. However, evidence suggesting that NCC do not represent the NK lineage has come through the development of multiple cytotoxic catfish cell lines that are morphologically and functionally similar to human NK cells and are distinct from NCC. In addition to characterizing cytotoxic cells from fish, recent work has identified the novel immune-type receptors (NITR) and cichlid killer leukocyte receptors (cKLR) that are structurally related to mammalian NK receptors and likely play a role in cytotoxic function in fish. This review summarizes the morphological and functional evidence for cytotoxic cells within bony fish and discusses future directions for examining cytotoxicity through genomics and transgenics. DA - 2004/7// PY - 2004/7// DO - 10.1016/j.cca.2004.03.008 VL - 138 IS - 3 SP - 271-280 J2 - Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology LA - en OP - SN - 1532-0456 UR - http://dx.doi.org/10.1016/j.cca.2004.03.008 DB - Crossref KW - C-type lectin KW - cytotoxicity KW - cytotoxic T lymphocyte KW - macrophage KW - natural killer cell KW - neutrophil KW - non-specific cytotoxic cells KW - novel immune-type receptors ER - TY - JOUR TI - Evaluation of a biologically active cover for mitigation of landfill gas emissions AU - Barlaz, MA AU - Green, RB AU - Chanton, JP AU - Goldsmith, CD AU - Hater, GR T2 - ENVIRONMENTAL SCIENCE & TECHNOLOGY AB - Landfills are the third largest source of anthropogenic CH4 in the United States, and there is potential for reduction in this source of greenhouse gases and other contaminants. The objective of this work was to contrast emissions of CH4 and non-methane organic compounds (NMOCs) from landfill cells covered with soil or a biologically active cover consisting of yard waste compost. On the basis of four field campaigns over 14 months, CH4 emissions from the biocover (BC) varied from −1.73 to 1.33 g m-2 d-1, with atmospheric uptake measured in 52% of tests. BC emissions did not increase when the gas collection system was turned off. Uptake of atmospheric CH4 was measured in 54% of tests on the soil cover (SC) when the gas collection was system active and 12% when the gas collection system was off. Many (26%) relatively high fluxes (>15 g m-2 d-1) were measured from the SC as were some dramatic effects due to deactivation of the gas collection system. In tests with positive emissions, stable isotope measurements showed that the BC and SC were responsible for oxidation of 55% and 21% of the CH4 reaching the bottom of the respective cover. Seven of the highest 10 NMOC emissions were measured in the SC, and 17 of 21 fluxes for speciated organic compounds were higher in the SC. The relationship between CH4, NMOC, and individual organic compound emissions suggested a correlation between CH4 and trace organic oxidation. BCs can reduce landfill gas emissions in the absence of a gas collection system and can serve as a polishing step in the presence of an active system. DA - 2004/9/15/ PY - 2004/9/15/ DO - 10.1021/es049605b VL - 38 IS - 18 SP - 4891-4899 SN - 1520-5851 ER - TY - BOOK TI - Algae detection and removal strategies for drinking water treatment plants AU - Knappe, D. R. U. CN - TD465 .A43 2004 DA - 2004/// PY - 2004/// PB - Denver Colo.: AWWA Research Foundation and American Water Works Association SN - 1583213074 ER - TY - JOUR TI - Effect of cellulose/hemicellulose and lignin on the bioavailability of toluene sorbed to waste paper AU - Chen, Y AU - Knappe, DRU AU - Barlaz, NA T2 - ENVIRONMENTAL SCIENCE & TECHNOLOGY AB - Paper constitutes about 38% of municipal solid waste, much of which is disposed of in landfills. Sorption to such lignocellulosic materials may limit the bioavailability of organic contaminants in landfills. The objective of this study was to identify the effect of individual biopolymers in paper on toluene sorption and bioavailability by subjecting fresh and anaerobically degraded office paper and newsprint to enzymatic hydrolysis and acid hydrolysis. Enzymatic degradation of cellulose and hemicellulose had no effect on toluene bioavailability. In contrast, acid-insoluble lignin controlled toluene sorption and