421 results on '"Thomas M. Harris"'
Search Results
152. The Formamidopyrimidine Derivative of 7-(2-Oxoethyl)-2′-deoxyguanosine
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Ivan D. Kozekov, Plamen P. Christov, Carmelo J. Rizzo, and Thomas M. Harris
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Magnetic Resonance Spectroscopy ,Time Factors ,Formamides ,Molecular Structure ,Stereochemistry ,Dado ,Epoxide ,General Medicine ,Toxicology ,Vinyl chloride ,Article ,Mass Spectrometry ,Adduct ,chemistry.chemical_compound ,DNA Adducts ,Pyrimidines ,chemistry ,Cyclization ,Deoxyguanosine ,AP site ,Crotonaldehyde ,Carcinogen ,Chromatography, High Pressure Liquid - Abstract
Vinyl chloride induces hepatic angiosarcomas, which are otherwise rare malignancies. The biochemical basis involves the formation of the epoxide, which reacts with DNA to give approximately 98% of the 7-(2-oxoethyl) adduct (4) of dGuo plus small amounts of the etheno derivatives of dGuo, dCyd, and dAdo. The carcinogenicity is generally ascribed to the etheno adducts, not 4, because 4 has been shown to disappear from cells rapidly and to have negligible mutagenicity, which argues against its biological importance, whereas etheno adducts are both persistent and mutagenic. It has also been shown that apurinic sites derived from 4 are unlikely to be crucial lesions. A confounding factor with regard to the etheno hypothesis is that etheno adducts arise in unexposed cells by reactions of various lipid peroxidation products. The present study explores the possibility that a major contributor to the carcinogenicity of vinyl chloride may be formamidopyrimidine (FAPy) 12, N-[2-amino-6-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-3,4-dihydro-4-oxo-5-pyrimidinyl]-N-(2-oxoethyl)-formamide, which can arise by ring opening of 4, although its formation has not been observed until the present study. N7 adduct 4 undergoes deglycosylation to give 7-(2-oxoethyl)-Gua (13) in acid and imidazolium ring-opening to 12 in base. At pH 7.4, both processes occur with the formation of 12 representing approximately 10% of the product mixture. FAPy 12 spontaneously cyclizes to 22, which upon mild acid treatment yields the deglycosylation product 2-amino-3,4,7,8-tetrahydro-7-hydroxy-4-oxopteridine-5(6H)-carbaldehyde (14). The structure of 14 has been established by NMR and mass spectroscopy and by independent synthesis. Reaction of the epoxide of crotonaldehyde with dGuo failed to give either 13 or 14, indicating that both compounds are unique products of the reactions of dGuo with the epoxides of vinyl monomers. Although FAPy 12 was found to be unstable, carbinolamine 22 arising from cyclization of 12 may be an important contributor to the carcinogenicity of vinyl chloride.
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- 2008
153. Interstrand DNA Cross-links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources
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Hai Huang, Hye-Young H. Kim, Michael P. Stone, Irina G. Minko, R. Stephen Lloyd, Young Jin Cho, Thomas M. Harris, Hao Wang, Albena Kozekova, Carmelo J. Rizzo, and Ivan D. Kozekov
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Models, Molecular ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Base pair ,Oligonucleotides ,Environment ,Photochemistry ,Article ,Adduct ,chemistry.chemical_compound ,DNA Adducts ,Molecule ,Transition Temperature ,Crotonaldehyde ,Aldehydes ,Molecular Structure ,Chemistry ,Oligonucleotide ,Acrolein ,General Medicine ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Amino Alcohols ,Cross-Linking Reagents ,Lipid Peroxidation ,DNA - Abstract
Significant levels of the 1, N(2)-gamma-hydroxypropano-dG adducts of the alpha,beta-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxy-2E-nonenal (HNE) have been identified in human DNA, arising from both exogenous and endogenous exposures. They yield interstrand DNA cross-links between guanines in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, as a result of opening of the 1,N(2)-gamma-hydroxypropano-dG adducts to form reactive aldehydes that are positioned within the minor groove of duplex DNA. Using a combination of chemical, spectroscopic, and computational methods, we have elucidated the chemistry of cross-link formation in duplex DNA. NMR spectroscopy revealed that, at equilibrium, the acrolein and crotonaldehyde cross-links consist primarily of interstrand carbinolamine linkages between the exocyclic amines of the two guanines located in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, that maintain the Watson-Crick hydrogen bonding of the cross-linked base pairs. The ability of crotonaldehyde and HNE to form interstrand cross-links depends upon their common relative stereochemistry at the C6 position of the 1,N(2)-gamma-hydroxypropano-dG adduct. The stereochemistry at this center modulates the orientation of the reactive aldehyde within the minor groove of the double-stranded DNA, either facilitating or hindering the cross-linking reactions; it also affects the stabilities of the resulting diastereoisomeric cross-links. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Reduced derivatives of these cross-links are useful tools for studying their biological processing.
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- 2008
154. Characterization of a Monoclonal Antibody Targeting Water‐Soluble Petroleum Compounds as Determined by an ELISA Method and by a Biosensor Technology
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Candace R. Spier, Erin Bromage, Michael A. Unger, Stephen L. Kaattari, and Thomas M. Harris
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Water soluble ,Chromatography ,Chemistry ,medicine.drug_class ,Genetics ,medicine ,Elisa method ,Monoclonal antibody ,Molecular Biology ,Biochemistry ,Biosensor ,Molecular biology ,Biotechnology - Published
- 2008
155. Butadiene-Mediated Mutagenesis and Carcinogenesis
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Thomas M. Harris, Priscilla H. Fernandes, Michael P. Stone, Jeffrey K. Wickliffe, Jonathan B. Ward, and R. Stephen Lloyd
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Molecular Toxicology ,Deletion mutation ,Library science ,Sociology - Abstract
Jonathan B. Ward, Jeffrey K. Wickliffe, Michael P. Stone, Thomas M. Harris, Priscilla H. Fernandes and R. Stephen Lloyd Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, TX 77555-1110, USA Department of Chemistry, Center for Molecular Toxicology, Vanderbilt University, Nashville, TN 37235, USA Center for Research on Occupational and Environmental Toxicology, Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97239, USA Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77021, USA e-mail: lloydst@ohsu.edu
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- 2008
156. Intercalation of aflatoxin B1 in two oligodeoxynucleotide adducts: comparative proton NMR analysis of d(ATCAFBGAT).cntdot.d(ATCGAT) and d(ATAFBGCAT)2
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Thomas M. Harris, Michael P. Stone, and S. Gopalakrishnan
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chemistry.chemical_compound ,Aflatoxin ,chemistry ,Deoxyribose ,Guanine ,Stereochemistry ,Intercalation (chemistry) ,Proton NMR ,Imidazole ,Nuclear Overhauser effect ,Biochemistry ,Adduct - Abstract
8,9-Dihydro-8-(N7-guanyl-[d(ATCGAT)])-9-hydroxyaflatoxin B1.d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1.8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1 were prepared by direct addition of afltoxin B1 8,9-epoxide to d(ATCGAT)2 and d(ATGCAT)2, respectively. In contrast to reaction of aflatoxin B1 8,9-epoxide with d(ATCGAT)2 which exhibits a limiting stoichiometry of 1:1 aflatoxin B1:d(ATCGAT)2 [Gopalakrishnan, S., Stone, M. P., & Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239], reaction of aflatoxin B1 8,9-epoxide with d(ATGCAT)2 exhibits a limiting stoichiometry of 2:1 aflatoxin B1:d(ATGCAT)2. 1H NOE experiments, nonselective 1H T1 relaxation measurements, and 1H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5'-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide 1H NOEs are observed between aflatoxin and the 5'-neighbor base pair and include both the major groove and the minor groove. The aflatoxin methoxy and cyclopentenone ring protons face into the minor groove; the furofuran ring protons face into the major groove. No NOE is observed between the imino proton of the modified base pair and the imino proton of the 5'-neighbor base pair; sequential NOEs between nucleotide base and deoxyribose protons are interrupted in both oligodeoxynucleotide strands on the 5'-side of the modified guanine. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons; chemical shift perturbations of the oligodeoxynucleotide protons are confined to the immediate vicinity of the adducted base pair. The imidazole proton of the modified guanine exchanges with water and is observed at 9.75 ppm. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B1 8,9-epoxide and B-DNA. Insertion of aflatoxin B1-8,9 epoxide above the 5'-face of guanine in d(ATCGAT)2 would prevent the binding of a second molecule of aflatoxin B1 8,9-epoxide. In contrast, two intercalation sites would be available with d(ATGCAT)2.(ABSTRACT TRUNCATED AT 400 WORDS)
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- 1990
157. Pipecolic acid biosynthesis in Rhizoctonia leguminicola. I. The lysine saccharopine, delta 1-piperideine-6-carboxylic acid pathway
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B.M. Wickwire, Thomas M. Harris, Harry P. Broquist, and Constance M. Harris
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chemistry.chemical_classification ,biology ,Stereochemistry ,Carboxylic acid ,Lysine ,Cell Biology ,biology.organism_classification ,complex mixtures ,Biochemistry ,chemistry.chemical_compound ,Swainsonine ,chemistry ,Biosynthesis ,Slaframine ,Saccharopine ,bacteria ,Rhizoctonia leguminicola ,Molecular Biology ,Pipecolic acid - Abstract
The biosynthesis of pipecolic acid from L-lysine in the fungal parasite, Rhizoctonia leguminicola has been reinvestigated. Pipecolate is then utilized to form the toxic octahydroindolizine alkaloids, slaframine and swainsonine. Incorporation studies of L-versus D-[U-14C]lysine into R. leguminicola metabolites confirmed earlier findings that L-lysine is the predominant substrate for pipecolate formation and D-lysine for alpha-N-acetyllysine (concerned in lysine catabolism). However [alpha-15N]lysine, not [epsilon-15N]lysine as previously reported, labeled pipecolate. Such findings implied that delta 1-piperideine-6-carboxylate, not delta 1-piperideine-2-carboxylate, was formed from lysine and was the immediate precursor of pipecolate. Evidence from cell-free enzyme systems established the following biosynthetic events: L-lysine A----saccharopine B----delta 1-piperideine-6-carboxylate C----pipecolate. Products of reactions A and C were identified from biological and chemical considerations. Reaction B was carried out by a previously undescribed flavin enzyme termed saccharopine oxidase. The product of reaction B, which reacted with p-dimethylaminobenzaldehyde, was reduced with Na-CNB2H3. Its NMR spectrum was identical with that of deuteriated pipecolate prepared from authentic delta 1-piperideine-6-carboxylate, but not from authentic delta 1-piperideine-2-carboxylate. Reaction B represents a branching of primary lysine metabolism from saccharopine to a secondary pathway leading to pipecolate and to octahydroindolizine alkaloids in R. leguminicola.
