TY - JOUR TI - Erratum: Effects of activated carbon surface chemistry and pore structure on the adsorption of organic contaminants from aqueous solution (Carbon (2002) 40 (2085-2100) PII: S0008622302000696) AU - Li, L. AU - Quinlivan, P.A. AU - Knappe, D.R.U. T2 - Carbon DA - 2003/// PY - 2003/// DO - 10.1016/S0008-6223(03)00121-0 VL - 41 IS - 8 SP - 1693 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0038617285&partnerID=MN8TOARS ER - TY - JOUR TI - Optimizing ferric sulfate coagulation of algae with streaming current measurements AU - Briley, D.S. AU - Knappe, D.R.U. T2 - Water Resources Research Institute News of the University of North Carolina DA - 2003/// PY - 2003/// IS - 341 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-40349083675&partnerID=MN8TOARS ER - TY - JOUR TI - NOM and MIB, who wins in the competition for activated carbon adsorption sites? AU - Newcombe, G. AU - Hepplewhite, C. AU - Knappe, D. T2 - Water DA - 2003/// PY - 2003/// VL - 30 IS - 1 SP - 33-36 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0037294576&partnerID=MN8TOARS ER - TY - JOUR TI - Estrogen signaling in trout liver: Activation of the ras p21/MAP-kinase pathway AU - Kullman, S.W. AU - Matsumura, F. AU - Hinton, D.E. T2 - Environmental Science DA - 2003/// PY - 2003/// VL - 10 SP - 223–227 ER - TY - JOUR TI - Analysis of medaka CYP3A gene regulation, promoter regulatory regions and cloning of the orphan nuclear receptor PXR AU - Kullman, S.W. AU - Hinton, D.E. T2 - The MDIBL Bulletin DA - 2003/// PY - 2003/// VL - 42 SP - 29–31 ER - TY - JOUR TI - ras oncogene mutations in diethylnitrosamine-induced hepatic tumors in medaka (Oryzias latipes), a teleost fish AU - Liu, Zi AU - Kullman, Seth W. AU - Bencic, David C. AU - Torten, Michael AU - Hinton, David E. T2 - Mutation Research/Genetic Toxicology and Environmental Mutagenesis AB - Medaka fish are an established non-mammalian research model for the study of liver carcinogenesis and exposure to environmental pollutants. Studies have emphasized the development of hepatic neoplasms in medaka following exposure to model carcinogens. To date however, little information is known regarding the mechanisms underlying initiation of hepatic tumors in this species. The aim of this study was to relate our understanding of diethylnitrosamine (DEN)-induced tumor formation to ras gene activation in hepatic neoplasms of exposed medaka. Initial studies were conducted to identify medaka ras exons 1 and 2 by reverse transcriptase polymerase chain reaction (RT-PCR). Amplification of ras exons 1 and 2 from untreated medaka liver resulted in the identification of three polymorphic ras sequence variants exhibiting a high degree of homology to other teleost and mammalian ras genes. Exposure of medaka to 159 ppm of DEN resulted in a wide range of hepatic neoplasms including: hepatocellular adenomas, hepatocellular carcinomas, cholangiomas, and mixed hepatocholangiocellular carcinomas. Individual liver tumors were examined for oncogenically activating ras mutations by probing genomic DNA with probes specific for activating point mutations or by direct cloning and sequencing of ras transcripts using RT-PCR. Using allele-specific oligonucleotide (ASO) analysis, a single point mutation was detected in codon 12 position two in 8/25 (32%) tumors examined. Mutated ras alleles were additionally detected in 12 of 39 (30%) medaka liver tumors by sequence analysis. Ten of the 12 mutations identified contained a single point mutation at codon 12 resulting in a Gly to Asp amino acid substitution. Two unique mutations were identified at codon 16 resulting in either Lys to Asn or Lys to Thr amino acid substitutions. Our results show that ras mutations are induced by DEN and are present in over 30% of the fish that developed tumors. A ras mutation incidence of 30% is similar to that reported in mammalian species exposed to DEN. While mutations at codon 12 have previously been reported, the present study is the first in vivo report of ras point mutations at codon 16. DA - 2003/8// PY - 2003/8// DO - 10.1016/s1383-5718(03)00133-5 VL - 539 IS - 1-2 SP - 43–53 SN - 1383-5718 UR - http://dx.doi.org/10.1016/s1383-5718(03)00133-5 KW - ras mutations KW - diethylnitrosamine KW - medaka KW - hepatic carcinogenesis ER - TY - JOUR TI - Method of treating alopecia AU - Smart, R. C. AU - Oh, H.-S. DA - 2003/// PY - 2003/// VL - 6,555,532 IS - 2003 Apr. 29 ER - TY - CONF TI - Performance-Based System For Post-Closure Care At MSW Landfills AU - Morris, J.W.F. AU - Houlihan, M.F. AU - Clark, S. AU - Barlaz, M.A. AU - Repa, E. AU - Sullivan, P.S. AU - Burt, D. T2 - Solid Waste Association of North America (SWANA) Landfill Symposium C2 - 2003/// CY - Atlantic City, NJ DA - 2003/// PY - 2003/6/16/ ER - TY - CONF TI - Factors Affecting the Bioavailability of Tetrachloroethylene Sorbed to Municipal Solid Waste Components AU - Zhang, Z. AU - Barlaz, M.A. AU - Knappe, D.R.U. T2 - 103rd General Meeting of the American Society for Microbiology C2 - 2003/// CY - Washington, DC DA - 2003/// PY - 2003/5/18/ ER - TY - CONF TI - Production of Non-Methane Organic Compounds (NMOCs) During the Decomposition of Refuse and Individual Waste Components Under Various Operating Conditions AU - Barlaz, M.A. AU - Cowie, S.J. AU - Hater, G.R. T2 - Solid Waste Association of North America (SWANA) Landfill Gas Symposium C2 - 2003/// CY - Tampa, FL DA - 2003/// PY - 2003/3/24/ ER - TY - CONF TI - Performance-Based System For Post-Closure Care At MSW Landfills- A New Approach To The Current 30-Year Time-Based System Of Subtitle D AU - Morris, J.W.F. AU - Houlihan, M.F. AU - Barlaz, M.A. AU - Sullivan, P.S. AU - Bonaparte, R. AU - Gallinatti, J.D. AU - Durant, N.D. AU - Gibbons, R.D. AU - Burt, D.M. AU - Clarke, H.S. AU - Baker, J.A. T2 - WasteTech C2 - 2003/// CY - New Orleans, LA DA - 2003/// PY - 2003/2/17/ ER - TY - SOUND TI - Landfill Chemistry and Microbiology and End of Post-Closure AU - Barlaz, M.A. DA - 2003/// PY - 2003/// M3 - Invited lecture ER - TY - RPRT TI - Effects of Activated Carbon Surface Chemistry and Pore Structure on the Adsorption of Trichloroethene and Methyl Tertiary-Butyl Ether from Natural Water AU - Knappe, D.R.U. AU - Li, L. AU - Quinlivan, P.A. AU - Wagner, T.B. A3 - American Water Works Association Research Foundation DA - 2003/// PY - 2003/// M3 - Executive Summary PB - American Water Works Association Research Foundation ER - TY - JOUR TI - Using Life-Cycle Analysis To Compare Solid Waste Management Alternatives Involving Recycling, Composting And Landfills AU - Barlaz, M.A. AU - Kaplan, P.O. AU - Ranjithan, S.R. T2 - MSW Management DA - 2003/// PY - 2003/// VL - 13 IS - 3 SP - 42–43 ER - TY - JOUR TI - Cancer Biomarkers: How Proteomics is Leading to the Discovery of New Markers AU - Muddiman, D.C. AU - Cliby, W.A. AU - Bergen, H.R., III T2 - Clinical Laboratory News DA - 2003/// PY - 2003/// VL - 29 SP - 12–16 ER - TY - JOUR TI - MGD: the Mouse Genome Database AU - Blake, J.A. AU - Richardson, J.E. AU - Bult, C.J. AU - Kadin, J.A. AU - Eppig, J.T. AU - Baldarelli, R.M. AU - Beal, J.S. AU - Bradt, D.W. AU - Burkart, D.L. AU - Butler, N.E. AU - Campbell, J. AU - Chu, T. AU - Corbani, L.E. AU - Cousins, S. AU - Drabkin, H.J. AU - Dahmen, D. AU - Frazer, K. AU - Garippa, D.M. AU - Goldsmith, C.W. AU - Grant, P.L. AU - Lennon-Pierce, M. AU - Lewis, J. AU - Lu, I. AU - Lutz, C.M. AU - Maltais, L.J. AU - Mani, P. AU - McKenzie, L.M. AU - Li, N. AU - Ormsby, J.E. AU - Planchart, Antonio AU - Ramachandran, S. AU - Reed, D.J. AU - Shaw, D.R. AU - Smith, C.L. AU - Szauter, P. AU - Borre, P. Vanden AU - Washburn, L. AU - Winslow, J. T2 - Nucleic Acids Research AB - The Mouse Genome Database (MGD) (http://www.informatics.jax.org) one component of a community database resource for the laboratory mouse, a key model organism for interpreting the human genome and for understanding human biology. MGD strives to provide an extensively integrated information resource with experimental details annotated from both literature and on-line genomic data sources. MGD curates and presents the consensus representation of genotype (sequence) to phenotype information including highly detailed information about genes and gene products. Primary foci of integration are through representations of relationships between genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse. Recent developments include a general implementation of database structures for controlled vocabularies and the integration of a phenotype classification system. DA - 2003/1/1/ PY - 2003/1/1/ DO - 10.1093/nar/gkg047 VL - 31 IS - 1 SP - 193–195 SN - 1362-4962 UR - http://dx.doi.org/10.1093/nar/gkg047 ER - TY - JOUR TI - Estrogens and ICI182,780 (Faslodex) modulate mitosis and cell death in immature cerebellar neurons via rapid activation of p44/p42 mitogen-activated protein kinase AU - Wong, J.K. AU - Le, H.H. AU - Zsarnovszky, A. AU - Belcher, S.M. T2 - Journal of Neuroscience DA - 2003/// PY - 2003/// VL - 23 IS - 12 SP - 4984-4995 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0038790428&partnerID=MN8TOARS ER - TY - JOUR TI - Enzymatic strategies for the characterization of nucleic acids by electrospray ionization mass spectrometry AU - Null, Allison P. AU - Benson, Linda M. AU - Muddiman, David C. T2 - Rapid Communications in Mass Spectrometry AB - Abstract Electrospray ionization mass spectrometry (ESI‐MS) is a powerful technique used for the identification and characterization of DNA polymorphisms. Continual improvement in instrument design assures high mass measurement accuracy, sensitivity, and resolving power. This work describes an eclectic array of enzymatic strategies we have invoked in order to detect single‐nucleotide polymorphisms by ESI‐MS, although other applications may be envisioned. One strategy combines the use of two enzymes, exonuclease III and lambda exonuclease, to provide a ladder of single‐stranded DNA fragments for straightforward sequence identification by mass spectrometry. A second strategy combines restriction enzymes to screen for polymorphisms present within specific amplicons. Finally, we describe the use of stable‐isotope‐labeled nucleotides for the determination of length and base composition of a PCR product. Copyright © 2003 John Wiley & Sons, Ltd. DA - 2003/// PY - 2003/// DO - 10.1002/rcm.1255 VL - 17 IS - 24 SP - 2699-2706 J2 - Rapid Commun. Mass Spectrom. LA - en OP - SN - 0951-4198 1097-0231 UR - http://dx.doi.org/10.1002/rcm.1255 DB - Crossref ER - TY - JOUR TI - 1H NMR and electrospray mass spectrometry of the mono-ionized bis(2,2′-bis(4,5-dimethylimidazole)chloronitrosylruthenium(II) complex ([Ru(NO)Cl(LH2)2]+, LH2=2,2′-bis(4,5-dimethylimidazole) AU - Stringfield, Thomas W AU - Somayajula, Kasi V AU - Muddiman, David C AU - Flora, Jason W AU - Shepherd, Rex E T2 - Inorganica Chimica Acta AB - The reaction of RuCl3(NO)(H2O)2 with 2 equiv. of LH2=2,2′-bis(4,5-dimethylimidazole) in refluxing ethanol generates cis-[Ru(NO)Cl(LH2)2]2+ (1), analogous in structure to cis-[Ru(NO)Cl(bpy)2]2+, as the chloride salt. In 1, the methyl groups are differentiated into eight different 1H NMR magnetic environments. Substitution of 4,5-dimethyl-2,2′-biimidazole (L′H2), an 11.1% impurity in commercial LH2, occurred randomly with the undecorated ring diminishing the intensity of all eight resonances equally. The methyl group of the “out-of-plane” ring that hangs over the π orbitals of the NO+ ligand (CH3(6)) is shifted most upfield from 2.19 ppm of free LH2 and is assigned to the 1.23 ppm resonance. With the aid of 2-D NMR methods, we assign the following shifts. The ring donor opposite the {Ru(NO)}3+, CH3(7), should experience the greatest withdrawing influence, and is assigned as the origin of the 2.50 ppm resonance. Its ring partner, CH3(5), placed below another ring current, is assigned to the resonance at 2.13 ppm. 2-D NMR methods support the assignments of 1.39 ppm to CH3(1) and 2.09 ppm to CH3(2), the “in-plane-near” CH3 groups; 2.36 and 2.34 ppm to “in-plane-remote” CH3(3) and CH3(4); a 2.47 ppm shift is assigned to the remaining CH3(8). Because of the presence of the impurity L′H2 which has a substitution rate advantage (ca. 2.14-fold) due to a lower steric barrier for the unmethylated ring, the isolated product contained 62.4% (1a) [Ru(NO)Cl(LH2)2]Cl2, 31.9% (1b) [Ru(NO)Cl(LH2)(L′H2)]Cl2, and 5.7% (1c) [Ru(NO)Cl(L′H2)2]Cl2. The ESI MS spectrum exhibits parent 1+ ions (2a–c) in which one of the imidazole rings has been deprotonated. Thus, eight-line patterns for RuCl-containing fragments appear for m/z with z=1+ centered about 546 (2a), 518 (2b) and 490 (2c) in the intensity ratios of 1.000:0.511:0.091, respectively. The atomic composition for 2a was shown to be RuC20H27N9OCl (m/z=540.109049) by checking the “540” line of the isotopic bundle around m/z=546. The atomic composition of 2b was established from the “512” mass as RuC18H23N9OCl (m/z=512.077748) from the isotopic bundle around m/z=518. 