bioavailability for both fresh and degraded newsprint. Acid-insoluble lignin could explain only 54% of the toluene sorption capacity of degraded office paper however, suggesting that crude protein and/or lipophilic organic matter were also important sorbent phases. Toluene sorbed to degraded office paper was also less bioavailable than toluene sorbed to an equivalent mass of lignin extracted from this sorbent. The latter result suggests that a fraction of toluene sorbed to degraded office paper may have been sequestered by lipophilic organic matter. The sorption and bioavailability data indicate that the preferential decomposition of cellulose and hemicellulose relative to lignin in landfills should not decrease the overall toluene sorption capacity of paper waste or increase the bioavailability of sorbed toluene. DA - 2004/7/1/ PY - 2004/7/1/ DO - 10.1021/es035286x VL - 38 IS - 13 SP - 3731-3736 SN - 1520-5851 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-3042787671&partnerID=MN8TOARS ER - TY - JOUR TI - Chemical characterization and sorption capacity measurements of degraded newsprint from a landfill AU - Chen, LX AU - Nanny, MA AU - Knappe, DRU AU - Wagner, TB AU - Ratasuk, N T2 - ENVIRONMENTAL SCIENCE & TECHNOLOGY AB - Newsprint samples collected from 12−16 ft (top layer (TNP)), 20−24 ft (middle layer (MNP)), and 32−36 ft (bottom layer (BNP)) below the surface of the Norman Landfill (NLF) were characterized by infrared (IR) spectroscopy, cross-polarization, magic-angle spinning 13C nuclear magnetic resonance (CP-MAS 13C NMR) spectroscopy, and tetramethylammonium hydroxide (TMAH) thermochemolysis gas chromatography/mass spectrometry (GC/MS). The extent of NLF newsprint degradation was evaluated by comparing the chemical composition of NLF newsprint to that of fresh newsprint (FNP) and newsprint degraded in the laboratory under methanogenic conditions (DNP). The O-alkyl/alkyl, cellulose/lignin, and lignin/resin acid ratios showed that BNP was the most degraded, and that all three NLF newsprint samples were more degraded than DNP. 13C NMR and TMAH thermochemolysis data demonstrated selective enrichment of lignin over cellulose, and TMAH thermochemolysis further exhibited selective enrichment of resin acids over lignin. In addition, the crystallinity of cellulose in NLF newsprint samples was significantly lower relative to that of FNP and DNP as shown by 13C NMR spectra. The yield of lignin monomers from TMAH thermochemolysis suggested that hydroxyl groups were removed from the propyl side chain of lignin during the anaerobic decomposition of newsprint in the NLF. Moreover, the vanillyl acid/aldehyde ratio, which successfully describes aerobic lignin degradation, was not a good indicator of the anaerobic degradation of lignin on the basis of the TMAH data. The toluene sorption capacity increased as the degree of newsprint degradation increased or as the O-alkyl/alkyl ratio of newsprint decreased. The results of this study further verified that the sorbent O-alkyl/alkyl ratio is useful for predicting sorption capacities of natural organic materials for hydrophobic organic contaminants. DA - 2004/7/1/ PY - 2004/7/1/ DO - 10.1021/es0305914 VL - 38 IS - 13 SP - 3542-3550 SN - 1520-5851 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-3042751476&partnerID=MN8TOARS ER - TY - JOUR TI - C/EBP alpha is a DNA damage-inducible p53-regulated mediator of the G(1) checkpoint in keratinocytes AU - Yoon, K AU - Smart, RC T2 - MOLECULAR AND CELLULAR BIOLOGY AB - The basic leucine zipper transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha), is abundantly expressed in keratinocytes of the skin; however, its function in skin is poorly characterized. UVB radiation is responsible for the majority of human skin cancers. In response to UVB-induced DNA damage, keratinocytes activate cell cycle checkpoints that arrest cell cycle progression and prevent replication of damaged DNA, allowing time for DNA repair. We report here that UVB radiation is a potent inducer of C/EBPalpha in human and mouse keratinocytes, as well as in mouse skin in vivo. UVB irradiation of keratinocytes resulted in the