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- 1990
158. Ontogeny and sexual dimorphism of sonic muscle in the oyster toadfish
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Michael L. Fine, Noelle M. Burns, and Thomas M. Harris
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Oyster toadfish ,medicine.medical_specialty ,Fiber diameter ,biology ,Ontogeny ,biology.organism_classification ,Muscle hypertrophy ,Sexual dimorphism ,Endocrinology ,Opsanus ,Internal medicine ,Swim bladder ,medicine ,Animal Science and Zoology ,Fiber ,Ecology, Evolution, Behavior and Systematics - Abstract
Previous work has shown that neurons in the sonic motor nucleus of the oyster toadfish, Opsanus tau, grow larger in males than in females and increase in size and number for 7–8 years. In order to correlate postnatal motoneuron development with growth of target muscle fibers, we examined the ontogeny of sonic muscle growth. Both the swim bladder and attached sonic muscles increased in size for life and were, respectively, 20 and 44% larger in males than in females. The muscle and swim bladder grew at an equivalent rate in males, whereas in females, muscle growth did not keep up with bladder growth. The number of muscle fibers increased about 16-fold (31 000 to 488 000), and mean minimum fiber diameter increased almost 3-fold (11.5 to 28.6 μm) as fish grew. Fibers were 15.3% larger in females than in males (adjusted means of 21.9 and 19.0 μm, respectively), but males had 47% more fibers per muscle (adjusted means of 307 000 and 209 000). Muscle fibers also exhibited morphological changes. Most of the fibers in two juveniles had yet to differentiate the core of sarcoplasm characteristic of sonic muscle, whereas the largest cells in mature males and females tended to have multiple pockets of sarcoplasm and a contractile cylinder split into fragments. Multiple pockets in large fibers and the presence of smaller fibers in males than females are interpreted as adaptations for increased speed and fatigue resistance.
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- 1990
159. Alteration of the aflatoxin cyclopentenone ring to a .delta.-lactone reduces intercalation with DNA and decreases formation of guanine N7 adducts by aflatoxin epoxides
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Kevin D. Raney, S. Gopalakrishnan, Michael P. Stone, Suzanne Byrd, and Thomas M. Harris
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Cyclopentenone ,Aflatoxin ,Aflatoxin B1 ,Guanine ,Stereochemistry ,Molecular Sequence Data ,Oligonucleotides ,Epoxide ,Thymus Gland ,Toxicology ,Binding, Competitive ,Adduct ,chemistry.chemical_compound ,Stereospecificity ,Aflatoxins ,Animals ,heterocyclic compounds ,Base Sequence ,DNA, Superhelical ,Oligonucleotide ,technology, industry, and agriculture ,food and beverages ,DNA ,General Medicine ,biological factors ,Gene Expression Regulation ,Solubility ,chemistry ,Nucleic Acid Conformation ,Cattle - Abstract
The regio- and stereospecificity exhibited by reaction of aflatoxin B1 8,9-epoxide with DNA as well as the efficiency of reaction are remarkable and suggests that a specific orientation of bound epoxide facilitates formation of the transition state leading to guanine N7 adducts. We have compared aflatoxins B1 and B2 with aflatoxins G1 and G2 as to their binding with calf thymus DNA, d(ATGCAT)2, d(GCATGC)2, and plasmid pBR322. Aflatoxins B1 and B2 contain a cyclopentenone ring fused to the lactone ring of the coumarin. They have similar DNA association constants and intercalate with B-DNA, as demonstrated by NMR analysis of association with d(ATGCAT)2 and d(GCATGC)2, alteration of pBR322 electrophoretic mobility, and flow dichroism using linearly oriented calf thymus DNA. The less planar delta-lactone ring of aflatoxins G1 and G2 reduces DNA binding affinity by approximately 1 order of magnitude. Nevertheless, binding studies with d(ATGCAT)2 and d(GCATGC)2 suggest that aflatoxins G1 and G2 also bind B-DNA by intercalation. To establish the existence of a relationship between the association of these aflatoxins with DNA and adduct formation induced by aflatoxin epoxides, we compared the yield of guanine N7 adduct from aflatoxin B1 8,9-epoxide and from aflatoxin G1 9,10-epoxide at three concentrations of calf thymus DNA. As DNA concentration is decreased, two observations are made: (1) the number of adducts formed by either aflatoxin B1 8,9-epoxide or aflatoxin G1 9,10-epoxide is reduced with a concomitant increase in formation of the respective dihydrodiols, and (2) the ratio of adducts formed by aflatoxin G1 9,10-epoxide to those formed by an equivalent concentration of aflatoxin B1 8,9-epoxide decreases.(ABSTRACT TRUNCATED AT 250 WORDS)
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- 1990
160. Formation of thiol conjugates of 9-deoxy-.DELTA.9,.DELTA.12(E)-prostaglandin D2 and .DELTA.12(E)-prostaglandin D2
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Thomas M. Harris, Jacob Atsmon, L. Jackson Roberts, Brian J. Sweetman, and Steven W. Baertschi
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Male ,Delta ,Magnetic Resonance Spectroscopy ,Chemical Phenomena ,Double bond ,Stereochemistry ,In Vitro Techniques ,Microtubules ,Biochemistry ,chemistry.chemical_compound ,Albumins ,Prostaglandins, Synthetic ,Animals ,Cysteine ,Sulfhydryl Compounds ,Glutathione Transferase ,chemistry.chemical_classification ,Nucleophilic addition ,Prostaglandin D2 ,Rats, Inbred Strains ,Glutathione ,Rats ,Chemistry ,chemistry ,Thiol ,Conjugate - Abstract
Albumin catalyzes the transformation of prostaglandin D2 to 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and to isomeric prostaglandin D2 compounds including delta 12(E)-prostaglandin D2. Both of these compounds are alpha,beta-unsaturated ketones, which should render them susceptible to nucleophilic addition. We therefore examined the ability of the compounds to form conjugates with thiols glutathione and cysteine. During incubation with excess glutathione, both 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2 formed a conjugate. Conjugation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 occurred very rapidly; approximately 70% was conjugated within 2 min. In contrast, conjugation of delta 12(E)-prostaglandin D2 with glutathione proceeded at a much slower rate; only 38% was conjugated at 60 min. The formation of both conjugates was enhanced by glutathione S-transferase. Conjugation of both compounds with cysteine was found to occur more rapidly than with glutathione. This effect was more pronounced with delta 12(E)-prostaglandin D2 in which 60% conjugated with cysteine within 2 min. These differences are likely attributed to greater steric hindrance for conjugation across the delta 12 double bond compared to that across the delta 9 bond. Analysis by fast atom bombardment mass spectrometry confirmed the formation of the glutathione conjugate of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2. Following prolonged incubation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 with excess glutathione in the presence of glutathione S-transferase, a small quantity of a bis conjugate of this compound was also detected by mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)
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- 1990
161. Formation of prostaglandin A analogues via an allene oxide
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Steven W. Baertschi, Alan R. Brash, and Thomas M. Harris
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biology ,Stereochemistry ,Allene ,Diastereomer ,Oxide ,Prostaglandin ,Cell Biology ,Biochemistry ,chemistry.chemical_compound ,Lipoxygenase ,chemistry ,Biosynthesis ,biology.protein ,Prostaglandin a ,Enantiomer ,Molecular Biology - Abstract
One potential biosynthetic route to the prostaglandins involves the participation of lipoxygenase and allene oxide synthase enzymes, giving a hydroxylated allene oxide, which then might cyclize to form prostaglandin A or a close analogue. We have tested a model of this type of transformation using 8-hydroxy-15S-hydroperoxy eicosanoids as substrates for the dehydrase (allene oxide synthase) in flaxseed. Four of these substrates, each with a 9E,11Z,13E-conjugated triene, gave an observable rate of reaction. The two derived from eicosapentaenoic acid reacted more rapidly than the corresponding arachidonic acid analogues. Also, the 8S-hydroxy-15S-hydroperoxy diastereomers reacted more rapidly than their 8R-hydroxy analogues. Products were characterized by high pressure liquid chromatography, UV, gas chromatography-mass specrometry, 1H NMR, and CD. Reaction of the (8S)-hydroxy-(15S)-hydroperoxy-eicosapentaenoic acid gave two alpha-ketols [8S),15-dihydroxy-14-oxoeicosa-5Z,9E,11Z,17Z+ ++-tetraenoic acid and the corresponding 11E isomer in a 2:1 ratio), together with four prostaglandin A3 analogues which differed in the configurations of the side chains. Oxygen 18 labeling fully supported the intermediacy of an allene oxide in the biosynthesis. The corresponding "8R" substrate was converted to the enantiomers of these products plus three 13-hydroxy-14,15-epoxy derivatives. The arachidonate analogues formed the epoxy-hydroxy derivatives, the alpha-ketols, and two prostaglandin A2 analogues with trans configuration of the side chains. These results demonstrate (i) a feasible route of metabolism of lipoxygenase products to hydroxy allene oxide, (ii) the potential for the resulting allene oxide to cyclize to a prostaglandin A analogue, and (iii) the marked influence of the hydroxyl configuration of the rate of reaction and resulting profile of products. Some of these reactions may occur in a natural pathway of prostanoid biosynthesis in corals and other organisms.