2a–c undergo the loss of LH2 or L′H2 with low efficiency, but few other fragmentations were observed. Notably, the loss of NO or HCl are absent. 2a–c have good π-donating imidazole and imidazolato functionalities which suppress NO loss. IR laser loss experiments on 2a in the mass spectral cavity were unable to identify a frequency value that selectively dissociates NO. Rather, complete fragmentation of the complexes 2a–c occurred at energies sufficient to induce any ligand dissociation; the parent ions being very robust. The 1H NMR data are supported by an MMFF94 energy-minimized structure for 1 and its theoretical trans isomer. DA - 2003/1// PY - 2003/1// DO - 10.1016/s0020-1693(02)01243-4 VL - 343 SP - 317-328 J2 - Inorganica Chimica Acta LA - en OP - SN - 0020-1693 UR - http://dx.doi.org/10.1016/s0020-1693(02)01243-4 DB - Crossref KW - ruthenium complexes KW - nitrosyl complexes KW - imidazole complexes KW - electrospray mass spectrometry ER - TY - JOUR TI - Naturally processed measles virus peptide eluted from class II HLA-DRB1*03 recognized by T lymphocytes from human blood AU - Ovsyannikova, Inna G AU - Johnson, Kenneth L AU - Naylor, Stephen AU - Muddiman, David C AU - Poland, Gregory A T2 - Virology AB - This is the first report of the direct identification of a HLA-DRB1*03 measles-derived peptide from measles virus infected EBV-transformed B cells. We purified HLA-DR3-peptide complexes from EBV-B cells infected with measles virus (Edmonston strain) and sequenced the HLA-DR3-peptides by mass spectrometry. A class II peptide, derived from a measles phosphoprotein, ASDVETAEGGEIHELLRLQ (P1, residues 179-197), exhibited the capacity to stimulate peripheral blood mononuclear cells to proliferate. Our data provides direct evidence that the antigenic peptide of measles virus was processed by antigen-presenting cells, presented in the context of HLA class II molecules, and was recognized by peripheral blood T cells from healthy individuals previously immunized with measles vaccine. The approach described herein provides a useful methodology for the future identification of HLA-presented pathogen-derived epitopes using mass spectrometry. The study of cell-mediated immune responses to the measles-derived peptide in immune persons should provide significant insight into the design and development of new vaccines. DA - 2003/8// PY - 2003/8// DO - 10.1016/s0042-6822(03)00281-2 VL - 312 IS - 2 SP - 495-506 J2 - Virology LA - en OP - SN - 0042-6822 UR - http://dx.doi.org/10.1016/s0042-6822(03)00281-2 DB - Crossref KW - measles-derived peptide KW - HLA-DRB1*03 KW - measles virus KW - mass spectrometry KW - lymphocyte proliferation ER - TY - JOUR TI - Coordination-Driven Self-Assembly of Supramolecular Cages:  Heteroatom-Containing and Complementary Trigonal Prisms AU - Kryschenko, Yury K. AU - Seidel, S. Russell AU - Muddiman, David C. AU - Nepomuceno, Angelito I. AU - Stang, Peter J. T2 - Journal of the American Chemical Society AB - The self-assembly of three nanoscopic prisms of approximate size 1 x 4 nm is reported. Tetrahedral carbon, silicon, and phosphorus were used as structure-defining elements in these coordination-based cages. A carbon-based assembly completes a pair of nanoscopic complementary 3-D structures. The formation of the structures is supported by multinuclear NMR, ESI FT-ICR mass spectrometry, and elemental analysis data. DA - 2003/8// PY - 2003/8// DO - 10.1021/ja030209n VL - 125 IS - 32 SP - 9647-9652 J2 - J. Am. Chem. Soc. LA - en OP - SN - 0002-7863 1520-5126 UR - http://dx.doi.org/10.1021/ja030209n DB - Crossref ER - TY - JOUR TI - Dual Electrospray Ionization Source for Confident Generation of Accurate Mass Tags Using Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry AU - Nepomuceno, Angelito I. AU - Muddiman, David C. AU - Bergen, H. Robert AU - Craighead, James R. AU - Burke, Michael J. AU - Caskey, Patrick E. AU - Allan, Jonathan A. T2 - Analytical Chemistry AB - Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has rapidly established a prominent role in proteomics because of its unparalleled resolving power, sensitivity and ability to achieve high mass measurement accuracy (MMA) simultaneously. However, space-charge effects must be quantitatively, routinely, and confidently corrected because they are known to profoundly influence MMA. We argue that the most effective way to account for space-charge effects is to introduce an internal mass calibrant (IMC) using a dual electrospray ionization (ESI) source where the IMC is added from a separate ESI emitter. The major disadvantage of our initial dual ESI source to achieve high MMA, and arguably the only one, was the time required to switch between the analyte emitter and IMC emitter (i.e., >300 ms). While this "switching time" was acceptable for direct infusion experiments, it did not lend itself to high-throughput applications or when conducting on-line liquid separations. In this report, we completely redesigned the dual ESI source and demonstrate several key attributes. First, the new design allows for facile alignment of ESI emitters, undetectable vibration, and the ability to extend to multiple emitters. Second, the switching time was reduced to <50 ms, which allowed the analyte and IMC to be accumulated "simultaneously" in the external ion reservoir and injected as a single ion packet into the ion cyclotron resonance cell, eliminating the need for a separate accumulation and ion injection event for the IMC. Third, by using a high concentration of the IMC, the residence time on this emitter could be reduced to approximately 80 ms, allowing for more time spent accumulating analyte ions of significantly lower concentration. Fourth, multiplexed on-line separations can be carried out providing increased throughput. Specifically, the new dual ESI source has demonstrated its ability to produce a stable ion current over a 45-min time period at 7 T resulting in mass accuracies of 1.08 ppm +/- 0.11 ppm (mean +/- confidence interval of the mean at 95% confidence; N = 160). In addition, the analysis of a tryptic digest of apomyoglobin by nanoLC-dual ESI-FT-ICR afforded an average MMA of -1.09 versus -74.5 ppm for externally calibrated data. Furthermore, we demonstrate that the amplitude of a peptide being electrosprayed at 25 nM can be linearly increased, ultimately allowing for dynamic analyte/IMC abundance modulation. Finally, we demonstrate that this source can reliably be used for multiplexing measurements from two (eventually more) flow streams. DA - 2003/7// PY - 2003/7// DO - 10.1021/ac0342471 VL - 75 IS - 14 SP - 3411-3418 J2 - Anal. Chem. LA - en OP - SN - 0003-2700 1520-6882 UR - http://dx.doi.org/10.1021/ac0342471 DB - Crossref ER - TY - JOUR TI - Determination of a correction to improve mass measurement accuracy of isotopically unresolved polymerase chain reaction amplicons by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry AU - Null, Allison P. AU - Muddiman, David C. T2 - Rapid Communications in Mass Spectrometry AB - The experimental determination of average mass by mass spectrometry is limited for large molecules due to the negative bias introduced by the natural distribution of isotopic abundances. This results in the measurement of the top-of-centroid (ToC) as opposed to the true centroid. We have developed a practical correction factor that is applied to the ToC measurement to largely remove the systematic bias introduced by nature. The correction factor is calculated easily using the average molecular mass (<100 kDa) of the analyte molecule and the full-width half maximum resolving power (<3,500) of the measurement. In addition, an approach to calculating resolving power is described that accurately predicts resolving power achievable for Fourier transform ion cyclotron resonance (FT-ICR) mass analysis of large molecules. A combination of internal calibration with a dual-electrospray source and application of the correction factor to average mass measurements improved the mass error from 192.5 to -35.0 ppm for a 44 kDa PCR amplicon. DA - 2003/// PY - 2003/// DO - 10.1002/rcm.1111 VL - 17 IS - 15 SP - 1714-1722 J2 - Rapid Commun. Mass Spectrom. LA - en OP - SN - 0951-4198 1097-0231 UR - http://dx.doi.org/10.1002/rcm.1111 DB - Crossref ER - TY - JOUR TI - Mass spectral determination of skeletal/cardiac actin isoform ratios in cardiac muscle AU - Bergen, H. Robert AU - Ajtai, Katalin AU - Burghardt, Thomas P. AU - Nepomuceno, Angelito I. AU - Muddiman, David C. T2 - Rapid Communications in Mass Spectrometry AB - Skeletal and cardiac muscle contains actin isoforms that vary by two juxtaposed amino acids and two amino acid substitutions (Met299Leu and Ser358Thr). This close sequence homology does not allow cardiac and skeletal actin isoforms to be resolved in traditional SDS-PAGE analysis as the molecular weights (Deltamass = 32 Da) are not significantly different and the pIs are identical (5.2). Although cardiac actin is the predominant form in cardiac muscle, there appears to be a specific skeletal/cardiac actin ratio in a normal heart that may vary in a compromised or diseased heart. In an effort to ascertain the validity of this hypothesis we developed a mass spectrometric technique to measure the ratio of skeletal to cardiac actin. The technique involves purification of muscle actin and subsequent liquid chromatography coupled with electrospray ionization Fourier transform ion cylcotron resonance (LC/FTICR-MS) mass spectrometry. A 7 Tesla FTICR mass spectrometer was utilized to compare skeletal/cardiac actin isoform ratios. Additionally, a new dual electrospray ionization source was employed to determine accurate masses of the alpha-skeletal and alpha-cardiac actins. DA - 2003/// PY - 2003/// DO - 10.1002/rcm.1075 VL - 17 IS - 13 SP - 1467-1471 J2 - Rapid Commun. Mass Spectrom. LA - en OP - SN - 0951-4198 1097-0231 UR - http://dx.doi.org/10.1002/rcm.1075 DB - Crossref ER - TY - JOUR TI - Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses AU - Benson, Linda M. AU - Null, Allison P. AU - Muddiman, David C. T2 - Journal of the American Society for Mass Spectrometry AB - The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z. DA - 2003/6// PY - 2003/6// DO - 10.1016/s1044-0305(03)00148-x VL - 14 IS - 6 SP - 601-604 J2 - J Am Soc Mass