transcriptional up-regulation of C/EBPalpha mRNA, producing a >70-fold increase in C/EBPalpha protein levels. N-Methyl-N'-nitro-N-nitrosoguanidine, etoposide, and bleomycin also induced C/EBPalpha. UVB-induced C/EBPalpha was accompanied by an increase in p53 protein and caffeine, an inhibitor of ataxia-telangiectasia-mutated kinase, and ataxia-telangiectasia-mutated and Rad3-related kinase inhibited UVB-induced increases in both C/EBPalpha and p53. UVB irradiation of p53-null or mutant p53-containing keratinocytes failed to induce C/EBPalpha. UVB irradiation of C/EBPalpha knockdown keratinocytes displayed a greatly diminished DNA damage G(1) checkpoint, and this was associated with increased sensitivity to UVB-induced apoptosis. Our results uncover a novel role for C/EBPalpha as a p53-regulated DNA damage-inducible gene that has a critical function in the DNA damage G(1) checkpoint response in keratinocytes. DA - 2004/12// PY - 2004/12// DO - 10.1128/MCB.24.24.10650-10660.2004 VL - 24 IS - 24 SP - 10650-10660 SN - 1098-5549 ER - TY - JOUR TI - An evolutionary algorithm to generate alternatives (EAGA) for engineering optimization problems AU - Zechman, EM AU - Ranjithan, , SR T2 - ENGINEERING OPTIMIZATION AB - Typically for a real optimization problem, the optimal solution to a mathematical model of that real problem may not always be the ‘best’ solution when considering unmodeled or unquantified objectives during decision-making. Formal approaches to explore efficiently for good but maximally different alternative solutions have been established in the operations research literature, and have been shown to be valuable in identifying solutions that perform expectedly well with respect to modeled and unmodeled objectives. While the use of evolutionary algorithms (EAs) to solve real engineering optimization problems is becoming increasingly common, systematic alternatives-generation capabilities are not fully extended for EAs. This paper presents a new EA-based approach to generate alternatives (EAGA), and illustrates its applicability via two test problems. A realistic airline route network design problem was also solved and analyzed successfully using EAGA. The EAGA promises to be a flexible procedure for exploring alternative solutions that could assist when making decisions for real engineering optimization problems riddled with unmodeled or unquantified issues. DA - 2004/10// PY - 2004/10// DO - 10.1080/03052150410001704863 VL - 36 IS - 5 SP - 539-553 SN - 1029-0273 KW - genetic algorithm KW - evolutionary algorithm KW - modeling to generate alternatives KW - niching ER - TY - JOUR TI - Resolution of the novel immune-type receptor gene cluster in zebrafish AU - Yoder, J. A. AU - Litman, R. T. AU - Mueller, M. G. AU - Desai, S. AU - Dobrinski, K. P. AU - Montgomery, J. S. AU - Buzzeo, M. P. AU - Ota, T. AU - Amemiya, C. T. AU - Trede, N. S. AU - Wei, S. AU - Djeu, J. Y. AU - Humphray, S. AU - Jekosch, K. AU - Hernandez Prada, J. A. AU - Ostrov, D. A. AU - Litman, G. W. T2 - Proceedings of the National Academy of Sciences AB - The novel immune-type receptor (NITR) genes encode a unique multigene family of leukocyte regulatory receptors, which possess an extracellular Ig variable (V) domain and may function in innate immunity. Artificial chromosomes that encode zebrafish NITRs have been assembled into a contig spanning approximately 350 kb. Resolution of the complete NITR gene cluster has led to the identification of eight previously undescribed families of NITRs and has revealed the presence of C-type lectins within the locus. A maximum haplotype of 36 NITR genes (138 gene sequences in total) can be grouped into 12 distinct families, including inhibitory and activating receptors. An extreme level of interindividual heterozygosity is reflected in allelic polymorphisms, haplotype variation, and family-specific isoform complexity. In addition, the exceptional diversity of NITR sequences among species suggests divergent evolution of this multigene family with a birth-and-death process of member genes. High-confidence modeling of Nitr V-domain structures reveals a significant shift in the