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- 1990
162. Mutagenic potential of DNA–peptide crosslinks mediated by acrolein-derived DNA adducts
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Irina G. Minko, Albena Kozekova, Thomas M. Harris, Carmelo J. Rizzo, Ivan D. Kozekov, and R. Stephen Lloyd
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DNA repair ,DNA damage ,Health, Toxicology and Mutagenesis ,medicine.disease_cause ,Transfection ,Article ,chemistry.chemical_compound ,DNA Adducts ,Deoxyadenosine ,Shuttle vector ,Chlorocebus aethiops ,Genetics ,medicine ,Deoxyguanosine ,Animals ,Acrolein ,Molecular Biology ,Mutation ,Deoxyadenosines ,Mutagenesis ,Molecular biology ,Cross-Linking Reagents ,chemistry ,Biochemistry ,COS Cells ,Peptides ,DNA ,DNA Damage - Abstract
Current data suggest that DNA-peptide crosslinks are formed in cellular DNA as likely intermediates in the repair of DNA-protein crosslinks. In addition, a number of naturally occurring peptides are known to efficiently conjugate with DNA, particularly through the formation of Schiff-base complexes at aldehydic DNA adducts and abasic DNA sites. Since the potential role of DNA-peptide crosslinks in promoting mutagenesis is not well elucidated, here we report on the mutagenic properties of Schiff-base-mediated DNA-peptide crosslinks in mammalian cells. Site-specific DNA-peptide crosslinks were generated by covalently trapping a lysine-tryptophan-lysine-lysine peptide to the N(6) position of deoxyadenosine (dA) or the N(2) position of deoxyguanosine (dG) via the aldehydic forms of acrolein-derived DNA adducts (gamma-hydroxypropano-dA or gamma-hydroxypropano-dG, respectively). In order to evaluate the potential of DNA-peptide crosslinks to promote mutagenesis, we inserted the modified oligodeoxynucleotides into a single-stranded pMS2 shuttle vector, replicated these vectors in simian kidney (COS-7) cells and tested the progeny DNAs for mutations. Mutagenic analyses revealed that at the site of modification, the gamma-hydroxypropano-dA-mediated crosslink induced mutations at only approximately 0.4%. In contrast, replication bypass of the gamma-hydroxypropano-dG-mediated crosslink resulted in mutations at the site of modification at an overall frequency of approximately 8.4%. Among the types of mutations observed, single base substitutions were most common, with a prevalence of G to T transversions. Interestingly, while covalent attachment of lysine-tryptophan-lysine-lysine at gamma-hydroxypropano-dG caused an increase in mutation frequencies relative to gamma-hydroxypropano-dG, similar modification of gamma-hydroxypropano-dA resulted in decreased levels of mutations. Thus, certain DNA-peptide crosslinks can be mutagenic, and their potential to cause mutations depends on the site of peptide attachment. We propose that in order to avoid error-prone replication, proteolytic degradation of proteins covalently attached to DNA and subsequent steps of DNA repair should be tightly coordinated.
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- 2007
163. Stereochemistry Modulates Stability of Reduced Inter-Strand Cross-Links Arising From R- and S-α-CH3-γ-OH-1,N2-propano-2′-Deoxyguanosine in the 5′-CpG-3′ DNA Sequence
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Ivan D. Kozekov, Carmelo J. Rizzo, Young Jin Cho, Michael P. Stone, and Thomas M. Harris
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Torsion Abnormality ,Base Sequence ,Chemistry ,Stereochemistry ,Hydrogen bond ,Molecular Conformation ,Alpha (ethology) ,Deoxyguanosine ,Sequence (biology) ,Hydrogen Bonding ,DNA ,Biochemistry ,Solution structure ,DNA sequencing ,Article ,chemistry.chemical_compound ,Cross-Linking Reagents ,CpG site ,Nucleic Acid Conformation ,CpG Islands ,Protons - Abstract
The solution structures of 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' interstrand DNA cross-links in the 5'-CpG-3' sequence were determined by NMR spectroscopy. These were utilized as chemically stable surrogates for the corresponding carbinolamine interstrand cross-links arising from the crotonaldehyde- and acetaldehyde-derived R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts. The results provide an explanation for the observation that interstrand cross-link formation in the 5'-CpG-3' sequence by the R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts is dependent upon stereochemistry, favoring the R-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adduct [Kozekov, I. D., Nechev, L. V., Moseley, M. S., Harris, C. M., Rizzo, C. J., Stone, M. P., and Harris, T. M. (2003) J. Am. Chem. Soc. 125, 50-61]. Molecular dynamics calculations, restrained by NOE-based distances and empirical restraints, revealed that both the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-links were located in the minor groove and retained Watson-Crick hydrogen bonds at the tandem cross-linked C.G base pairs. However, for the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the center of the minor groove, whereas for the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the 3' direction, showing steric interference with the DNA helix. The 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link exhibited a lower thermal stability as evidenced by NMR spectroscopy as a function of temperature. The two cross-links also exhibited apparent differences in the conformation of the interstrand three-carbon cross-link, which may also contribute to the lower apparent thermodynamic stability of the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link.
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- 2007
164. Structure of the 1,4-Bis(2'-deoxyadenosin-N(6)-yl)-2S,3S-butanediol intrastrand DNA cross-link arising from butadiene diepoxide in the human N-ras codon 61 sequence
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R. Stephen Lloyd, Michael P. Stone, Lubomir V. Nechev, W. Keither Merritt, Thomas M. Harris, Wen Xu, and Constance M. Harris
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Models, Molecular ,Dna duplex ,Magnetic Resonance Spectroscopy ,Base pair ,Stereochemistry ,Toxicology ,Sensitivity and Specificity ,Article ,chemistry.chemical_compound ,DNA Adducts ,Structure-Activity Relationship ,Alkane stereochemistry ,Humans ,Butylene Glycols ,Codon ,Deoxyadenosines ,Molecular Structure ,Chemistry ,Hydrogen bond ,Cross-link ,General Medicine ,Reference Standards ,Genes, ras ,Butanediol ,Oligodeoxyribonucleotides ,Duplex (building) ,Epoxy Compounds ,DNA - Abstract
The 1,4-bis(2'-deoxyadenosin-N(6)-yl)-2S,3S-butanediol intrastrand DNA cross-link arises from the bis-alkylation of tandem N(6)-dA sites in DNA by R,R-butadiene diepoxide (BDO(2)). The oligodeoxynucleotide 5'-d(C(1)G(2)G(3)A(4)C(5)X(6)Y(7)G(8)A(9)A(10)G(11))-3'.5'-d(C(12)T(13)T(14)C(15)T(16)T(17)G(18)T(19)C(20)C(21)G(22))-3' contains the BDO(2) cross-link between the second and third adenines of the codon 61 sequence (underlined) of the human N-ras protooncogene and is named the (S,S)-BD-(61-2,3) cross-link (X,Y = cross-linked adenines). NMR analysis reveals that the cross-link is oriented in the major groove of duplex DNA. Watson-Crick base pairing is perturbed at base pair X(6).T(17), whereas base pairing is intact at base pair Y(7).T(16). The cross-link appears to exist in two conformations, in rapid exchange on the NMR time scale. In the first conformation, the beta-OH is predicted to form a hydrogen bond with T(16) O(4), whereas in the second, the beta-OH is predicted to form a hydrogen bond with T(17) O(4). In contrast to the (R,R)-BD-(61-2,3) cross-link in the same sequence (Merritt, W. K., Nechev, L. V., Scholdberg, T. A., Dean, S. M., Kiehna, S. E., Chang, J. C., Harris, T. M., Harris, C. M., Lloyd, R. S., and Stone, M. P. (2005) Biochemistry 44, 10081-10092), the anti-conformation of the two hydroxyl groups at C(beta) and C(gamma) with respect to the C(beta)-C(gamma) bond results in a decreased twist between base pairs X(6).T(17) and Y(7).T(16), and an approximate 10 degrees bending of the duplex. These conformational differences may account for the differential mutagenicity of the (S,S)- and (R,R)-BD-(61-2,3) cross-links and suggest that stereochemistry plays a role in modulating biological responses to these cross-links (Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580).