Spectrom LA - en OP - SN - 1044-0305 1879-1123 UR - http://dx.doi.org/10.1016/s1044-0305(03)00148-x DB - Crossref ER - TY - JOUR TI - Implications of Hydrophobicity and Free Energy of Solvation for Characterization of Nucleic Acids by Electrospray Ionization Mass Spectrometry AU - Null, Allison P. AU - Nepomuceno, Angelito I. AU - Muddiman, David C. T2 - Analytical Chemistry AB - Electrospray ionization (ESI) is a dynamic process that, when coupled with mass spectrometry (MS), serves as an invaluable tool for analysis of biomolecules. Our group, as well as others, has observed that there is a bias in signal intensity for one strand of a PCR amplicon over the complementary strand in an ESI mass spectrum. In this report, we have investigated the contributions of hydrophobicity and free energy of solvation to relative signal intensities in ESI-MS spectra of nucleic acids. We developed approaches for predicting which strand of the PCR amplicon will be the most intense: one based on a rate equation for calculating ion flux using values from the literature for hydrophobicity and free energy of solvation and the other based on the percentage of the relatively hydrophilic guanines present in the strand. A trend in signal intensity for deoxyribonucleotide triphosphates, oligonucleotides, and PCR amplicons was observed that was consistent with our model. On the basis of the observation that increased hydrophobicity correlates with greater signal intensity, we selectively enhanced the signal intensity of a 20-mer with the addition of an alkyl chain to the 5' terminus, which subsequently improved the limit of detection to 1 nM, an improvement by 1 order of magnitude. This was extended to a 53-bp PCR amplicon by modifying one primer with the hydrophobic moiety, which resulted in a 16% increase in signal intensity. We capitalized on this result to determine allele frequencies from pooled DNA for single-nucleotide polymorphisms down to 1%. DA - 2003/3// PY - 2003/3// DO - 10.1021/ac026217o VL - 75 IS - 6 SP - 1331-1339 J2 - Anal. Chem. LA - en OP - SN - 0003-2700 1520-6882 UR - http://dx.doi.org/10.1021/ac026217o DB - Crossref ER - TY - JOUR TI - Animal production and wheeze in the Agricultural Health Study: interactions with atopy, asthma, and smoking AU - Hoppin, J A T2 - Occupational and Environmental Medicine AB - Exposure to animals, their feeds, and by-products contribute to respiratory symptoms among farmers.To investigate the role of animal exposures and wheeze, and to assess whether their impact differs among susceptible subgroups, including atopics, asthmatics, and smokers.Using the Agricultural Health Study, a cohort of pesticide applicators in Iowa and North Carolina enrolled in 1994-97, wheeze associated with animal production was evaluated and interactions among susceptible subgroups assessed. Logistic regression models were used to examine risk factors for wheeze in the past year among 20 468 farmers.Individuals raising animals requiring direct contact had the highest odds ratios (OR) for wheeze (OR(dairy) = 1.26; OR(eggs) = 1.70). A significant dose response was observed for both the number of poultry and the number of livestock on the farm. Farmers who performed veterinary procedures on a daily basis had an OR of 1.51. The odds of wheeze associated with poultry production was greater among atopic than non-atopic individuals. Milking cows daily increased the odds of wheeze in all individuals, with the largest association observed among atopic asthmatic individuals. The impact of dairy, poultry, and egg production varied among smoking groups. Past smokers had the highest odds ratios, followed by never smokers, and then current smokers. The OR(eggs) was 2.88 among past smokers but only 1.46 for never smokers. The OR(eggs) for current smokers of 0.80 might reflect self selection of exposure among smokers.Results are consistent with animal production and respiratory symptoms, and suggest that subgroups may respond differently to exposure. DA - 2003/8/1/ PY - 2003/8/1/ DO - 10.1136/oem.60.8.e3 VL - 60 IS - 8 SP - 3e-3 J2 - Occupational and Environmental Medicine LA - en OP - SN - 1351-0711 UR - http://dx.doi.org/10.1136/oem.60.8.e3 DB - Crossref ER - TY - JOUR TI - Application of ion mobility to the gas-phase conformational analysis of polyhedral oligomeric silsesquioxanes (POSS) T2 - International Journal of Mass Spectrometry AB - Ion mobility experiments and molecular modeling calculations were used to investigate the gas-phase conformational properties of various polyhedral oligomeric silsesquioxanes (POSS) cationized by sodium. POSS, (RSiO3/2)n, has a rigid SiO cage with organic substituents attached to each Si atom. Na+POSS ions were formed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) and their collision cross-sections were measured in helium using ion mobility methods. Calculated cross-sections of theoretical models of the POSS ions, generated by molecular mechanics (MM) calculations, were compared to experiment for conformational identification. The calculations predict that the Na+ ion remains outside of the SiO cage and binds to one to two oxygen atoms in the cage or interacts with two neighboring organic substituents. Cross-sections of X-ray structures were also compared to the experimental and theoretical data to determine if any changes occur to POSS in the gas phase (compared to the condensed phase) and to provide a check for the geometries predicted by theory (which used untested Si parameters). Several types of POSS compounds were investigated that had different SiO cage sizes (Si6O9, Si8O12, Si10O15, Si12O18, …) and different “R” substituents such as cyclohexyl, cyclopropyl, vinyl, and phenyl groups. Experimental, theoretical, and X-ray cross-sections differed by <2% for each POSS compound. DA - 2003/// PY - 2003/// DO - 10.1016/S1387-3806(02)00951-X VL - 222 IS - 1-3 SP - 63-73 UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000180104600007&KeyUID=WOS:000180104600007 KW - POSS KW - ion mobility KW - gas-phase conformational analysis ER - TY - JOUR TI - 3-dimensional structural characterization of cationized polyhedral oligomeric silsesquioxanes (POSS) with styryl and phenylethyl capping agents AU - Baker, E.S. AU - Gidden, J. AU - Fee, D.P. AU - Kemper, P.R. AU - Anderson, S.E. AU - Bowers, M.T. T2 - International Journal of Mass Spectrometry AB - The 3-dimensional gas-phase conformations of polyhedral oligomeric silsesquioxanes (POSS), R8Si8O12, capped with styryl and phenylethyl substituents (R) and cationized by sodium were examined. MALDI was used to generate sodiated styryl–POSS (Na+Sty8T8) and phenylethyl–POSS (Na+PhEt8T8) ions and their collision cross-sections in helium were measured using ion mobility-based methods. Five distinct conformers with different collision cross-sections were experimentally observed for Na+Sty8T8 while only one conformer was detected for Na+PhEt8T8. Theoretical modeling of Na+Sty8T8, using molecular mechanics/dynamics calculations, predicts three low-energy conformations. In each conformer, the Na+ ion binds to four oxygens on one side of the SiO cage and the styryl groups extend away from the cage. However, different numbers of styryl groups “pair” together (forming 2, 3 or 4 pairs), yielding three different conformations. The calculated cross-sections of these conformers match the largest three cross-sections obtained from the ion mobility experiments (∼2% error). If, however, one or two of the styryl groups are rotated so that the phenyl groups are “cis” with respect to the Si atom on the cage (i.e., the SiCCC dihedral angle changes from 180 to 0°) two smaller conformers are predicted by theory whose cross-sections match the smallest two values obtained from the ion mobility experiments (1–2% error). Theoretical modeling of Na+PhEt8T8 yields one low-energy conformation in which the Na+ ion binds to one oxygen on the SiO cage and is sandwiched between two phenyl groups. The remaining phenylethyl groups fold toward the SiO cage, yielding a significantly more compact structure than Na+Sty8T8 (∼20% smaller cross-section). The calculated cross-section of the predicted Na+PhEt8T8 structure agrees very well with the experimental cross-section obtained from the ion mobility experiments (∼1% error). DA - 2003/// PY - 2003/// DO - 10.1016/S1387-3806(03)00068-X VL - 227 IS - 1 SP - 205-216 UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000182724200018&KeyUID=WOS:000182724200018 ER - TY - JOUR TI - Use of Agricultural Pesticides and Prostate Cancer Risk in the Agricultural Health Study Cohort AU - Alavanja, M. C. R. AU - Samanic, C. AU - Dosemeci, M. AU - Lubin, J. AU - Tarone, R. AU - Lynch, C.F. AU - Knott, C. AU - Thomas, K. AU - Hoppin, J.A. AU - Barker, J. AU - Coble, J. AU - Sandler, D.P. AU - Blair, A. T2 - American Journal of Epidemiology AB - The authors examined the relation between 45 common agricultural pesticides and prostate cancer incidence in a prospective cohort study of 55,332 male pesticide applicators from Iowa and North Carolina with no prior history of prostate cancer. Data were collected by means of self-administered questionnaires completed at enrollment (1993-1997). Cancer incidence was determined through population-based cancer registries from enrollment through December 31, 1999. A prostate cancer standardized incidence ratio was computed for the cohort. Odds ratios were computed for individual pesticides and for pesticide use patterns identified by means of factor analysis. A prostate cancer standardized incidence ratio of 1.14 (95% confidence interval: 1.05, 1.24) was observed for the Agricultural Health Study cohort. Use of chlorinated pesticides among applicators over 50 years of age and methyl bromide use were significantly associated with prostate cancer risk. Several other pesticides showed a significantly increased risk of prostate cancer among study subjects with a family history of prostate cancer but not among those with no family history. Important family history-pesticide interactions were observed. DA - 2003/5/1/ PY - 2003/5/1/ DO - 10.1093/aje/kwg040 VL - 157 IS - 9 SP - 800–814 SN - 0002-9262 UR - http://dx.doi.org/10.1093/aje/kwg040 KW - agrochemicals KW - fungicides, industrial KW - herbicides KW - insecticides KW - pesticides KW - prostatic neoplasms KW - risk ER - TY - JOUR TI - Male reproductive effects of phthalates: An emerging picture AU - Hoppin, J.A. T2 - Epidemiology AB - Like so many other byproducts of human enterprise, phthalates, without much attention paid to their possible health effects, have become widely distributed among people. In 2000, the Centers for Disease Control and Prevention (CDC) published the first data on phthalate levels in the U.S. population. The highest levels were still less than 1% of the lowest observable effect level in animals, though limited human health data were available. 1,2 However, three recent reports 3–5 (including one in this issue of Epidemiology by Susan Duty and colleagues 3) suggest that phthalates at current population levels may have measurable effects on male reproductive health. “…there is an emerging pattern of adverse semen parameters in the presence of high phthalate levels.” Just as “plastics” was the key word of advice to Dustin Hoffman in The Graduate, “phthalates” may become the key word for environmental epidemiologists in coming years. Phthalate esters are common in PVC plastics, paints and cosmetics. Exposure of laboratory animals as fetuses, pups and adults to phthalate esters can cause reproductive harm. In 1999, the U.S. National Toxicology Program commissioned an expert panel to assess seven phthalate esters and their risk to human reproduction. The candidates included two prime suspects: DEHP (diethylhexyl phthalate) and DBP (dibutyl phthalate). The primary metabolite of DEHP is MEHP (monoethylhexyl phthalate), an antiandrogen that disrupts male reproductive development in animals. Based on the limited scientific literature, which 2 years ago included virtually no human data, the panel identified “serious concern” for neonatal males exposed to DEHP. 6 Preterm babies are highly exposed to DEHP via intravenous and other medical tubing. 