spatial orientation of the Ig fold, in the region of highest interfamily variation, compared with Ig V domains. These studies resolve a complete immune gene cluster in zebrafish and indicate that the NITRs represent the most complex family of activating/inhibitory surface receptors thus far described. DA - 2004/10/20/ PY - 2004/10/20/ DO - 10.1073/pnas.0405242101 VL - 101 IS - 44 SP - 15706-15711 J2 - Proceedings of the National Academy of Sciences LA - en OP - SN - 0027-8424 1091-6490 UR - http://dx.doi.org/10.1073/pnas.0405242101 DB - Crossref ER - TY - JOUR TI - NOM and MIB, who wins in the competition for activated carbon adsorption sites? AU - Hepplewhite, C AU - Newcombe, G AU - Knappe, DRU T2 - WATER SCIENCE AND TECHNOLOGY AB - The adsorption of an odour compound common in drinking water, 2-methylisoborneol (MIB), was studied on two activated carbons in the presence of 13 well-characterised natural organic matter (NOM) solutions. It was found that, although the carbons and the NOM solutions had a wide range of characteristics, the major competitive mechanism was the same in all cases. The low molecular weight NOM compounds were the most competitive, participating in a direct competition with the MIB molecule for adsorption sites. Equivalent background concentration (EBC) calculations indicated a relatively low concentration of directly competing compounds in the NOM. Some evidence of pore restriction was also seen, with microporous carbons most affected by low molecular weight NOM, and mesoporous carbons impacted by the higher molecular weight compounds. DA - 2004/// PY - 2004/// DO - 10.2166/wst.2004.0584 VL - 49 IS - 9 SP - 257-265 SN - 1996-9732 KW - activated carbon KW - adsorption KW - adsorption competition KW - MIB KW - natural organic material KW - tastes and odours ER - TY - JOUR TI - Intraparticle diffusion and adsorption of arsenate onto granular ferric hydroxide (GFH) AU - Badruzzaman, M AU - Westerhoff, P AU - Knappe, DRU T2 - WATER RESEARCH AB - Porous iron oxides are being evaluated and selected for arsenic removal in potable water systems. Granular ferric hydroxide, a typical porous iron adsorbent, is commercially available and frequently considered in evaluation of arsenic removal methods. GFH is a highly porous (micropore volume ∼0.0394±0.0056 cm3 g−1, mesopore volume ∼0.0995±0.0096 cm3 g−1) adsorbent with a BET surface area of 235±8 m2 g−1. The purpose of this paper is to quantify arsenate adsorption kinetics on GFH and to determine if intraparticle diffusion is a rate-limiting step for arsenic removal in packed-bed treatment systems. Data from bottle-point isotherm and differential column batch reactor (DCBR) experiments were used to estimate Freundlich isotherm parameters (K and 1/n) as well as kinetic parameters describing mass transfer resistances due to film diffusion (kf) and intraparticle surface diffusion (Ds). The pseudo-equilibrium (18 days of contact time) arsenate adsorption density at pH 7 was 8 μg As/mg dry GFH at a liquid phase arsenate concentration of 10 μg As/L. The homogeneous surface diffusion model (HSDM) was used to describe the DCBR data. A non-linear relationship (DS=3.0−9×Rp1.4) was observed between Ds and GFH particle radius (RP) with Ds values ranging from 2.98×10−12 cm2 s−1 for the smallest GFH mesh size (100×140) to 64×10−11 cm2 s−1 for the largest GFH mesh size (10×30). The rate-limiting process of intraparticle surface diffusion for arsenate adsorption by porous iron oxides appears analogous to organic compound adsorption by activated carbon despite differences in adsorption mechanisms (inner-sphere complexes for As versus hydrophobic interactions for organic contaminants). The findings are discussed in the context of intraparticle surface diffusion affecting packed-bed treatment system design and application of rapid small-scale column tests (RSSCTs) to simulate the performance of pilot- or full-scale systems at the bench-scale. DA - 2004/11// PY - 2004/11// DO - 10.1016/j.watres.2004.07.007 VL - 38 IS - 18 SP - 4002-4012 SN - 0043-1354 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-4544343747&partnerID=MN8TOARS KW - arsenic