- Published
- 2007
165. Corrigendum to 'Novel monobactams utilizing a siderophore uptake mechanism for the treatment of Gram-negative infections' [Bioorg. Med. Chem. Lett. 22/18 (2012) 5989–5994]
- Author
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Chao Li, Mark S. Plummer, Veerabahu Shanmugasundaram, Thuy Hoang, Seungil Han, Lisa Mullins, Jennifer A. Young, Mark C. Noe, Thomas M. Harris, Steven M. Finegan, John P. Mueller, Joel R. Hardink, Jian Lin, Joel T. Arcari, Hongying Gao, Jennifer Winton, Manjinder S. Lall, Jeffrey M. Casavant, Jianmin Sun, Hud Lawrence Risley, Mark Edward Flanagan, Joseph Penzien, Brandon P. Schuff, Mark Niosi, Brian S. Gerstenberger, Mark J. Mitton-Fry, Jeremy T. Starr, Rebecca Irvine, David M. George, Matthew Frank Brown, M. Megan Lemmon, Sandra P. McCurdy, Michael D. Huband, and David Pattavina
- Subjects
Siderophore ,Mechanism (biology) ,Chemistry ,Stereochemistry ,Organic Chemistry ,Clinical Biochemistry ,Drug Discovery ,Pharmaceutical Science ,Molecular Medicine ,Monobactams ,Molecular Biology ,Biochemistry ,Gram - Published
- 2015
166. Abstract 185: MicroRNA-375 suppresses extracellular matrix degradation and invadopodial activity in head and neck squamous cell carcinoma
- Author
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Ved P. Sharma, Lizandra Jimenez, John S. Condeelis, Michael B. Prystowsky, Geoffrey J. Childs, Jeffrey E. Segall, Thomas J. Ow, and Thomas M. Harris
- Subjects
Cancer Research ,Pathology ,medicine.medical_specialty ,medicine.diagnostic_test ,Chemistry ,Tyrosine phosphorylation ,medicine.disease ,Head and neck squamous-cell carcinoma ,Extracellular matrix ,stomatognathic diseases ,chemistry.chemical_compound ,Oncology ,Western blot ,Cell culture ,microRNA ,Invadopodia ,Cancer research ,medicine ,Extracellular Matrix Degradation - Abstract
Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer having an association with locoregional recurrence and lymph node metastasis. We previously reported that patients with lower microRNA-375 (miR-375) tumor:normal expression levels showed significantly decreased disease-specific survival, increased locoregional recurrence and distant metastasis. We also previously described that HNSCC cells with increased miR-375 showed significantly diminished invasion in vitro. The ability of cancer cells to degrade extracellular matrix (ECM) through secreted and membrane-bound proteases allows local invasion into the surrounding stroma, as well as possible metastasis. Invadopodia are specialized actin-rich structures, which mediate ECM degradation. Our objective was to determine whether elevated miR-375 expression in HNSCC cells also affects invadopodia formation and activity. For the evaluation of the matrix degradation properties of UMSCC1 and OSC19 cell lines, an invadopodial matrix degradation assay was used. Western blot analyses were conducted to measure the total protein levels of selected invadopodia-associated proteins. The tyrosine phosphorylation states of cortactin were evaluated by immunoprecipitation experiments. For the detection of levels of secreted proteases in the conditioned cell growth medium, Human Protease Arrays were used. Quantitative RT-PCR measurements were used to evaluate the mRNA expression levels of the commonly regulated proteases. We observed that the HNSCC cell lines with elevated miR-375 expression showed significant reductions in ECM degradation. We further identified that HNSCC cells expressing increased miR-375 expression had significantly suppressed amounts of mature invadopodia, although the levels of invadopodium precursors were not significantly altered. We determined that increased miR-375 expression in the HNSCC cell lines did not reduce cellular levels of invadopodia components, such as Tks5, cortactin and fascin. We also did not observe significant alterations to the tyrosine phosphorylation states of cortactin in the HNSCC cell lines. However, we found that the HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression and secreted levels of specific proteases, including Kallikrein 6, Kallikrein 10, and MMP-9. In summary, we have extended our understanding of the impact of miR-375 expression levels on HNSCC invasion. We demonstrated that increased miR-375 expression in HNSCC cells reduced ECM degradation and invadopodial activity. Our data suggest that reduced miR-375 expression in HNSCC patients can contribute to the invasive properties of head and neck cancer through increased invadopodial activity. Citation Format: Lizandra Jimenez, Ved P. Sharma, John S. Condeelis, Thomas M. Harris, Thomas J. Ow, Michael B. Prystowsky, Geoffrey J. Childs, Jeffrey E. Segall. MicroRNA-375 suppresses extracellular matrix degradation and invadopodial activity in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 185. doi:10.1158/1538-7445.AM2015-185
- Published
- 2015
167. Spectroscopic Characterization of Interstrand Carbinolamine Crosslinks Formed in the 5'-CpG-3' Sequence by the Acrolein-Derived γ-OH-1,N2-Propano-2'-deoxyguanosine DNA Adduct
- Author
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Thomas M. Harris, Alvira Slutsky, Lubomir V. Nechev, R. Stephen Lloyd, Ivan D. Kozekov, Hai Huang, Markus Voehler, Hao Wang, Carmelo J. Rizzo, Irina G. Minko, Young Jin Cho, Hye-Young H. Kim, Michael P. Stone, Pamela Tamura, Albena Kozekova, and Jaison Jacob
- Subjects
Models, Molecular ,Base pair ,Stereochemistry ,Diol ,Oligonucleotides ,macromolecular substances ,Biochemistry ,Aldehyde ,Catalysis ,Article ,Adduct ,Substrate Specificity ,Deoxyribonucleotide ,chemistry.chemical_compound ,DNA Adducts ,Colloid and Surface Chemistry ,DNA adduct ,Acrolein ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Geminal diol ,Carbon Isotopes ,Nitrogen Isotopes ,technology, industry, and agriculture ,Deoxyguanosine ,Hydrogen Bonding ,Stereoisomerism ,General Chemistry ,DNA ,Cross-Linking Reagents ,chemistry ,Isotope Labeling ,CpG Islands ,Oligopeptides - Abstract
The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.
- Published
- 2005
168. Formation of inter- and intrastrand imine type DNA-DNA cross-links through secondary reactions of aldehydic adducts
- Author
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Ivan D. Kozekov, Thomas M. Harris, Ana M. Sanchez, and R. Stephen Lloyd
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Aldehydes ,Chemistry ,DNA damage ,Stereochemistry ,Guanine ,Imine ,General Medicine ,DNA ,Toxicology ,chemistry.chemical_compound ,DNA Adducts ,CpG site ,Biochemistry ,Deoxyadenosine ,Duplex (building) ,Deoxyguanosine ,DNA Damage - Abstract
Acrolein-derived DNA adducts of guanine have previously been detected in tissues of several species, including humans, and have been shown to be mutagenic in mammalian cells and potentially carcinogenic in higher organisms. In duplex DNA, the predominant acrolein-derived lesion, gamma-hydroxy-1,N2-propanodeoxyguanosine (gamma-HOPdG), exists in an equilibrated mixture of ring-opened and ring-closed forms. We have previously shown that the acyclic form can undergo secondary chemical reactions to form both interstrand DNA-DNA cross-links in a CpG sequence context and DNA-protein and DNA-peptide cross-links. Investigations using duplex DNAs containing gamma-HOPdG in a CpG sequence context reversibly created at least two cross-linked species: an imine, which is a minor species but could be readily reduced by NaBH4, and a major heat labile, nonreducible species that we formulate as a carbinolamine cross-link. The DNA came to equilibrium over several days with the carbinolamine species being significantly more abundant than the reducible imine. In an effort to find other types of DNA-DNA cross-links, we have developed a high throughput screen to evaluate the effects of DNA sequence and lesion structure on the formation of reducible interstrand and intrastrand cross-links. These data reveal that four different lesions, two involving deoxyguanosine and two involving deoxyadenosine, can react with nearby bases to form inter- and intrastrand DNA cross-links.
- Published
- 2005
169. Site Specific Synthesis and Polymerase Bypass of Oligonucleotides Containing a 6-Hydroxy-3,5,6,7-tetrahydro-9H-imidazo[1,2-a]purin-9-one Base, an Intermediate in the Formation of 1N2-Etheno-2′-deoxyguanosine
- Author
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Ivan D. Kozekov, F. Peter Guengerich, Hong Zang, Jeong-Yun Choi, Angela K. Goodenough, Carmelo J. Rizzo, and Thomas M. Harris
- Subjects
chemistry.chemical_classification ,Base (chemistry) ,biology ,Oligonucleotide ,Stereochemistry ,Oligonucleotides ,Temperature ,Deoxyguanosine ,General Medicine ,Toxicology ,Etheno adducts ,Article ,chemistry.chemical_compound ,chemistry ,Drug Stability ,Chemical agents ,biology.protein ,Chloroacetaldehyde ,heterocyclic compounds ,DNA ,Polymerase ,Chromatography, High Pressure Liquid - Abstract
The reaction of DNA with certain bis-electrophiles such as chlorooxirane and chloroacetaldehyde produces etheno adducts. These lesions are highly miscoding, and some of the chemical agents that produce them have been shown to be carcinogenic in laboratory animals and in humans. An intermediate in the formation of 1,N2-ethenoguanine is 6-hydroxy-3,5,6,7-tetrahydro-9H-imidazo[1,2-a]purin-9-one (6-hydroxyethanoguanine), which undergoes conversion to the etheno adduct. The chemical properties and miscoding potential of the hydroxyethano adduct have not been previously studied. A synthesis of the hydroxyethano-adducted nucleoside was developed, and it was site specifically incorporated into oligonucleotides. This adduct had a half-life of between 24 and 48 h at neutral pH and 25 degrees C at the nucleoside and oligonucleotide levels. The miscoding potential of the hydroxyethano adduct was examined by primer extension reactions with the DNA polymerases Dpo4 and pol T7-, and the results were compared to the corresponding etheno-adducted oligonucleotide. Dpo4 preferentially incorporated dATP opposite the hydroxyethano adduct and dGTP opposite the etheno adduct; pol T7- preferentially incorporated dATP opposite the etheno adduct while dGTP and dATP were incorporated opposite the hydroxyethano adduct with nearly equal catalytic efficiencies. Collectively, these results indicate that the hydroxyethano adduct has a sufficient lifetime and miscoding properties to contribute to the mutagenic spectrum of chlorooxirane and related genotoxic species.
- Published
- 2005
170. Evidence for Escherichia coli polymerase II mutagenic bypass of intrastrand DNA crosslinks
- Author
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Lubomir V. Nechev, Constance M. Harris, R. Stephen Lloyd, Pamela J. Tamura, Manorama Kanuri, Thomas M. Harris, and Sarah E. Kiehna
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DNA Replication ,DNA polymerase ,DNA polymerase II ,RNA polymerase II ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Transformation, Genetic ,medicine ,Escherichia coli ,Molecular Biology ,Polymerase ,biology ,Mutagenesis ,Cell Biology ,DNA ,DNA Polymerase II ,biology.organism_classification ,Enterobacteriaceae ,Molecular biology ,chemistry ,biology.protein ,Epoxy Compounds - Abstract
The mutagenic potentials of DNAs containing site- and stereospecific intrastrand DNA crosslinks were evaluated in Escherichia coli cells that contained a full complement of DNA polymerases or were deficient in either polymerases II, IV, or V. Crosslinks were made between adjacent N 6 - N 6 adenines and consisted of R , R - and S , S -butadiene crosslinks and unfunctionalized 2-, 3-, and 4-carbon tethers. Although replication of single-stranded DNAs containing the unfunctionalized 3- and 4-carbon tethers were non-mutagenic in all strains tested, replication past all the other intrastrand crosslinks was mutagenic in all E. coli strains, except the one deficient in polymerase II in which no mutations were ever detected. However, when mutagenesis was analyzed in cells induced for SOS, mutations were not detected, suggesting a possible change in the overall fidelity of polymerase II under SOS conditions. These data suggest that DNA polymerase II is responsible for the in vivo mutagenic bypass of these lesions in wild-type E. coli .