7 If phthalates damage male reproduction, preterm male infants are a group likely to experience problems. However, the ability to evaluate this group is limited. It is difficult to assemble a comparison group for a group of infants having extensive medical interventions. Although this remains a vital research question, we will have to look elsewhere for clues about potential reproductive damage from phthalates. In 2000, the CDC released the first population-based data on phthalate exposure. Four phthalate monoesters, including MEHP and MBP (the primary metabolite of DBP), were detectable in nearly all 289 people in an NHANES III sample from 1988–1994 2 and in most of the 1029 people from the NHANES data of 1999. 8 By far the highest levels were for monoethyl phthalate (MEP), a chemical not evaluated by the National Toxicology Program because of its apparent low toxicity in laboratory animals. The extent of human exposure is troubling. Detectable exposures range over two orders of magnitude, and there are practically no data on possible human health effects. In the past few months, the first results from epidemiologic studies have become available. 4–5 It appears that the reproductive effects of phthalates may not be limited to highly exposed animals. In studies from infertility clinics in India 4 and Boston, 5 there is an emerging pattern of adverse semen parameters in the presence of high phthalate levels. “Researchers…should not neglect the possibility that phthalates might affect women as well.” In India, Rozati and colleagues 4 measured phthalate levels in seminal fluid of community controls and patients being treated at an infertility clinic. Phthalates were associated with adverse morphology, sperm head defects and a higher percentage of single-strand deoxyribonucleic acid (DNA) in sperm. These authors used a measure of total phthalate diesters, which does not permit the responsible agent to be specifically identified. Because the treatment protocol was not mentioned, it is possible that these patients may have been exposed to specific phthalates in the course of their medical treatment. If the patients had the same pattern of phthalate exposure as the U.S. population, the main phthalate would be MEP. Among men at a Boston infertility clinic, urinary levels of MEP, but not other phthalates, were associated with adverse integrity of sperm DNA. 5 Although MEP is not known to be genotoxic, 9 taken together these studies suggest the need for further research into its potential to cause DNA damage in sperm. In this issue, Susan Duty and colleagues 3 provide further evidence from the Boston study of infertile men. This new paper reports an association of high urinary MBP levels with low sperm concentration and sperm motility. There was a similar association between MBzP (monobenzylphthalate) and sperm concentration. Surprisingly, no association was observed with MEHP, the phthalate of most concern in the report from the expert panel. Perhaps this is not so unexpected. As the authors note, 3 the animal literature suggests that DEHP (with its metabolite MEHP) has its effects on the male reproductive system only when exposure is before birth. DEHP exposure later in life apparently has no effect. 6 In contrast, DBP can apparently disrupt male reproduction at all stages of life. Thus, the apparent association of MBP (DBP’s active metabolite) with low sperm count and motility in humans is not implausible. Even so, these are all preliminary findings. These studies have been carried out within selected groups of men with known or suspected fertility problems. Both of the analyses by Duty 3,7 have been conducted among men from subfertile couples, so the spectrum of infertility represented is broader than among participants in the study by Rozati, 6 in which all cases had documented low sperm counts. All three studies relied on one sperm sample and one phthalate sample from each subject. As Duty notes, “an accurate measurement of sperm count is difficult using one specimen.” Although urinary phthalate levels appear reproducible from one day to the next, 10 no data are available to assess the long-term reliability of these urinary measures with a short half-life. The stage is now set for more detailed and comprehensive studies of men who are more representative of the general population. Finally, we should not overlook the fact that MBP levels are higher in women than men, 2 perhaps owing to exposures through cosmetics. The animal data suggest that phthalates can also have reproductive effects in females, including impaired uterine function. 11 As researchers attempt to replicate Duty’s findings in men, they should not neglect the possibility that phthalates might affect women as well. About the Author JANE HOPPIN is an environmental epidemiologist at the National Institute of Environmental Health Sciences of the National Institutes of Health. She has conducted studies on the health effects of lead and pesticides. Her current work includes the investigation of human exposure to phthalates, particularly with respect to patterns of exposure. DA - 2003/// PY - 2003/// DO - 10.1097/00001648-200305000-00002 VL - 14 IS - 3 SP - 259-260 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0038672685&partnerID=MN8TOARS ER - TY - JOUR TI - Animal production and wheeze in the Agricultural Health Study: interactions with atopy, asthma, and smoking. AU - Hoppin, J.A. AU - Umbach, D.M. AU - London, S.J. AU - Alavanja, M.C. AU - Sandler, D.P. T2 - Occupational and environmental medicine DA - 2003/// PY - 2003/// VL - 60 IS - 8 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0141454811&partnerID=MN8TOARS ER - TY - JOUR TI - The Comparative Toxicogenomics Database (CTD). AU - Mattingly, Carolyn J AU - Colby, Glenn T AU - Forrest, John N AU - Boyer, James L T2 - Environmental Health Perspectives AB - The Mount Desert Island Biological Laboratory in Salsbury Cove, Maine, USA, is developing the Comparative Toxicogenomics Database (CTD), a community-supported genomic resource devoted to genes and proteins of human toxicologic significance. CTD will be the first publicly available database to a) provide annotated associations among genes, proteins, references, and toxic agents, with a focus on annotating data from aquatic and mammalian organisms; b) include nucleotide and protein sequences from diverse species; c) offer a range of analysis tools for customized comparative studies; and d) provide information to investigators on available molecular reagents. This combination of features will facilitate cross-species comparisons of toxicologically significant genes and proteins. These comparisons will promote understanding of molecular evolution, the significance of conserved sequences, the genetic basis of variable sensitivity to environmental agents, and the complex interactions between the environment and human health. CTD is currently under development, and the planned scope and functions of the database are described herein. The intent of this report is to invite community participation in the development of CTD to ensure that it will be a valuable resource for environmental health, molecular biology, and toxicology research. DA - 2003/5// PY - 2003/5// DO - 10.1289/ehp.6028 VL - 111 IS - 6 SP - 793-795 J2 - Environmental Health Perspectives LA - en OP - SN - 0091-6765 1552-9924 UR - http://dx.doi.org/10.1289/ehp.6028 DB - Crossref KW - aquatic KW - comparative KW - database KW - environmental health KW - fishes KW - genomic KW - health KW - toxicogenomics KW - toxicology ER - TY - BOOK TI - Complex function sets improve symbolic discriminant analysis of microarray data AU - Reif, D.M. AU - White, B.C. AU - Olsen, N. AU - Aune, T. AU - Moore, J.H. DA - 2003/// PY - 2003/// VL - 2724 SE - 2277-2287 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-33744780676&partnerID=MN8TOARS ER - TY - JOUR TI - Complex Function Sets Improve Symbolic Discriminant Analysis of Microarray Data AU - Reif, David M. AU - White, Bill C. AU - Olsen, Nancy AU - Aune, Thomas AU - Moore, Jason H. T2 - Genetic and Evolutionary Computation — GECCO 2003 AB - Our ability to simultaneously measure the expression levels of thousands of genes in biological samples is providing important new opportunities for improving the diagnosis, prevention, and treatment of common diseases. However, new technologies such as DNA microarrays are generating new challenges for variable selection and statistical modeling. In response to these challenges, a genetic programming-based strategy called symbolic discriminant analysis (SDA) for the automatic selection of gene expression variables and mathematical functions for statistical modeling of clinical endpoints has been developed. The initial development and evaluation of SDA has focused on a function set consisting of only the four basic arithmetic operators. The goal of the present study is to evaluate whether adding more complex operators such as square root to the function set improves SDA modeling of microarray data. The results presented in this paper demonstrate that adding complex functions to the terminal set significantly improves SDA modeling by reducing model size and, in some cases, reducing classification error and runtime. We anticipate SDA will be an important new evolutionary computation tool to be added to the repertoire of methods for the analysis of microarray data. DA - 2003/// PY - 2003/// DO - 10.1007/3-540-45110-2_121 SP - 2277–2287 ER - TY - JOUR TI - The Zebrafish as a Model Organism to Study Development of the Immune System AU - TRAVER, DAVID AU - HERBOMEL, PHILIPPE AU - PATTON, E.ELIZABETH AU - MURPHEY, RYAN D. AU - Yoder, Jeffrey AU - LITMAN, GARY W. AU - CATIC, ANDRÉ AU - AMEMIYA, CHRIS T. AU - LEONARD, I. ZON AU - TREDE, NIKOLAUS S. AU - al. T2 - Advances in Immunology AB - Early events in the development of the primitive and definitive blood forming system are still poorly understood. Additionally, the specification of both B and T cells occurs during embryogenesis and, given the completion of this process before birth, are difficult to study in mammals by forward genetics. Historically, the major strength of the zebrafish has been the opportunity it offered to carry forward genetic screens in a vertebrate organism in a relatively restricted space. Establishing the zebrafish as a model system for the study of the immune system will provide an alternative and complementary tool to the use of forward genetic screens in mice. Rapid advances in a variety of fields have allowed the zebrafish to become a more versatile tool for immunology. DA - 2003/// PY - 2003/// DO - 10.1016/s0065-2776(03)81007-6 VL - 81 SP - 254-330 ER - TY - JOUR TI - Novel Immune-type Receptor Genes and the Origins of Adaptive and Innate Immune Recognition AU - Litman, G.W. AU - Yoder, J.A. AU - Cannon, J.P. AU - Haire, R.N. T2 - Integrative and Comparative Biology AB - The prototypic forms of teleost novel immune-type receptors (NITRs) consist of a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs). NITRs encode diversified