KW - iron KW - adsorption KW - surface diffusion KW - water treatment ER - TY - JOUR TI - Biodegradation of 1,4-dioxane using trickling filter AU - Zenker, M. J. AU - Borden, R. C. AU - Barlaz, Morton T2 - Journal of Environmental Engineering (New York, N.Y.) AB - The ability of a laboratory-scale trickling filter to biodegrade cyclic ethers was investigated and a simple kinetic model was developed to predict ether biodegradation. The trickling filter received a feed solution designed to mimic ether concentrations typically encountered in contaminated groundwater. The reactor was operated for approximately 1 year and was capable of biodegrading 93–97% of 1,4-dioxane at various loading rates in the obligate presence of tetrahydrofuran (THF) as the growth substrate. A simple tanks-in-series hydraulic model combined with a kinetic model that incorporated cometabolism was utilized to simulate removal of THF and 1,4-dioxane. Model simulations of THF removal were satisfactory for all loading rates analyzed. However, the model somewhat over predicted 1,4-dioxane removal. This research demonstrates the ability to treat groundwater contaminated with low concentrations of ethers in attached growth reactors. DA - 2004/// PY - 2004/// DO - 10.1061/(asce)0733-9372(2004)130:9(926) VL - 130 IS - 9 SP - 926–931 ER - TY - JOUR TI - Cell cycle-dependent phosphorylation of C/EBP beta mediates oncogenic cooperativity between C/EBP beta and H-Ras(V12) AU - Shuman, JD AU - Sebastian, T AU - Kaldis, P AU - Copeland, TD AU - Zhu, SY AU - Smart, RC AU - Johnson, PF T2 - MOLECULAR AND CELLULAR BIOLOGY AB - CCAAT/enhancer binding protein beta (C/EBPbeta) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPbeta is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with RasV12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPbeta in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogen-activated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBPbeta activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G0) cells but was strongly increased in mid-G1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBPbeta, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited RasV12-induced focus formation. Our findings support a role for C/EBPbeta as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPbeta on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells. DA - 2004/9// PY - 2004/9// DO - 10.1128/MCB.24.17.7380-7391.2004 VL - 24 IS - 17 SP - 7380-7391 SN - 1098-5549 ER - TY - JOUR TI - Bioreactor landfills: progress continues AU - Barlaz, MA AU - Reinhart, D T2 - WASTE MANAGEMENT AB - Landfill bioreactors are based on an acceleration of in-situ waste biodegradation by performing leachate recirculation. To quantify the water content and to evaluate the leachate injection system, in-situ methods are required to obtain spatially distributed information, usually electrical resistivity tomography (ERT). In a previous study, the MICS (multiple inversions and clustering strategy) methodology was proposed to improve the hydrodynamic interpretation of ERT results by a precise delimitation of the infiltration area. In this study, MICS was applied on two ERT time-lapse data sets recorded on different waste deposit cells in order to compare the hydrodynamic behaviour of leachate flow between the two cells. This comparison is based on an analysis of: (i) the volume of wetted waste assessed by MICS and the wetting rate, (ii) the infiltration shapes and (iii) the pore volume used by the leachate flow. This paper shows that leachate hydrodynamic behaviour is comparable from one waste deposit cell to another with: (i) a high leachate infiltration speed at the beginning of the infiltration, which decreases with time, (ii) a horizontal anisotropy of the leachate infiltration shape and (iii) a very small fraction of the pore volume used by the leachate flow. This hydrodynamic information derived from MICS results can be useful for subsurface flow modelling used to predict leachate flow at the landfill scale. DA - 2004/// PY - 2004/// DO - 10.1016/j.wasman.2004.09.001 VL - 24 IS - 9 SP - 859-860 SN - 0956-053X ER -