- Published
- 2005
171. Centronuclear myopathy in mice lacking a novel muscle-specific protein kinase transcriptionally regulated by MEF2
- Author
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Ichizo Nishino, Michael Arnold, Thomas M. Harris, Hajime Kusano, Osamu Nakagawa, Masayo Nakagawa, James A. Richardson, Geoffrey Childs, Kevin P. Campbell, Hideaki Hamada, Eric N. Olson, and John M. Shelton
- Subjects
Mef2 ,Transcription, Genetic ,Molecular Sequence Data ,Mice, Transgenic ,Biology ,Protein Serine-Threonine Kinases ,Gene Expression Regulation, Enzymologic ,Mice ,Genetics ,medicine ,Myocyte ,Animals ,Amino Acid Sequence ,Centronuclear myopathy ,Enhancer ,Protein kinase A ,Myopathy ,Muscle, Skeletal ,Mice, Knockout ,Base Sequence ,MEF2 Transcription Factors ,Gene Expression Profiling ,Skeletal muscle ,Gene Expression Regulation, Developmental ,DNA ,medicine.disease ,Molecular biology ,Research Papers ,DNA-Binding Proteins ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Enhancer Elements, Genetic ,Myogenic Regulatory Factors ,Signal transduction ,medicine.symptom ,Developmental Biology ,Myopathies, Structural, Congenital ,Signal Transduction ,Transcription Factors - Abstract
Myocyte enhancer factor 2 (MEF2) plays essential roles in transcriptional control of muscle development. However, signaling pathways acting downstream of MEF2 are largely unknown. Here, we performed a microarray analysis using Mef2c-null mouse embryos and identified a novel MEF2-regulated gene encoding a muscle-specific protein kinase, Srpk3, belonging to the serine arginine protein kinase (SRPK) family, which phosphorylates serine/arginine repeat-containing proteins. The Srpk3 gene is specifically expressed in the heart and skeletal muscle from embryogenesis to adulthood and is controlled by a muscle-specific enhancer directly regulated by MEF2. Srpk3-null mice display a new entity of type 2 fiber-specific myopathy with a marked increase in centrally placed nuclei; while transgenic mice overexpressing Srpk3 in skeletal muscle show severe myofiber degeneration and early lethality. We conclude that normal muscle growth and homeostasis require MEF2-dependent signaling by Srpk3.
- Published
- 2005
172. Use of laser-capture microdissection for the identification of marker genes for the ventromedial hypothalamic nucleus
- Author
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Joel K. Elmquist, Charlotte E. Lee, Keith L. Parker, Jeremy P. Segal, Agnes Viale, Nancy R. Stallings, Nicholas D. Socci, Jeffrey M. Friedman, Marcelo B. Soares, Thomas M. Harris, Liping Zhao, and Geoffrey Childs
- Subjects
Steroidogenic factor 1 ,Receptors, Cytoplasmic and Nuclear ,Nerve Tissue Proteins ,In situ hybridization ,Biology ,Steroidogenic Factor 1 ,Mice ,Animals ,RNA, Messenger ,Protein Precursors ,Gene ,Transcription factor ,Microdissection ,In Situ Hybridization ,Laser capture microdissection ,Homeodomain Proteins ,Mice, Knockout ,Reverse Transcriptase Polymerase Chain Reaction ,General Neuroscience ,Lasers ,Blotting, Northern ,Flow Cytometry ,Microarray Analysis ,Molecular biology ,Mice, Inbred C57BL ,Nuclear receptor ,Gene Expression Regulation ,Ventromedial Hypothalamic Nucleus ,Gene chip analysis ,Female ,Biomarkers ,Transcription Factors ,Cellular/Molecular - Abstract
The ventromedial hypothalamic nucleus (VMH) plays an important role in the control of feeding and energy homeostasis. In contrast to other hypothalamic nuclei that are also known to regulate energy balance, there is a paucity of nucleus-specific marker genes for the VMH, limiting the application of molecular approaches for analyzing VMH information processing, function, and circuitry. Here, we report the use of laser-capture microdissection to isolate a set of cDNAs that are enriched in the VMH relative to two adjacent hypothalamic nuclei, the arcuate and dorsomedial hypothalamus. The relative expression levels of nine of the 12 most robustly expressed VMH-enriched genes were confirmed by real-time PCR analysis using separate RNAs from these three nuclei. Three of these VMH-enriched genes were further characterized byin situhybridization histochemistry, includingpituitary adenylate cyclase activating polypeptide,cerebellin 1, and an expressed sequence tag namedLBH2. Finally, to test whether some of these genes were coordinately regulated, we monitored their expression insteroidogenic factor 1(SF-1) knock-out mice. SF-1 is a transcription factor that controls the development of the VMH. The RNA levels for four of these genes were reduced in these knock-out animals, further suggesting that they are direct or indirect targets of this orphan nuclear receptor. The VMH-enriched genes identified here provide a basis for a functional analysis of VMH neuronal subpopulations via the use of bacterial artificial chromosome transgenics and related technologies. These results also demonstrate the utility of laser-capture microdissection coupled with microarray technology to identify nucleus-specific transcriptional networks.
- Published
- 2005
173. Initiation of repair of DNA-polypeptide cross-links by the UvrABC nuclease
- Author
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R. Stephen Lloyd, Irina G. Minko, Thomas M. Harris, Bennett Van Houten, Andrew J. Kurtz, and Deborah L. Croteau
- Subjects
DNA Repair ,Proteolysis ,Molecular Sequence Data ,Biology ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,DNA Adducts ,medicine ,Amino Acid Sequence ,Endodeoxyribonucleases ,medicine.diagnostic_test ,Base Sequence ,Escherichia coli Proteins ,Deoxyguanosine ,DNA ,Recombinant Proteins ,Metabolic pathway ,Kinetics ,UvrABC nuclease ,Cross-Linking Reagents ,chemistry ,Peptides ,Oligopeptides - Abstract
Although the biochemical pathways that repair DNA-protein cross-links have not been clearly elucidated, it has been proposed that the partial proteolysis of cross-linked proteins into smaller oligopeptides constitutes an initial step in removal of these lesions by nucleotide excision repair (NER). To test the validity of this repair model, several site-specific DNA-peptide and DNA-protein cross-links were engineered via linkage at (1) an acrolein-derived gamma-hydroxypropanodeoxyguanosine adduct and (2) an apurinic/apyrimidinic site, and the initiation of repair was examined in vitro using recombinant proteins UvrA and UvrB from Bacillus caldotenax and UvrC from Thermotoga maritima. The polypeptides cross-linked to DNA were Lys-Trp-Lys-Lys, Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, and the 16 kDa protein, T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase. For the substrates examined, DNA incision required the coordinated action of all three proteins and occurred at the eighth phosphodiester bond 5' to the lesion. The incision rates for DNA-peptide cross-links were comparable to or greater than that measured on fluorescein-adducted DNA, an excellent substrate for UvrABC. Incision rates were dependent on both the site of covalent attachment on the DNA and the size of the bound peptide. Importantly, incision of a DNA-protein cross-link occurred at a rate approximately 3.5-8-fold slower than the rates observed for DNA-peptide cross-links. Thus, direct evidence has been obtained indicating that (1) DNA-peptide cross-links can be efficiently incised by the NER proteins and (2) DNA-peptide cross-links are preferable substrates for this system relative to DNA-protein cross-links. These data suggest that proteolytic degradation of DNA-protein cross-links may be an important processing step in facilitating NER.
- Published
- 2005
174. Mammalian cell mutagenesis of the DNA adducts of vinyl chloride and crotonaldehyde
- Author
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Lubomir V. Nechev, Thomas M. Harris, Priscilla H. Fernandes, R. Stephen Lloyd, and Manorama Kanuri
- Subjects
Epidemiology ,Health, Toxicology and Mutagenesis ,Genetic Vectors ,Oligonucleotides ,Vinyl Chloride ,medicine.disease_cause ,Vinyl chloride ,Adduct ,chemistry.chemical_compound ,DNA Adducts ,Chlorocebus aethiops ,medicine ,Deoxyguanosine ,Animals ,Crotonaldehyde ,Genetics (clinical) ,Carcinogen ,Aldehydes ,Chemistry ,Mutagenicity Tests ,Mutagenesis ,Nucleic Acid Hybridization ,Biochemistry ,COS Cells ,Autoradiography ,DNA ,Genotoxicity - Abstract
Vinyl chloride and crotonaldehyde are known mutagens and carcinogens that, through their reaction with DNA, form specific deoxyguanosine adducts. To investigate the mutagenic potential of a subset of the possible deoxyguanosine lesions, site-specific adducts of vinyl chloride and crotonaldehyde were synthesized, inserted into a shuttle vector, and replicated in mammalian cells. Mutation yields of the DNA adducts of vinyl chloride and crotonaldehyde were found to be 2% and 5-6%, respectively, thus suggesting that these adducts could contribute to the overall genotoxicity and carcinogenicity associated with exposure to these chemicals.
- Published
- 2005
175. Efficient and Error-Free Replication past a Minor-Groove N2-Guanine Adduct by the Sequential Action of Yeast Rev1 and DNA Polymerase ζ
- Author
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Thomas M. Harris, Satya Prakash, M. Todd Washington, Irina G. Minko, Louise Prakash, Lajos Haracska, R. Stephen Lloyd, and Robert E. Johnson
- Subjects
DNA Replication ,Guanine ,Saccharomyces cerevisiae Proteins ,DNA Repair ,Base pair ,DNA polymerase ,DNA damage ,Recombinant Fusion Proteins ,DNA-Directed DNA Polymerase ,Saccharomyces cerevisiae ,DNA polymerase delta ,DNA Adducts ,Humans ,Acrolein ,Molecular Biology ,DNA clamp ,biology ,Molecular Structure ,DNA replication ,Cell Biology ,DNA ,DNA Dynamics and Chromosome Structure ,Nucleotidyltransferases ,Biochemistry ,biology.protein ,Biophysics ,REV1 ,Nucleic Acid Conformation ,Primer (molecular biology) ,DNA Damage - Abstract
Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase zeta (Pol zeta). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Pol zeta-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N(2)-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the gamma-hydroxy-1,N(2)-propano-2'deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N(2) of guanine. Acrolein, an alpha,beta-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Pol zeta subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N(2)-guanine DNA minor-groove adducts that form in DNA.