V regions in large multigene families but do not undergo somatic rearrangement. Studies in four different bony fish model systems have identified a number of different organizational forms of NITRs. Specifically, NITR genes encode N-terminal ectodomains of the V-type but otherwise vary in the: total number of extracellular immunoglobulin domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues within transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. V region-containing NITRs constitute a far more complex family than recognized originally and currently include individual members that potentially function through inhibitory as well as activating mechanisms. The genomic organization of the NITR gene cluster as well as the structural diversity and overall architecture of the NITR proteins is reminiscent of genes encoded at the mammalian leukocyte receptor cluster (LRC); however, there presently is no functional evidence to support an orthologous relationship between NITR and LRC gene products. Comparisons of the predicted structures of the NITRs have identified several short regions of sequence identity and a novel cloning strategy has been devised that selects for secretory and transmembrane proteins that encode these short motifs. Using this approach, related genes termed immune-type receptors (ITRs) have been identified in cartilaginous fish. Taken together, these studies indicate that leukocyte regulatory receptors, including those that mediate natural killer function, might have emerged early in vertebrate evolution and that the NITR/ITR genes represent a new and potentially highly significant link between innate and adaptive immune responses. DA - 2003/4/1/ PY - 2003/4/1/ DO - 10.1093/icb/43.2.331 VL - 43 IS - 2 SP - 331-337 SN - 1540-7063 1557-7023 UR - http://dx.doi.org/10.1093/icb/43.2.331 ER - TY - CHAP TI - Using the asphalt pavement layer condition assessment program - Case studies AU - Xu, B. AU - Ranjithan, S. R. AU - Kim, Y. R. T2 - Pavement assessment, monitoring and evaluation 2003 AB - The Asphalt Pavement Layer Condition Assessment Program (APLCAP) is developed in this research to help highway agencies assess layer conditions of asphalt pavements. APLCAP implements a new integrated procedure for condition assessment from falling-weight deflectometer (FWD) deflections. The main components of this procedure include screening of FWD raw deflections, predictions of condition indicators from FWD measurements, structural adjustments for the predicted condition indicators, and layer condition evaluation based on the adjusted condition indicators. This procedure was developed on the basis of dynamic nonlinear finite element analysis and calibrated using field measurements. The three case studies presented show that the APLCAP algorithms can predict the asphalt concrete modulus, pavement critical strains, and strengths of the base and subgrade quite well, but not the compressive strain in the aggregate base layer. Although the APLCAP procedure includes the complicated dynamic effect of FWD loading and nonlinear behavior of unbound materials, the time to obtain results from this procedure is insignificant and therefore suitable for real-time evaluation of pavement conditions. CN - TE7 .H5 no.1860 PY - 2003/// DO - 10.3141/1860-08 SP - 66-75 PB - Washington, DC: Transportation Research Board SN - 0309085977 ER - TY - JOUR TI - Relationship of compressibility parameters to municipal solid waste decomposition AU - Hossain, M. S. AU - Gabr, Mohammed AU - Barlaz, Morton T2 - Journal of Geotechnical and Geoenvironmental Engineering AB - Recently, there has been substantial interest in the enhancement of refuse decomposition in landfills, which results in increased settlement. In this paper, changes in waste compressibility as a function of the state of decomposition are reported. Samples representative of residential refuse were decomposed under conditions designed to simulate decomposition in both control and bioreactor landfills. Twenty four one-dimensional oedometer tests (63.5 mm cell) were performed on refuse prepared in laboratory-scale reactors for measurement of primary (Cc) and secondary (Cαi, representing creep, and Cβi, representing biological) compression indices. The state of decomposition was quantified by the methane yield and the cellulose (C) plus hemicellulose (H) to lignin (L) ratio. The magnitude of compressibility was shown to increase as refuse decomposed and compressibility parameters were correlated with the state of decomposition. Initial settlement increased with decreasing (C+H)/L ratio while the creep index was fairly independent of the state of decomposition. The coefficients of primary compression (Cc) for bioreactor samples showed an increasing trend with decreasing (C+H)/L ratios. Cc increased from 0.16 to 0.36 as (C+H)/L decreased from 1.29 to 0.25, and similar values of Cc were obtained with control samples at similar (C+H)/L ratios. The creep index range was estimated at 0.02–0.03 for control and bioreactor samples in various states of decomposition. The magnitude of the biological degradation index (Cβi) depended on the degradation phase with the highest value of 0.19 obtained during the phase of accelerated methane production. Proposing a single Cc for landfill settlement calculations may lead to inaccurate predictions. Properties of each waste sublayer will change as a function of the decomposition stage, and dominating processes with appropriate compressibility parameters should be applied to individual sublayers. DA - 2003/// PY - 2003/// DO - 10.1061/(ASCE)1090-0241(2003)129:12(1151) VL - 129 IS - 12 SP - 1151–1158 ER - TY - JOUR TI - Lithium stabilizes the CCAAT/enhancer-binding protein alpha (C/EBP alpha) through a glycogen synthase kinase 3 (GSK3)-independent pathway involving direct inhibition of proteasomal activity AU - Shim, M AU - Smart, RC T2 - JOURNAL OF BIOLOGICAL CHEMISTRY AB - CCAAT/enhancer-binding protein α (C/EBPα), a basic leucine zipper transcription factor, is involved in mitotic growth arrest and differentiation. Given that numerous proteins involved in cell cycle regulation are degraded via the ubiquitin-proteasome system, we examined whether the C/EBPα protein is degraded via a proteasomal mechanism. In cycloheximide-treated BALB/MK2 keratinocytes we found that C/EBPα is a short-lived protein with a half-life of ∼1 h. Treatment with proteasome inhibitors, MG-132 or lactacystin, blocked the degradation of the C/EBPα protein. Higher molecular weight species of ubiquitinated C/EBPα were detected in BALB/MK2, and in vitro studies confirmed that C/EBPα is degraded by the proteasome in an ATP- and ubiquitin-dependent manner. GSK3 is a known C/EBPα kinase and treatment of keratinocytes with LiCl, an inhibitor of GSK3 resulted in: (i) a 5-fold increase in C/EBPα protein levels, (ii) increased electrophoretic mobility of C/EBPα, and (iii) no increase in C/EBPα mRNA levels suggesting that GSK3-mediated phosphorylation of C/EBPα may target it for proteasomal degradation. However, a mutant C/EBPα containing T to A mutations in the GSK3 phosphorylation sites (T222A and T226A) retained its response to LiCl, and additional pharmacological inhibitors of GSK3 did not alter C/EBPα levels indicating the effects of LiCl on C/EBPα are GSK3-independent. LiCl treatment of BALB/MK2 cells inhibited C/EBPα degradation and produced a 6-fold increase in the half-life of C/EBPα protein. In vitro studies revealed that LiCl inhibited proteasome activity and the ensuing degradation of C/EBPα. These results demonstrate C/EBPα is degraded via a ubiquitin-dependent proteasomal pathway, and LiCl stabilizes C/EBPα through a GSK3-independent pathway involving direct inhibition of proteasome activity. CCAAT/enhancer-binding protein α (C/EBPα), a basic leucine zipper transcription factor, is involved in mitotic growth arrest and differentiation. Given that numerous proteins involved in cell cycle regulation are degraded via the ubiquitin-proteasome system, we examined whether the C/EBPα protein is degraded via a proteasomal mechanism. In cycloheximide-treated BALB/MK2 keratinocytes we found that C/EBPα is a short-lived protein with a half-life of ∼1 h. Treatment with proteasome inhibitors, MG-132 or lactacystin, blocked the degradation of the C/EBPα protein. Higher molecular weight species of ubiquitinated C/EBPα were detected in BALB/MK2, and in vitro studies confirmed that C/EBPα is degraded by the proteasome in an ATP- and ubiquitin-dependent manner. GSK3 is a known C/EBPα kinase and treatment of keratinocytes with LiCl, an inhibitor of GSK3 resulted in: (i) a 5-fold increase in C/EBPα protein levels, (ii) increased electrophoretic mobility of C/EBPα, and (iii) no increase in C/EBPα mRNA levels suggesting that GSK3-mediated phosphorylation of C/EBPα may target it for proteasomal degradation. However, a mutant C/EBPα containing T to A mutations in the GSK3 phosphorylation sites (T222A and T226A) retained its response to LiCl, and additional pharmacological inhibitors of GSK3 did not alter C/EBPα levels indicating the effects of LiCl on C/EBPα are GSK3-independent. LiCl treatment of BALB/MK2 cells inhibited C/EBPα degradation and produced a 6-fold increase in the half-life of C/EBPα protein. In vitro studies revealed that LiCl inhibited proteasome activity and the ensuing degradation of C/EBPα. These results demonstrate C/EBPα is degraded via a ubiquitin-dependent proteasomal pathway, and LiCl stabilizes C/EBPα through a GSK3-independent pathway involving direct inhibition of proteasome activity. The CCAAT/enhancer-binding protein α (C/EBPα) 1The abbreviations used are: C/EBPα, CCAAT/enhancer-binding protein α; GSK3, glycogen synthase kinase 3; EMEM, Eagle's minimal essential medium; hEGF, human epidermal growth factor; ERS, energy regeneration system. is a member of the basic leucine zipper (bZIP) class of transcription factors. There are six members of the C/EBP family (C/EBPα, C/EBPβ, C/EBPδ, C/EBPϵ, C/EBPγ, and C/EBPζ) (1Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1401) Google Scholar, 2Williams S.C. Cantwell C.A. Johnson P.F. Genes Dev. 1991; 5: 1553-1567Crossref PubMed Scopus (464) Google Scholar, 3Wedel A. Ziegler-Heitbrock H.W. Immunobiology. 1995; 193: 171-185Crossref PubMed Scopus (214) Google Scholar). The C-terminal region of C/EBP contains a basic domain that is responsible for binding specific DNA sequences and a leucine zipper domain that functions in dimerization (4Vinson C.R. Sigler P.B. McKnight S.L. Science. 1989; 246: 911-916Crossref PubMed Scopus (824) Google Scholar). C/EBPα can form homodimers as well as heterodimers with other members of the C/EBP family. The N-terminal region of C/EBPα contains three transactivation elements and an additional highly conserved region (CR4) that is thought to have a regulatory function. C/EBPα is highly expressed in liver, fat, lung, peripheral leukocytes, epidermis, intestine, and skeletal muscle (5Birkenmeier E.H. Gwynn B. Howard S. Jerry J. Gordon J.I. Landschulz W.H. McKnight S.L. Genes Dev. 1989; 3: 1146-1156Crossref PubMed Scopus (529) Google Scholar, 6Oh H.S. Smart R.C. J. Invest. Dermatol. 1998; 110: 939-945Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Antonson P. Xanthopoulos K.G. Biochem. Biophys. Res. Commun. 1995; 215: 106-113Crossref PubMed Scopus (83) Google Scholar). In numerous cell types, including preadipocytes (8Umek R.M. Friedman A.D. McKnight S.L. Science. 1991; 251: 288-292Crossref PubMed Scopus (601) Google Scholar, 9Lin F.T. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8757-8761Crossref PubMed Scopus (390) Google Scholar), various myeloid cells (10Radomska H.S. Huettner C.S. Zhang P. Cheng T. Scadden D.T. Tenen D.G. Mol. Cell. Biol. 1998; 18: 4301-4314Crossref PubMed Scopus (415) Google Scholar, 11Wang X. Scott E. Sawyers C.L. Friedman A.D. Blood. 1999; 94: 560-571Crossref PubMed Google Scholar), hepatocytes (12Diehl A.M. Johns D.C. Yang S. Lin H. Yin M. Matelis L.A. Lawrence J.H. J. Biol. Chem. 1996; 271: 7343-7350Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and keratinocytes (6Oh H.S. Smart R.C. J. Invest. Dermatol. 1998; 110: 939-945Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 13Zhu S. Oh H.S. Shim M. Sterneck E. Johnson P.F. Smart R.C. Mol. Cell. Biol. 1999; 19: 7181-7190Crossref PubMed Google Scholar), C/EBPα is involved in the regulation of mitotic growth arrest and/or differentiation. Consistent with this, C/EBPα-null mice display cell proliferation defects in liver and lung (14Wang N.D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (850) Google Scholar, 15Flodby P. Barlow C. Kylefjord H. Ahrlund-Richter L. Xanthopoulos K.G. J. Biol. Chem. 1996; 271: 24753-24760Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). C/EBPα also plays a key role in energy homeostasis. C/EBPα-null mice die shortly after birth because of altered hepatic glucose and glycogen metabolism and also display defects in white adipose tissue differentiation (14Wang N.D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (850) Google Scholar). The importance of C/EBPα in the regulation of growth arrest and differentiation is exemplified by recent studies in which dominant-negative mutations in C/EBPα were found to be associated with human acute myeloid leukemia (16Pabst T. Mueller B.U. Zhang P. Radomska H.S. Narravula S. Schnittger S. Behre G. Hiddemann W. Tenen D.G. Nat. Genet. 2001; 27: 263-270Crossref PubMed Scopus (759) Google Scholar, 17Pabst T. Mueller B.U. Harakawa N. Schoch C. Haferlach T. Behre G. Hiddemann W. Zhang D.E. Tenen D.G. Nat. Med. 2001; 7: 444-451Crossref PubMed Scopus (401) Google Scholar). Such mutations in C/EBPα are thought to result in a differentiation block of the granulocytic blasts and have implicated C/EBPα as a tumor suppressor gene. The growth and differentiation regulatory functions of C/EBPα are complex and multifaceted. For example, C/EBPα has been proposed to regulate p21 expression (18Timchenko N.A. Wilde M. Nakanishi M. Smith J.R. Darlington G.J. Genes Dev. 1996; 10: 804-815Crossref PubMed Scopus (347) Google Scholar, 19Cram E.J. Ramos R.A. Wang E.C. Cha H.H. Nishio Y. Firestone G.L. J. Biol. Chem. 1998; 273: 2008-2014Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and interact with retinoblastoma (Rb) family proteins (20Timchenko N.A. Wilde M. Darlington G.J. Mol. Cell. Biol. 1999; 19: 2936-2945Crossref PubMed Google Scholar, 21Chen P.L. Riley D.J. Chen Y. Lee W.H. Genes Dev. 1996; 10: 2794-2804Crossref PubMed Scopus (397) Google Scholar). C/EBPα has been shown to directly repress E2F function through its physical associations with E2F, and this repression is necessary for growth arrest and adipocyte and granulocyte differentiation (22Porse B.T. Pedersen T.A. Xu X. Lindberg B. Wewer U.M. Friis-Hansen L. Nerlov C. Cell. 2001; 107: 247-258Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). However, recent studies indicate that C/EBPα can block growth independent of its DNA binding and transcriptional activity by forming a complex with cdk2 and cdk4, thereby blocking cyclin-cdk interactions and cell cycle progression (23Wang H. Iakova P. Wilde M. Welm A. Goode T. Roesler W.J. Timchenko N.A. Mol. Cell. 2001; 8: 817-828Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Thus, it appears that in addition to its DNA binding/transcription factor activity, C/EBPα can modulate growth arrest and differentiation by protein-protein interactions with cell cycle regulatory proteins independent of its transcription activity. Therefore, the identification of cellular processes that impinge on the regulation of C/EBPα protein stability may be critical in understanding the regulation and/or deregulation of C/EBPα-induced growth arrest and differentiation. Ubiquitin-proteasome degradation system plays an important role in the degradation of cellular proteins, which are involved in regulating various cellular processes, including cell cycle regulation, differentiation, and apoptosis (24Myung J. Kim K.B. Crews C.M. Med. Res. Rev. 2001; 21: 245-273Crossref PubMed Scopus (375) Google Scholar, 25Ciechanover A. Orian A. Schwartz A.L. Bioessays. 2000; 22: 442-451Crossref PubMed Scopus (717) Google Scholar). Recent studies have indicated that GSK3 phosphorylates a number of cell cycle regulatory proteins including p21 (26Rossig L. Badorff C. Holzmann Y. Zeiher A.M. Dimmeler S. J. Biol. Chem. 2002; 277: 9684-9689Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), β-catenin (27Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2201) Google Scholar), cyclin D (28Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1882) Google Scholar), and c-Myc (29Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (1003) Google Scholar) and in doing so targets these molecules for proteasomal degradation. Lithium, an inhibitor of GSK3 (30Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar, 31Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Crossref PubMed Scopus (457) Google Scholar), blocked the proteasomal degradation of these proteins. C/EBPα can be phosphorylated by GSK3 on Thr222 and Thr226, and this phosphorylation can be blocked by lithium (32Ross S.E. Erickson R.L. Hemati N. MacDougald O.A. Mol. Cell. Biol. 1999; 19: 8433-8441Crossref PubMed Google Scholar, 33Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Science. 2000; 289: 950-953Crossref PubMed Scopus (1572) Google Scholar). However, the functional significance of GSK3-mediated phosphorylation is not known, nor is it known whether C/EBPα is degraded via a proteasomal pathway or whether GSK3 or lithium treatment can alter C/EBPα protein degradation/stability. While the effect of lithium on the inhibition of proteasomal degradation of proteins that are GSK3 substrates has generally been attributed to its inhibition of GSK3, lithium also inhibits a number of other cellular enzymes and more recently lithium has been shown to a be an inhibitor of chymotryptic activity of both the 20 S and 26 S proteasome (34Rice A.M. Sartorelli A.C. J. Biol. Chem. 2001; 276: 42722-42727Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Therefore, we have examined whether C/EBPα is degraded via a proteasomal mechanism and investigated the role of GSK3 and lithium on C/EBPα protein stability. We demonstrate that C/EBPα is degraded via an ubiquitin-dependent proteasomal pathway and that lithium stabilizes the C/EBPα protein through a GSK3-independent pathway involving direct inhibition of proteasomal activity. Materials—Fetal bovine serum, trypsin, human recombinant epidermal growth factor (hEGF), lipofectin, and Tris-glycine gels were purchased from Invitrogen. Ca2+-free Eagle's minimal essential medium (EMEM) was purchased from BioWhittaker. The enhanced chemiluminescence (ECL) reagents were purchased from PerkinElmer Life Sciences. Horseradish peroxidase-linked donkey anti-rabbit IgG was purchased from Amersham Biosciences. Ubiquitin, HeLa S-100 fraction, energy regeneration system (ERS), ubiquitin-aldehyde, MG-132, and lactacystin were purchased from Boston Biochem. Cycloheximide and monoclonal anti-FLAG antibody were purchased from Sigma. Bio-Rad protein assay reagent was purchased from Bio-Rad. Construction of pcDNA3-C/EBPα has been previously described (13Zhu S. Oh H.S. Shim M. Sterneck E. Johnson P.F. Smart R.C. Mol. Cell. Biol. 1999; 19: 7181-7190Crossref PubMed Google Scholar). λ-phosphatase was purchased from New England Biolabs. GSK3 inhibitor SB216763 and SB415286 were from GlaxoSmithKline. pCMV-FLAG expression vector was a kind gift from Dr. Jun Tsuji (North Carolina State University, Raleigh, NC). Rabbit polyclonal anti-ubiquitin antibody, rabbit polyclonal anti-C/EBPα antibody, rabbit polyclonal anti-p53 antibody, and protein A/G-plus agarose were purchased from Santa Cruz Biotechnology. Transfection and Lithium Treatment—BALB/MK2 keratinocytes (a gift from Dr. Weissman, University of North Carolina, Chapel Hill, NC) were plated at 2.5 × 105 cells/60-mm culture dish in Ca2+-free EMEM supplemented with 8% Chelex-treated fetal bovine serum, 4 ng of hEGF per ml, and 0.05 mm calcium chloride. Two days after plating, BALB/MK2 keratinocytes were transfecetd with 2 μg of pcDNA3-C/EBPα and 12 μg of lipofectin in 2 ml of serum-free EMEM containing 4 ng of hEGF per ml, and 0.05 mm calcium chloride according to the manufacturer's protocol. Twenty-four hours following transfection, cells were refed with EMEM supplemented with 8% Chelex-treated fetal bovine serum, 4 ng of hEGF per ml, and 0.05 mm calcium chloride, and incubated with 20 mm lithium chloride for 24 h. Preparation of Nuclear and Whole Cell Lysates—Nuclear extracts were prepared as previously described by Schreiber et al. (35Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3978) Google Scholar). For the preparation of whole cell lysates, cells were washed with cold phosphate-buffered saline, harvested by scraping, collected by brief centrifugation. Cells were lysed in lysis buffer (10 mm Hepes, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 100 μg/ml aprotinin, 100 μg/ml leupeptin, 1 mm sodium orthovanadate, 0.6% Nonidet P-40) by sonication, and then one-tenth volume of 5 m NaCl was added. The incubation mixture was vortexed, incubated for 15 min on ice, and centrifuged (14,000 × g, 10 min, 4 °C). Supernatants were stored at –80 °C until use. Protein concentration was determined by the Bio-Rad protein assay reagent. Western Blot Analysis—Equal amounts of protein were precipitated by adding equal volume of 20% trichloroacetic acid and washed with acetone (–20 °C). Protein samples were solubilized and boiled in SDS sample buffer for 2 min and then separated by SDS-PAGE. The separated proteins were transferred to an Immobilon-P membrane (Millipore). Following incubation in blocking buffer (phosphate-buffered saline with 1% bovine serum albumin, 5% nonfat dry milk, and 0.1% Tween-20) for1hat room temperature, the membranes were probed for 2 h at room temperature with rabbit polyclonal IgG raised against C/EBPα (Santa Cruz Biotechnology). The membranes were washed and then probed with a horseradish peroxidase-linked secondary antibody for 1 h at room temperature. Detection was made with an enhanced chemiluminescence reagent followed by exposure of the membrane to film. Luciferase Assay—BALB/MK2 keratinocytes were plated at 1 × 105 cells/well in a 12-well culture plate. Two days after plating, BALB/MK2 keratinocytes were transfected in triplicate with 100 ng of pcDNA3-C/EBPα and 400 ng of the specified C/EBP-dependent promoter/reporter plasmid as described in the text and 3 μg of lipofectin in 0.5 ml of serum-free EMEM according to the manufacturer's protocol. Forty-eight hours later, cells were harvested, and the luciferase activity was determined by using the luciferase assay kit (Promega). Protein concentration was determined with the Bio-Rad protein assay reagent. Dephosphorylation of C/EBPα Protein by Phosphatase Treatment— BALB/MK2 cells were transfected with pcDNA3-C/EBPα, and 48 h following transfection, nuclear extracts were isolated as described above, except that the phosphatase inhibitor (sodium orthovanadate) was omitted. 