- Published
- 2004
176. Structure of a site specific major groove (2S,3S)-N6-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl DNA adduct of butadiene diol epoxide
- Author
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Tandace A, Scholdberg, Lubomir V, Nechev, W Keither, Merritt, Thomas M, Harris, Constance M, Harris, R Stephen, Lloyd, and Michael P, Stone
- Subjects
Poly U ,DNA Adducts ,Glycols ,Genes, ras ,Magnetic Resonance Spectroscopy ,Alkylation ,Deoxyadenosines ,Molecular Structure ,Oligodeoxyribonucleotides ,Mutagenesis, Site-Directed ,Epoxy Compounds ,Hydrogen Bonding ,DNA-Directed DNA Polymerase - Abstract
The solution structure of the (2S,3S)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl adduct arising from the alkylation of adenine N(6) at position X(6) in d(CGGACXAGAAG).d(CTTCTTGTCCG), by butadiene diol epoxide, was determined. This oligodeoxynucleotide contains codon 61 (underlined) of the human N-ras protooncogene. This oligodeoxynucleotide, containing the adenine N(6) adduct butadiene triol (BDT) adduct at the second position of codon 61, was named the ras61 S,S-BDT-(61,2) adduct. NMR spectroscopy revealed modest structural perturbations localized to the site of adduction at X(6).T(17), and its nearest-neighbor base pairs C(5).G(18) and A(7).T(16). All sequential NOE connectivities arising from DNA protons were observed. Torsion angle analysis from COSY data suggested that the deoxyribose sugar at X(6) remained in the C2'-endo conformation. Molecular dynamics calculations using a simulated annealing protocol restrained by a total of 442 NOE-derived distances and J coupling-derived torsion angles refined structures in which the BDT moiety oriented in the major groove. Relaxation matrix analysis suggested hydrogen bonding between the hydroxyl group located at the beta-carbon of the BDT moiety and the T(17) O(4) of the modified base pair X(6).T(17). The minimal perturbation of DNA induced by this major groove adduct correlated with its facile bypass by three Escherichia coli DNA polymerases in vitro and its weak mutagenicity [Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol. Mutagen. 35, 48-56]. Overall, the structure of this adduct is consistent with an emerging pattern in which major groove adenine N(6) alkylation products of styrene and butadiene oxides that do not strongly perturb DNA structure are not strongly mutagenic.
- Published
- 2004
177. MicroRNA-375 Expression Correlates With Increased Radiosensitivity, Decreased Invasion, and Improved Patient Survival in Head-and-Neck Squamous Cell Carcinoma
- Author
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Jeffrey E. Segall, Chandan Guha, Geoffrey J. Childs, Michael B. Prystowsky, Lizandra Jimenez, Thomas M. Harris, P. Boimel, and Alan A. Alfieri
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,Radiation ,business.industry ,Patient survival ,medicine.disease ,Head and neck squamous-cell carcinoma ,Internal medicine ,microRNA ,Medicine ,Radiology, Nuclear Medicine and imaging ,Radiosensitivity ,business - Published
- 2012
178. Competing Reactions of Aflatoxin B1 Dialdehyde: Enzymatic Reduction versus Adduction with Lysine
- Author
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Zhenwu Deng, F. Peter Guengerich, Markus Voehler, W. W. Johnson, Kyle O. Arneson, Thomas R. Sutter, John D. Hayes, Kevin M. Williams, and Thomas M. Harris
- Subjects
chemistry.chemical_classification ,Reduction (complexity) ,Aflatoxin ,Enzyme ,Biochemistry ,chemistry ,Lysine ,Organic chemistry - Published
- 2003
179. 2-n-Butyl-2-methylcyclohexanone
- Author
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Charles R. Hauser, Thomas M. Harris, and Sandra Boatman
- Subjects
Acylation ,chemistry.chemical_compound ,Ammonia ,Potassium amide ,Chemistry ,Bromide ,Potassium ,chemistry.chemical_element ,Organic chemistry ,Alkylation ,Cleavage (embryo) ,Ethyl formate - Abstract
2-n-Butyl-2-methylcyclohexanone reactant: 123 g. (1.10 moles) of 2-methylcyclohexanone reactant: 81.4 g. (1.10 moles) of ethyl formate intermediate: Sodio-2-formyl-6-methylcyclohexanone reactant: 28.2 g. (0.21 mole) of n-butyl bromide product: 2-n-butyl-2-methylcyclohexanone byproduct: 2-formyl-6-n-butyl-6-methylcyclohexanone product: 2,2-Dimethylcyclohexanone product: 2-benzyl-2-methylcyclohexanone Keywords: acylation; alkylation, C-alkylation; cleavage, ketonic; ethyl formate; potassium amide, preparation of; ammonia; potassium
- Published
- 2003
180. Phenylation with Diphenyliodonium Chloride: 1-Phenyl-2,4-Pentanedione
- Author
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Thomas M. Harris, Charles R. Hauser, and K. Gerald Hampton
- Subjects
chemistry.chemical_compound ,Ammonia ,Chemistry ,Product (mathematics) ,Sodium amide ,Diphenyliodonium chloride ,Iodobenzene ,Organic chemistry - Abstract
Phenylation with diphenyliodonium chloride: 1-phenyl-2,4-pentanedione reactant: 40.0 g. (0.400 mole) of 2,4-pentanedione product: 1-phenyl-2,4-pentanedione byproduct: iodobenzene product: 1-phenyl-1,3-butanedione product: 2,4-heptanedione product: 2,4-nonanedione product: 2,4-tridecanedione product: 3,5-heptanedione product: 1-(4-chlorophenyl)-2,4-pentanedione product: 4-(4-methylphenyl)-1-phenyl-1,3-butanedione product: 1-(4-methylphenyl)-2,4-nonanedione Keywords: arylation; 2,4-pentanedione; sodium amide, preparation of; ammonia
- Published
- 2003
181. Directed Lithiation of Aromatic Compounds: (2-Dimethylamino-5-Methylphenyl)Diphenylcarbinol
- Author
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Thomas M. Harris and J. V. Hay
- Subjects
chemistry.chemical_compound ,Chemistry ,Metalation ,Butyllithium ,chemistry.chemical_element ,Organic chemistry ,Lithium ,Tetramethylethylenediamine ,Menthol ,Catalysis - Abstract
Directed lithiation of aromatic compounds: (2-dimethylamino-5-methylphenyl)diphenylcarbinol product: (2-dimethylamino-5-methylphenyl)diphenylcarbinol catalyst: 8.8 g. (0.076 mole of N,N,N′,N′-tetramethylethylenediamine Keywords: arylation; metalation reactions, lithium; assay methods, for butyllithium, with 2-butanol, menthol, 2-pentanol, or 2-propanol, 1,10-phenanthroline indicator; tetramethylethylenediamine
- Published
- 2003
182. 1-(p-Methoxyphenyl)-5-Phenyl-1,3,5-Pentanetrione
- Author
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Marion L. Miles, Thomas M. Harris, and Charles R. Hauser
- Subjects
Acylation ,chemistry.chemical_compound ,chemistry ,Product (mathematics) ,Organic chemistry ,Sodium hydride - Abstract
1-(p-Methoxyphenyl)-5-phenyl-1,3,5-pentanetrione product: 1-(p-methoxyphenyl)-5-phenyl-1,3,5-pentanetrione reactant: benzoylacetone was obtained from Eastman Kodak (Eastman grade) product: 1-phenyl-1,3,5-hexanetrione product: 1,7-diphenyl-1,3,5,7-heptanetetraone product: 1,5-diphenyl-1,3,5-pentanetrione product: 1,5-di(p-methoxyphenyl)-1,3,5-pentanetrione Keywords: acylation; 1,2-dimethoxyethane, purification of; sodium hydride, handling of; sodium hydride, handling
- Published
- 2003
183. NMR determination of the conformation of a trimethylene interstrand cross-link in an oligodeoxynucleotide duplex containing a 5'-d(GpC) motif
- Author
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Thomas M. Harris, Patricia A. Dooley, Lubomir V. Nechev, Michael P. Stone, Gregory A. Korbel, Mingzhou Zhang, and Constance M. Harris
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Cyclopropanes ,Models, Molecular ,Chemical Phenomena ,Guanine ,Stereochemistry ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Malondialdehyde ,Nucleotide ,Computer Simulation ,Base Pairing ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Base Sequence ,Oligonucleotide ,Chemistry, Physical ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,DNA ,Cross-Linking Reagents ,chemistry ,CpG site ,Models, Chemical ,Oligodeoxyribonucleotides ,Duplex (building) ,Covalent bond ,Nucleic Acid Conformation ,Thermodynamics ,CpG Islands - Abstract
Malondialdehyde interstrand cross-links in DNA show strong preference for 5'-d(CpG) sequences. The cross-links are unstable and a trimethylene cross-link has been used as a surrogate for structural studies. A previous structural study of the 5'-d(CpG) cross-link in the sequence 5'-d(AGGCGCCT), where G is the modified nucleotide, by NMR spectroscopy and molecular dynamics using a simulated annealing protocol showed the guanine residues and the tether lay approximately in a plane such that the trimethylene tether and probably the malondialdehyde tether, as well, could be accommodated without major disruptions of duplex structure [Dooley et al. J. Am Chem. Soc. 2001, 123, 1730-1739]. The trimethylene cross-link has now been studied in a GpC motif using the reverse sequence. The structure lacks the planarity seen with the 5'-d(CpG) sequence and is skewed about the trimethylene cross-link. Melting studies indicate that the trimethylene cross-link is thermodynamically less stable in the GpC motif than in the 5-d(CpG). Furthermore, lack of planarity of the GpC cross-link precludes making an isosteric replacement of the trimethylene tether by malondialdehyde. A similar argument can be used to explain the 5'-d(CpG) preference for interchain cross-linking by acrolein.
- Published
- 2003
184. DNA interchain cross-links formed by acrolein and crotonaldehyde
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M Scott Moseley, Thomas M. Harris, Lubomir V. Nechev, Carmelo J. Rizzo, Ivan D. Kozekov, Constance M. Harris, and Michael P. Stone
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Spectrometry, Mass, Electrospray Ionization ,Chemical Phenomena ,Stereochemistry ,Imine ,Biochemistry ,Catalysis ,Adduct ,Substrate Specificity ,chemistry.chemical_compound ,DNA Adducts ,Colloid and Surface Chemistry ,DNA adduct ,Deoxyguanosine ,Organic chemistry ,Crotonaldehyde ,Acrolein ,Nuclear Magnetic Resonance, Biomolecular ,Chromatography, High Pressure Liquid ,Aldehydes ,Chemistry, Physical ,Diastereomer ,Electrophoresis, Capillary ,Stereoisomerism ,General Chemistry ,DNA ,Cross-Linking Reagents ,chemistry ,CpG Islands ,Methyl group - Abstract
Acrolein and higher alpha,beta-unsaturated aldehydes are bifunctional genotoxins. The deoxyguanosine adduct of acrolein, 3-(2-deoxy-beta-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purin-10(3H)-one (8-hydroxy-1,N(2)-propanodeoxyguanosine, 2a), is a major DNA adduct formed by acrolein. The potential for oligodeoxynucleotide duplexes containing 2a to form interchain cross-links was evaluated by HPLC, CZE, MALDI-TOF, and melting phenomena. Interchain cross-links represent one of the most serious types of damage in DNA since they are absolute blocks to replication. In oligodeoxynucleotides containing the sequence 5'-dC-2a, cross-linking occurred in a slow, reversible manner to the extent of approximately 50%. Enzymatic digestion to form 3-(2-deoxy-beta-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-(N(2)-2'-deoxyguanosinyl)pyrimido[1,2-a]purin-10(3H)one (5a) and reduction with NaCNBH(3) followed by enzymatic digestion to give 1,3-bis(2'-deoxyguanosin-N(2)-yl)propane (6a) established that cross-linking had occurred with the exocyclic amino group of deoxyguanosine. It is concluded that the cross-link is a mixture of imine and carbinolamine structures. With oligodeoxynucleotide duplexes containing the sequence 5'-2a-dC, cross-links were not detected by the techniques enumerated above. In addition, (15)N-(1)H HSQC and HSQC-filtered NOESY spectra carried out with a duplex having (15)N-labeling of the target amino group established unambiguously that a carbinolamine cross-link was not formed. The potential for interchain cross-link formation by the analogous crotonaldehyde adduct (2b) was evaluated in a 5'-dC-2b sequence. Cross-link formation was strongly dependent on the configuration of the methyl group at C6 of 2b. The 6R diastereomer of 2b formed a cross-link to the extent of 38%, whereas the 6S diastereomer cross-linked only 5%.
- Published
- 2003
185. Reaction of aflatoxin B(1) oxidation products with lysine
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Kevin M. Williams, F. Peter Guengerich, Zhengwu Deng, Thomas M. Harris, and Kyle O. Arneson
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Aflatoxin ,Spectrometry, Mass, Electrospray Ionization ,Aflatoxin B1 ,Stereochemistry ,Lysine ,Buffers ,Toxicology ,High-performance liquid chromatography ,Binding, Competitive ,Adduct ,chemistry.chemical_compound ,Hydrolysis ,Animals ,Bovine serum albumin ,Chromatography, High Pressure Liquid ,Serum Albumin ,Aldehydes ,Binding Sites ,biology ,Methylamine ,Albumin ,Serum Albumin, Bovine ,General Medicine ,Hydrogen-Ion Concentration ,Glutathione ,Kinetics ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,biology.protein ,Cattle ,Spectrophotometry, Ultraviolet ,Oxidation-Reduction - Abstract
Aflatoxin (AF) B(1) exo-8,9-epoxide hydrolysis yields AFB(1) dihydrodiol, which undergoes base-catalyzed rearrangement to, and is in equilibrium with, AFB(1) dialdehyde. We investigated the reaction of AFB(1) dialdehyde with albumin to generate a Lys adduct, previously characterized by others [Sabbioni, G., Skipper, P. L., Büchi, G., and Tannenbaum, S. R. (1987) Carcinogenesis8, 819-824; Sabbioni, G. (1990) Chem.-Biol. Interact. 75, 1-15]. Pronase digestion of bovine albumin serum treated with AFB(1) dialdehyde and HPLC yielded the adduct, identified by its characteristic UV and mass spectra. The structure of the Lys-AFB(1) dialdehyde adduct is concluded to be (S)-alpha-amino-2,3-dihydro-2-oxo-4-(1,2,3,4-tetrahydro-7-hydroxy-9-methoxy-3,4-dioxocyclopenta[c][1]benzopyran-6-yl)-1H-pyrrole-1-hexanoic acid, structure B of the former paper and 8 of the latter, based on work with the methylamine adduct described in the following paper in this issue [Guengerich, F. P., Voehler, M., Williams, K. M., Deng, Z., and Harris, T. M. (2002) Chem. Res. Toxicol. 15, 793-798]. The time course of product formation at varying concentrations of AFB(1) dialdehyde could be described by complexation with albumin with a K(d) of 1.5 mM and a first-order reaction rate with the N6-amino group of Lys of 0.033 min(-)(1). The reaction of AFB(1) dialdehyde with N(2)-acetylLys was monitored by UV spectroscopy and yielded a final spectrum similar to that of the described Lys adduct. Kinetic analysis of the changes at pH 7.2 was best described with a scheme involving equilibrium of the dialdehyde with dihydrodiol and a rate-limiting reaction of AFB(1) dialdehyde with the N6 atom of N(2)-acetylLys, with an apparent second-order rate constant of 2.6 x 10(3) M(-)(1) min(-)(1), followed by putative carbinolamine formation and rearrangement, collectively described by a first-order rate constant of 7.6 min(-)(1). Competition experiments with the hydrolysis of AFB(1) exo-8,9-epoxide indicate that N2-acetylLys also reacts with the epoxide at pH 7.2 (k = 350 M(-)(1) min(-)(1)) and 9.5 (k = 1.8 x 10(3) M(-)(1) min(-)(1)). This reaction might contribute to the formation of protein Lys adducts, depending upon the local concentration of free or protein Lys. Mass spectral analysis of trypsin digests of bovine serum albumin modified with AFB(1) dialdehyde indicated selective modification of Lys455 and Lys548. Collectively, these results provide more insight into the mechanism of formation of AFB(1) dialdehyde-protein adducts and indicate that the formation of Lys adducts is a moderately efficient process. The binding of AFB(1) dialdehyde to albumin or the protonation of the N6-amino group retards the reaction with Lys residues.
- Published
- 2002
186. DNA adducts of acrolein: site-specific synthesis of an oligodeoxynucleotide containing 6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one, an acrolein adduct of guanine
- Author
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Lubomir V. Nechev, Angela K Brock, Thomas M. Harris, Carmelo J. Rizzo, and Ivan D. Kozekov
- Subjects
Phosphoramidite ,Guanine ,Silylation ,Stereochemistry ,Imine ,Acrolein ,Oligonucleotides ,Ether ,General Medicine ,Pyrimidinones ,Toxicology ,Adduct ,chemistry.chemical_compound ,DNA Adducts ,chemistry ,Purines ,Deoxyguanosine ,Nucleoside - Abstract
3-(2-Deoxy-beta-D-erythro-pentofuranosyl)-6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one is formed in low yield by the reaction of acrolein with 2'-deoxyguanosine. The nucleoside and an oligodeoxynucleotide containing it have been synthesized. For preparation of the nucleoside 2'-deoxyguanosine was alkylated at the N1 position using 1-bromo-3-butene to give 1-(3-butenyl)-2'-deoxyguanosine. Oxidation with OsO(4) and N-methylmorpholine-N-oxide to give the 3,4-dihydroxybutyl adduct followed by oxidation with NaIO(4) gave the 1-(3-oxopropyl) adduct which cyclized spontaneously to yield the title compound as a rapidly epimerizing mixture of two diastereomers. Reduction of the nucleoside with NaBH(4) gave the unfunctionalized compound plus 1-(3-hydroxypropyl)-2'-deoxyguanosine showing that epimerization was occurring via both the imine and the 1-(3-oxopropyl) adduct. Reduction with NaCNBH(3) gave exclusively unfunctionalized 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one. The phosphoramidite reagent needed for preparation of oligonucleotides was prepared from 1-(3-butenyl)-2'-deoxyguanosine by glycolation after protection of the 3' and 5' hydroxyl groups as silyl derivatives. Acetylation of the vicinal hydroxyl groups and the exocyclic amino group followed by removal of silyl protection gave the protected nucleoside. Protection of the 5' hydroxyl group as the 4,4'-dimethoxytrityl ether followed by phosphitylation with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite gave the prosphoramidite reagent which was used to prepare a 12-mer oligodeoxynucleotide.
- Published
- 2002
187. The aflatoxin B1 formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma
- Author
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Paul T. Henderson, John M. Essigmann, Constance M. Harris, Thomas M. Harris, Maryann E. Smela, and Michelle L. Hamm
- Subjects
Aflatoxin B1 ,Carcinoma, Hepatocellular ,Guanine ,DNA damage ,Biology ,medicine.disease_cause ,chemistry.chemical_compound ,DNA Adducts ,DNA adduct ,medicine ,Humans ,AP site ,Mutation frequency ,Mutation ,Multidisciplinary ,Molecular Structure ,Mutagenesis ,Liver Neoplasms ,DNA, Neoplasm ,Biological Sciences ,medicine.disease ,Molecular biology ,Pyrimidines ,chemistry ,Hepatocellular carcinoma ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,DNA, Viral ,DNA ,Bacteriophage M13 ,Mutagens - Abstract
A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin B 1 (AFB 1 ). Hypotheses have been put forth that AFB 1 , in concert with hepatitis B virus (HBV), may play a role in the formation of, and/or the selection for, this mutation. The primary DNA adduct of AFB 1 is 8,9-dihydro-8-( N 7 -guanyl)-9-hydroxyaflatoxin B 1 (AFB 1 -N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AFB 1 -formamidopyrimidine (AFB 1 -FAPY) adduct. AFB 1 -FAPY is detected at near maximal levels in rat DNA days to weeks after AFB 1 exposure, underscoring its high persistence in vivo . The present study reveals two striking properties of this DNA adduct: ( i ) AFB 1 -FAPY was found to cause a G to T mutation frequency in Escherichia coli approximately 6 times higher than that of AFB 1 -N7-Gua, and ( ii ) one proposed rotamer of AFB 1 -FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell. Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer.