50 μg of nuclear extracts were incubated with 400 units of λ-phosphatase in 50 μl of phosphatase buffer (50 mm Tris-HCl, pH 7.5, 0.1 mm EDTA, 5 mm dithiothreitol, 0.01% Brij 35, and 2 mm MnCl2) at 30 °C for 1 h. The reaction was stopped by adding equal volume of 20% trichloroacetic acid. Precipitated proteins were washed with acetone (–20 °C), solubilized and boiled in SDS sample buffer for 2 min. Protein samples were separated by SDS-PAGE and analyzed by Western blotting. Northern Blot Analysis—Total RNA was isolated from untransfected or pcDNA3-C/EBPα-transfected BALB/MK2 cells using Promega's SV total RNA isolation kit. C/EBPα cDNA was labeled with [α-32P]dCTP by using Ready-To-Go labeling beads (Amersham Biosciences). RNA was electrophoresed on agarose gel containing formaldehyde, transferred to zeta-probe GT membrane (BioRad), and UV cross-linked. Blots were incubated at 65 °C in hybridization buffer (0.25 m Na2HPO4, pH 7.2, 7% SDS) and sequentially washed with washing buffer 1 (20 mm Na2HPO4, pH 7.2, 5% SDS) and washing buffer 2 (20 mm Na2HPO4, 1% SDS) at room temperature. Films were exposed to membrane at –80 °C and developed. Inhibition of C/EBPα Degradation by Proteasomal Inhibitors— BALB/MK2 cells were transfected with pcDNA3-C/EBPα. Forty-six hours following transfection, cells were incubated with either 25 μm MG-132 or 10 μm lactacystin for 30 min prior to the addition of 50 μg/ml cycloheximide. Lysates were prepared at the indicated time points and subjected to Western blot analysis. MG-132 was prepared as a 25 mm stock solution in Me2SO, and lactacystin was prepared as a 5 mm stock solution in Me2SO. Control experiments were carried out with Me2SO. In Vitro Proteasomal Degradation Assay—Degradation reactions were carried out in a final volume of 10 μl and contained 30 μg of S-100 fraction as the source of ubiquitin-proteasomal system components. The reaction mixtures contained 50 mm Tris-HCl, pH 7.6, 5 mm MgCl2, 1 mm dithiothreitol, 1 mm ATP along with nuclear extracts from pcDNA3-C/EBPα-transfected BALB/MK2 cells as a proteasomal substrate (125 ng/reaction) supplemented with the ERS and 1 mm ubiquitin. For the inhibition of proteasomal activity, 25 μm MG-132 or 10 mm LiCl was included. Reactions were incubated at 37 °C for 2 h and were terminated by boiling after the addition of an equal volume of 2× SDS sample buffer. The reaction products were resolved by 10% SDS-PAGE and subjected to Western blot analysis. Effect of Li+on C/EBPα Stability—BALB/MK2 cells were transfected with pcDNA3-C/EBPα and treated with 20 mm lithium chloride as described above. Twenty-two hours after the lithium treatment, cells were incubated with 50 μg/ml cycloheximide. Cells were harvested at various time points, and extracts were subjected to Western blot analysis followed by densitometric analysis. Detection of Ubiquitinated C/EBPa—For the detection of ubiquitinated C/EBPα protein in the cell, BALB/MK2 keratinocytes were plated on a 60-mm culture dish and transfected with pCMV-FLAG-C/EBPα. Twenty-four hours later transfected cells were treated with 20 mm LiCl for 24 h. For MG-132 treatment, cells were incubated with 25 μm MG-132 for 2 h. Forty-eight hours following transfection, cells were washed with ice-cold phosphate-buffered saline, lysed in ice-cold immunoprecipitation buffer (20 mm Hepes pH 7.4, 150 mm NaCl, 12.5 mm β-glycerophosphate, 1.5 mm MgCl2, 2 mm EGTA, 10 mm NaF, 2 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 5 mmN-ethylmaleimide, and Mini Complete protease inhibitor tablet from Roche Applied Science) and rocked for 20 min at 4 °C. N-ethylmaleimide was included to inhibit isopeptidase activity. The lysed cells were scraped and centrifuged at 14,000 × g for 10 min at 4 °C. Anti-FLAG monoclonal antibody (1:150) and protein A/G-agarose plus were added to cleared lysates and rocked overnight at 4 °C. Immunoprecipitates were centrifuged at 2500 × g and washed three times with ice-cold lysis buffer. Immunoprecipitated proteins were boiled in 2× SDS sample buffer, electrophoresed, and analyzed by Western blot. In vitro ubiquitination reactions were carried out in a final volume of 15 μl and contained 40 μg of the S-100 fraction. The reaction mixtures contained 50 mm Tris-HCl, pH 7.6, 5 mm MgCl2, 1 mm dithiothreitol, 5 mm ATP along with nuclear extracts from pCMV-FLAG-C/EBPα transfected BALB/MK2 cells as a substrate (2 μg/reaction) supplemented with ERS, 1 mm ubiquitin, and 10 μm ubiquitin-aldehyde. To inhibit proteasomal activity, 25 μm MG-132 was included in reaction mixture. Reactions were incubated at 37 °C for 2 h and were terminated by adding 5 μl of 4% SDS. Reaction mixture was diluted 130 μl of immunoprecipitation buffer and FLAG-C/EBPα was immunoprecipitated using anti-FLAG antibody. Immunoprecipitated proteins were boiled in 2× SDS sample buffer, electrophoresed, and analyzed by Western blot. Site-directed Mutagenesis—Threonine to alanine mutations on Thr222 and Thr226 of pcDNA3-C/EBPα (Thr222-Pro-Pro-Pro-Thr226-Pro-Val-Pro-Ser230-Pro) were introduced using the QuickChange mutagenesis kit (Stratagene) according to the manufacturer's protocol. The following primer was used to generate the threonine to alanine mutations in T222A and T226A: 5′-648GCAGCCTGGCCACCCT(A→G)CGCCGCCGCCG(A→G)CGCC CGTGCCCAGCCCTC694-3′. Nucleotide changes from cDNA sequence are indicated. Specific mutations were confirmed by sequencing. C/EBPα Is a Short-lived Protein—Numerous proteins involved in regulation of the cell cycle are short-lived proteins that are degraded via an ubiquitin-dependent proteasomal pathway. Because C/EBPα protein is involved in the regulation of mitotic growth arrest and/or differentiation, we examined the half-life of the C/EBPα protein and whether proteasomal inhibitors can increase the half-life of C/EBPα. BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBPα, and 46 h later cells were treated with cycloheximide in the presence or absence of a proteasomal inhibitor. Cells were collected at 0, 0.5, 1, and 2 h after cycloheximide treatment, and C/EBPα protein levels were determined by Western blotting. As shown in Fig. 1A, treatment of BALB/MK2 keratinocytes with proteasome inhibitor, MG-132, blocked the degradation of C/EBPα. Treatment of BALB/MK2 cells with lactacystin, a highly specific proteasome inhibitor (36Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1507) Google Scholar), also blocked degradation of C/EBPα (Fig. 1B). Densitometric analysis of these blots revealed that the C/EBPα protein has a half-life of ∼1 h (Fig. 1C). These results demonstrate that C/EBPα is a short-lived protein and suggest that C/EBPα is degraded via a proteasomal mechanism. C/EBPα Is Ubiqutinated and Is a Proteasome Substrate— Because proteasome substrates are often polyubiquitinated before their degradation, we examined whether C/EBPα is ubiquitinated in BALB/MK2 cells. BALB/MK2 keratinocytes were transfected with FLAG-tagged C/EBPα, and 46 h later cells were left untreated or treated with proteasome inhibitor, MG-132 for 2 h. FLAG immunoprecipitates were prepared from pCMV-FLAG C/EBPα-transfected BALB/MK2 cells by using monoclonal antibodies to FLAG, followed by immunoblot with polyclonal antibody to ubiquitin. As shown in Fig. 2A (left panel), ubiquitin-immunoreactive higher molecular weight forms were detected in FLAG-C/EBPα-transfected cell lysates, and treatment of cells with MG-132 further increased ubiquitin-reactive higher molecular weight forms of C/EBPα. The membrane was stripped and reprobed with an antibody to C/EBPα. As shown in Fig. 2A (right panel), high molecular weight immunoreactive forms of C/EBPα were detected. To provide additional evidence for the proteasomal-mediated degradation of C/EBPα, we examined whether that C/EBPα is degraded via the proteasome and ubiquitinated in a cell-free assay. HeLa S-100 fraction contains proteasome and ubiquitin ligases and can be used to demonstrate whether a protein is degraded via an ubiquitin-proteasome pathway. As shown in Fig. 2B, C/EBPα protein was degraded by the HeLa S-100 fraction in an ATP- and ubiquitin-dependent manner. When MG-132 was included in the assay, it blocked the degradation of C/EBPα. While C/EBPα was not degraded in the absence of ATP and ubiquitin, its electrophoretic mobility was increased suggesting that C/EBPα is a substrate for cellular phosphatases in vitro. In order to examine whether C/EBPα is ubiquitinated in vitro, FLAG-tagged C/EBPα was incubated with HeLa S-100 fraction for 2 h with/without MG-132 in the presence of ubiquitin-aldehyde, an isopeptidase inhibitor. FLAG-tagged C/EBPα was immunoprecipitated from an in vitro ubiquitination mixture and subjected to immunoblot with ubiquitin antibody. As shown in Fig. 2C (left panel), ubiquitin-immunoreactive higher molecular weight forms of C/EBPα were detected and MG-132 further increased ubiquitin-immunoreactive higher molecular weight forms of C/EBPα. Similar results were obtained when membrane was stripped and reprobed with an antibody to C/EBPα (Fig. 2C, right panel). Collectively, these results demonstrate that the C/EBPα protein is degraded via an ubiquitin-dependent proteasomal pathway. Lithium Increases C/EBPα Protein Levels—Ross et al. (32Ross S.E. Erickson R.L. Hemati N. MacDougald O.A. Mol. Cell. Biol. 1999; 19: 8433-8441Crossref PubMed Google Scholar) showed that GSK3 is a C/EBPα kinase and phosphorylates Thr222 and Thr226 of C/EBPα, and lithium treatment can block this phosphorylation. Because phosphorylation by GSK3 is known to target cell cycle regulatory proteins such as p21 (26Rossig L. Badorff C. Holzmann Y. Zeiher A.M. Dimmeler S. J. Biol. Chem. 2002; 277: 9684-9689Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), β-catenin (27Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2201) Google Scholar), cyclin D1 (28Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1882) Google Scholar), and c-Myc (29Sears R. Nuckolls F. Haura E. Taya Y. Tamai K. Nevins J.R. Genes Dev. 2000; 14: 2501-2514Crossref PubMed Scopus (1003) Google Scholar) for proteasomal degradation, we examined whether treatment with lithium, an inhibitor of GSK3 (30Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar, 31Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Crossref PubMed Scopus (457) Google Scholar), can block C/EBPα degradation. BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBPα and treated with lithium chloride, a known inhibitor of GSK3. Lithium treatment produced a dose-dependent increase in C/EBPα levels (4–5-fold increase at 20 mm LiCl) as well as an electrophoretically faster migrating form of C/EBPα (Fig. 3, A and B). This increase in electrophoretic mobility is consistent with lithium inhibiting GSK3-mediated phosphorylation of C/EBPα. To confirm this notion, lysates from untreated cells were incubated with protein phosphatase prior to electrophoresis. As shown in Fig. 3C, phosphatase treatment, like lithium, resulted in a faster migrating form of C/EBPα. However, phosphatase treatment produced a faster migrating form of C/EBPα than lithium treatment suggesting that there are additional phosphorylation sites within C/EBPα that are not sensitive to lithium treatment. The effect of lithium on C/EBPα levels was specific as treatment with sodium, another cationic metal, did not increase C/EBPα levels (Fig. 3D). In contrast to the effect of lithium on C/EBPα protein levels, lithium had no effect on C/EBPα mRNA levels indicating that the effects of lithium are post-transcriptional (Fig. 3E). Lithium treatment of untransfected BALB/MK2 cells also resulted in an increase in the endogenous C/EBPα levels, produced a fast DA - 2003/5/30/ PY - 2003/5/30/ DO - 10.1074/jbc.M301356200 VL - 278 IS - 22 SP - 19674-19681 SN - 0021-9258 ER - TY - CHAP TI - Evolutionary multiobjective optimization in watershed water quality management AU - Dorn, J. L. AU - Ranjithan, S. R. T2 - Evolutionary multi-criterion optimization: Second international conference, EMO 2003, Faro, Portugal, April 8-11, 2003: proceedings AB - The watershed water quality management problem considered in this study involves the identification of pollution control choices that help meet water quality targets while sustaining necessary growth. The primary challenge is to identify nondominated management choices that represent the noninferior tradeoff between the two competing management objectives, namely allowable urban growth and water quality. Given the complex simulation models and the decision space associated with this problem, a genetic algorithm-based multiobjective optimization (MO) approach is needed to solve and analyze it. This paper describes the application of the Nondominated Sorting Algorithm II (NSGA-II) to this realistic problem. The effects of different population sizes and sensitivity to random seed are explored. As the water quality simulation run times can become prohibitive, appropriate stopping criteria to minimize the number of fitness evaluations are being investigated. To compare with the NSGA-II results, the MO watershed management problem was also analyzed via an iterative application of a hybrid GA/local-search method that solved a series of single objective ε-constraint formilations of the multiobjective problem. In this approach, the GA solutions were used as the starting points for the Nelder-Mead local search algorithm. The results indicate that NSGA-II offers a promising approach to solving this complex, real-world MO watershed management problem. CN - T57.95 .E48 2003 PY - 2003/// DO - 10.1007/3-540-36970-8_49 VL - 2632 SP - 692-706 PB - Berlin; New York: Springer SN - 3540018697 ER - TY - CHAP TI - Are the "best" solutions to a real optimization problem always found in the noninferior set? Evolutionary algorithm for generating alternatives (EAGA) AU - Zechman, E. M. AU - Ranjithan, S. R. T2 - Genetic and evolutionary computation--GECCO 2003: Genetic and Evolutionary Computation Conference, Chicago, IL, USA, July 12-16, 2003: Proceedings AB - Evolutionary algorithms (EAs) continue to offer an effective, powerful, and sometimes exclusive way to search for solutions to real optimization problems. While these algorithms can help solve a complex optimization problem, whether the results represent the “best” choices for making decisions about a solution to a real problem is questionable. In decision-making problems that are ill posed, all objectives may not be defined clearly and therefore not quantitatively captured in the optimization model [1]. The noninferior set of solutions to the optimization model being solved may not necessarily contain the best solution to the actual problem. CN - QA402.5 .G4563 2003 v.1-2 PY - 2003/// DO - 10.1007/3-540-45110-2_55 VL - 2724 SP - 1622-1623 PB - Berlin; New York: Springer SN - 3540406026 ER - TY - JOUR TI - Distributed model of solid waste anaerobic digestion - Effects of leachate recirculation and pH adjustment AU - Vavilin, VA AU - Rytov, SV AU - Lokshina, JY AU - Pavlostathis, SG AU - Barlaz, MA T2 - BIOTECHNOLOGY AND BIOENGINEERING AB - A distributed model of solid waste digestion in a 1-D bioreactor with leachate recirculation and pH adjustment was developed to analyze the balance between the rates of polymer hydrolysis/acidogenesis and methanogenesis during the anaerobic digestion of municipal solid waste (MSW). The model was calibrated on previously published experimental data generated in 2-L reactors filled with shredded refuse and operated with leachate recirculation and neutralization. Based on model simulations, both waste degradation and methane production were stimulated when inhibition was prevented rapidly from the start, throughout the reactor volume, by leachate recirculation and neutralization. An optimal strategy to reduce the time needed for solid waste digestion is discussed. DA - 2003/1/5/ PY - 2003/1/5/ DO - 10.1002/bit.10450 VL - 81 IS - 1 SP - 66-73 SN - 0006-3592 KW - solid waste KW - distributed model KW - hydrolysis KW - methanogenesis KW - leachate recirculation KW - pH adjustment ER - TY - JOUR TI - Integrated solid waste management in the United States AU - Barlaz, Morton AU - Cekander, G. C. AU - Vasuki, N. C. T2 - Journal of Environmental Engineering (New York, N.Y.) AB - In this editorial, we examine the meaning of integrated solid waste management, discuss its application in practice, and identify areas where both technological and regulatory development are needed. Let us recognize that like water and wastewater treatment, solid waste must be managed by virtually every community to protect human health and the environment. The U.S. EPA ~2002! defines municipal solid waste ~MSW! to include waste generated in the residential, commercial, and institutional sectors. Of the 232 million tons generated in 2000, approximately 55.4% was disposed of in landfills, 23% was recovered for recycling, 7.1% was recovered for composting ~primarily yard waste!, and 14.5% was combusted in waste-to-energy facilities. In addition to MSW, many other nonhazardous wastes are managed in these same facilities, including construction and demolition ~C&D! waste, water and wastewater treatment plant sludges, and nonhazardous industrial wastes ranging from foodprocessing wastes to foundry sands. We often find that the best way to analyze waste management is to understand how the money flows. For waste generated in the residential and institutional sectors, the cost of solid waste management is typically borne by residents through a unit of government, which means that the costs for collection, recycling, composting, combustion, and disposal are constantly competing for always scarce tax revenue. MSW management may also be funded through user fees. Some have advocated the implementation of ‘‘pay as you throw’’ ~PAYT! systems, in which waste generators are charged for refuse collection in proportion to the volume discarded. Conceptually, this should encourage people to reduce waste generation and to recycle wherever possible. The implementation of such systems is increasing in the United States. Of course, PAYT is standard practice for commercial waste generators. MSW is frequently managed by a combination of public and private entities. The local government’s responsibility for protection of public health and the local environment is cost-effectively discharged through public-private partnerships. Thus, the waste management infrastructure consists of numerous public-private partnerships that together are charged with protecting human health and the environment in a cost-effective manner. The EPA identified a hierarchy for waste management in which source reduction is considered to be most favorable, followed by recycling, treatment, and ultimately landfill disposal. While seemingly intuitive, we suggest that this hierarchy is most useful when both economic feasibility and environmental sustain- DA - 2003/// PY - 2003/// DO - 10.1061/(asce)0733-9372(2003)129:7(583) VL - 129 IS - 7 SP - 583–584 ER - TY - JOUR TI - Detention pond design and land use planning for watershed management AU - Harrell, L. J. AU - Ranjithan, S. R. T2 - Journal of Water Resources Planning and Management DA - 2003/// PY - 2003/// DO - 10.1061/(ASCE)0733-9486(2003)129:2(98) VL - 129 IS - 2 SP - 98-106 ER - TY - JOUR TI - Special issue: Second Intercontinental Landfill Research Symposium, Asheville, North Carolina, USA, October 2002 AU - Barlaz, M AU - Reinhart, D T2 - WASTE MANAGEMENT DA - 2003/// PY - 2003/// DO - 10.1016/s0956-053x(03)00115-6 VL - 23 IS - 7 SP - 557-559 SN - 0956-053X ER - TY - JOUR TI - Occurrence and treatment of 1,4-dioxane in aqueous environments AU - Zenker, MJ AU - Borden, RC AU - Barlaz, MA T2 - ENVIRONMENTAL ENGINEERING SCIENCE AB - 1,4-Dioxane is classified as a probable human carcinogen. It is used as a stabilizer for chlorinated solvents, particularly, 1,1,1-trichloroethane (TCA), and it is formed as a by-product during the manufacture of polyester and various polyethoxylated compounds. Improper disposal of industrial waste and accidental solvent spills have resulted in the contamination of groundwater with 1,4-dioxane. Volatilization and sorption are not significant attenuation mechanisms due to 1,4-dioxane's complete miscibility with water. At present, advanced oxidation processes (AOPs) are the only proven technology for 1,4-dioxane treatment. 1,4-Dioxane was believed to be very resistant to both abiotic and biologically mediated degradation due to its heterocyclic structure with two ether linkages. However, recent studies have shown that 1,4-dioxane can be biodegraded as a sole carbon and energy source, and that cost-effective biological treatment processes can be developed. Future work should be oriented towards the development of better information on the extent of 1,4-dioxane contamination in the environment and of full-scale biological treatment processes. In addition, the application of chemical oxidation for in situ treatment of 1,4-dioxane warrants further investigation. DA - 2003/// PY - 2003/// DO - 10.1089/109287503768335913 VL - 20 IS - 5 SP - 423-432 SN - 1092-8758 KW - 1,4-dioxane KW - ethers KW - biodegradation KW - remediation KW - occurrence ER - TY - JOUR TI - Nitrogen management in bioreactor landfills AU - Price, GA AU - Barlaz, MA AU - Hater, GR T2 - WASTE MANAGEMENT AB - One scenario for long-term nitrogen management in landfills is ex situ nitrification followed by denitrification in the landfill. The objective of this research was to measure the denitrification potential of actively decomposing and well decomposed refuse. A series of 10-l reactors that were actively producing methane were fed 400 mg NO3-N /l every 48 h for periods of 19-59 days. Up to 29 nitrate additions were either completely or largely depleted within 48 h of addition and the denitrification reactions did not adversely affect the leachate pH. Nitrate did inhibit methane production, but the reactors recovered their methane-producing activity with the termination of nitrate addition. In well decomposed refuse, the nitrate consumption rate was reduced but was easily stimulated by the addition of either acetate or an overlayer of fresh refuse. Addition of acetate at five times the amount required to reduce nitrate did not lead to the production of NH4+ by dissimilatory nitrate reduction. The most probable number of denitrifying bacteria decreased by about five orders of magnitude during refuse decomposition in a reactor that did not receive nitrate. However, rapid denitrification commenced immediately with nitrate addition. This study shows that the use of a landfill as a bioreactor for the conversion of nitrate to a harmless byproduct, nitrogen gas, is technically viable. DA - 2003/// PY - 2003/// DO - 10.1016/s0956-053x(03)00104-1 VL - 23 IS - 7 SP - 675-688 SN - 0956-053X ER - TY - PAT TI - Method of treating alopecia AU - Smart, R. C. AU - Oh, H.-S. C2 - 2003/// DA - 2003/// PY - 2003/// ER - TY - JOUR TI - Comparing recycling, composting and landfills AU - Barlaz, M. A. AU - Kaplan, P. O. AU - Ranjithan, S. R. AU - Rynk, R. T2 - BioCycle DA - 2003/// PY - 2003/// VL - 44 IS - 9 SP - 60- ER - TY - JOUR TI - Evaluating environmental impacts of solid waste management alternatives AU - Barlaz, M. A. AU - Kaplan, P. O. AU - Ranjithan, S. R. AU - Rynk, R. T2 - BioCycle DA - 2003/// PY - 2003/// VL - 44 IS - 10 SP - 52-56 ER -