- Published
- 2002
188. Global gene expression patterns during differentiation of F9 embryonal carcinoma cells into parietal endoderm
- Author
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Thomas M. Harris and Geoffrey Childs
- Subjects
Teratocarcinoma ,Embryonal Carcinoma Stem Cells ,Cellular differentiation ,Retinoic acid ,Gene Expression ,Tretinoin ,Biology ,chemistry.chemical_compound ,Mice ,Gene expression ,Genetics ,medicine ,Tumor Cells, Cultured ,Animals ,Humans ,Transcription factor ,Oligonucleotide Array Sequence Analysis ,Gene Expression Profiling ,Endoderm ,Cell Differentiation ,General Medicine ,Cell cycle ,Molecular biology ,Gene expression profiling ,medicine.anatomical_structure ,chemistry ,Bucladesine ,Multigene Family ,Neoplastic Stem Cells - Abstract
Expression levels of over 8,900 murine genes were examined, using cDNA microarrays, during the differentiation of F9 cells into parietal endoderm following exposure to retinoic acid/dibutyryl cAMP. Gene induction and repression over the time course exhibited a biphasic pattern consistent with a transition from undifferentiated F9 cells to primitive endoderm and finally parietal endoderm. A 6-h induction with retinoic acid/cAMP/cycloheximide resulted in 109 candidate immediate response genes. During a 9-day time course 516 genes were selected as being significantly induced/repressed. Several of these genes had been previously identified as having altered expression patterns in F9 cells undergoing differentiation by retinoic acid/cAMP. Functional characterization of these genes demonstrated that the majority were transcription factors while others included surface antigens and genes involved in intracellular transport. Cluster analysis, utilizing both a hierarchical algorithm and self-organizing map, resulted in very similar gene clusters. Our studies revealed an extremely complex set of interacting signals that decide between cell death, differentiation, cell cycle withdrawal, and ultimately the traits associated with the terminal differentiated parietal endoderm cell type. The sets of genes identified here can now be modulated in a rational way to try to understand their role in differentiation.
- Published
- 2002
189. Error prone translesion synthesis past gamma-hydroxypropano deoxyguanosine, the primary acrolein-derived adduct in mammalian cells
- Author
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Manorama Kanuri, Irina G. Minko, Thomas M. Harris, Lubomir V. Nechev, R. Stephen Lloyd, and Constance M. Harris
- Subjects
DNA Replication ,Molecular Sequence Data ,Error-prone translesion synthesis ,Biochemistry ,Adduct ,chemistry.chemical_compound ,DNA adduct ,Deoxyguanosine ,Animals ,Acrolein ,Molecular Biology ,biology ,DNA synthesis ,Base Sequence ,Mutagenesis ,Cell Biology ,DNA ,Molecular biology ,Proliferating cell nuclear antigen ,chemistry ,COS Cells ,biology.protein ,Nucleic Acid Conformation ,DNA Damage - Abstract
8-Hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin- 10(3H)-one,3-(2'-deoxyriboside) (1,N(2)-gamma-hydroxypropano deoxyguanosine, gamma-HOPdG) is a major DNA adduct that forms as a result of exposure to acrolein, an environmental pollutant and a product of endogenous lipid peroxidation. gamma-HOPdG has been shown previously not to be a miscoding lesion when replicated in Escherichia coli. In contrast to those prokaryotic studies, in vivo replication and mutagenesis assays in COS-7 cells using single stranded DNA containing a specific gamma-HOPdG adduct, revealed that the gamma-HOPdG adduct was significantly mutagenic. Analyses revealed both transversion and transition types of mutations at an overall mutagenic frequency of 7.4 x 10(-2)/translesion synthesis. In vitro gamma-HOPdG strongly blocks DNA synthesis by two major polymerases, pol delta and pol epsilon. Replicative blockage of pol delta by gamma-HOPdG could be diminished by the addition of proliferating cell nuclear antigen, leading to highly mutagenic translesion bypass across this adduct. The differential functioning and processing capacities of the mammalian polymerases may be responsible for the higher mutation frequencies observed in this study when compared with the accurate and efficient nonmutagenic bypass observed in the bacterial system.
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- 2002
190. Point mutations induced by 1,2-epoxy-3-butene N1 deoxyinosine adducts
- Author
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R. Stephen Lloyd, Constance M. Harris, Jonathan B. Ward, Thomas M. Harris, Agnieszka Kowalczyk, and David A. Rodriguez
- Subjects
Epidemiology ,DNA damage ,Health, Toxicology and Mutagenesis ,Deamination ,DNA, Single-Stranded ,Mutagen ,medicine.disease_cause ,chemistry.chemical_compound ,DNA Adducts ,Deoxyadenosine ,Isomerism ,medicine ,Escherichia coli ,Point Mutation ,Base Pairing ,Genetics (clinical) ,Carcinogen ,Mutagenicity Tests ,Point mutation ,Adenine ,fungi ,Mutagenesis ,Inosine ,Biochemistry ,chemistry ,Epoxy Compounds ,DNA ,Mutagens - Abstract
The National Toxicology Program has recently classified 1,3-butadiene (BD) as a human carcinogen. BD is metabolized to the intermediates 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-dihydroxy-3,4-epoxybutane. All three metabolites have been implicated in producing specific types of DNA damage and as genotoxic agents in mice, rat, and human cells. This study has focused on EB-induced N1 deoxyinosine lesions that are formed by deamination of deoxyadenosine following reaction of the epoxide at the N1 position. The R and S stereoisomers of this lesion were incorporated site-specifically within the context of an 11-mer oligodeoxynucleotide, incorporated into M13mp7L2 single-stranded DNA, and transfected into E. coli. Both stereoisomers modestly reduced plaque-forming ability, indicating that neither lesion presents a base modification that cannot be bypassed. The resulting plaques were assessed for point mutations using differential hybridization and DNA sequence analyses. The overall mutagenic spectrum revealed that the N1 adducts were highly mutagenic (∼90% per replication cycle), causing a predominance of A G transitions. Environ. Mol. Mutagen. 38:292–296, 2001. © 2001 Wiley-Liss, Inc.
- Published
- 2002
191. Efficient nonmutagenic replication bypass of DNAs containing beta-adducts of styrene oxide at adenine N(6)
- Author
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Jari Finneman, Manorama Kanuri, R. Stephen Lloyd, Constance M. Harris, and Thomas M. Harris
- Subjects
DNA Replication ,Epidemiology ,Mutagenicity Tests ,Health, Toxicology and Mutagenesis ,Adenine ,Replication (microscopy) ,Adduct ,Styrene ,chemistry.chemical_compound ,DNA Adducts ,chemistry ,Biochemistry ,Styrene oxide ,Escherichia coli ,Epoxy Compounds ,Genetics (clinical) ,DNA - Published
- 2002
192. The Mechanism of Phosphorus Incorporation during the Electrodeposition of Nickel‐Phosphorus Alloys
- Author
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Thomas M. Harris and Quoc D. Dang
- Subjects
inorganic chemicals ,Hypophosphorus Acid ,Hydrogen ,Renewable Energy, Sustainability and the Environment ,Phosphorus ,Inorganic chemistry ,chemistry.chemical_element ,Phosphorus acid ,Condensed Matter Physics ,Redox ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Ion ,chemistry.chemical_compound ,Nickel ,chemistry ,Materials Chemistry ,Electrochemistry ,Phosphine - Abstract
Phosphine was detected and quantified during the electrodeposition of nickel‐phosphorus alloys. The presence of supports the indirect mechanism of phosphorus incorporation: the phosphorus oxyacid in the bath is reduced to phosphine, which then undergoes a redox reaction with Ni2+ to produce both phosphorus and nickel in the elemental form. Phosphine is produced more readily from hypophosphorus acid than from phosphorus acid. Phosphine production from hypophosphorus acid is favored by low pH, and facilitated by the presence of nickelous ion. The latter effect may result from the release of hydrogen ions in the redox reaction involving and Ni2+.
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- 1993
193. Abstract 1452: MicroRNA-375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity
- Author
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Thomas M. Harris, Ved P. Sharma, Ruth Hogue Angeletti, Geoffrey Childs, Lizandra Jimenez, Jeffrey E. Segall, Michael B. Prystowsky, John S. Condeelis, and Jihyeon Lim
- Subjects
Cancer Research ,Pathology ,medicine.medical_specialty ,Invadopodium ,Cancer ,Biology ,medicine.disease ,Head and neck squamous-cell carcinoma ,Oncology ,Cell culture ,Stable isotope labeling by amino acids in cell culture ,Invadopodia ,microRNA ,medicine ,Cancer research ,biology.protein ,Cortactin - Abstract
Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer, with a five-year survival rate of around 50%. Our goal is to improve patient outcome through understanding the mechanisms determining HNSCC invasiveness. Our research group previously reported that miR-375 expression levels correlate with patient survival, recurrence and distant metastasis. We have previously observed diminished HNSCC invasion in vitro correlates with increased miR-375 expression. To assess whether the miR-375 over-expressing transductant lines have reduced matrix degradation properties, a fluorescent matrix degradation assay was used. We have observed that miR-375 over-expression in HNSCC cells suppresses extracellular matrix degradation. We further determined whether miR-375 over-expressing cells show reduced invadopodium formation and maturation. We observed miR-375 expression in HNSCC cell lines reduces the number of mature invadopodia, but does not affect levels of invadopodium precursors. We examined the phosphorylation and total levels of key invadopodia components (Tks5 and cortactin) to determine whether changes in regulation or expression of these components may lead impaired matrix degradation properties. We determined that miR-375 expression in HNSCC cell lines does not reduce cellular levels of cortactin and Tks5, nor is tyrosine phosphorylation of cortactin altered. To identify candidate proteins regulated by miR-375, we are utilizing stable isotope labeling of cells in cell culture (SILAC). Candidate target proteins are being validated as targets of miR-375 and tested for involvement in the diminished invasive properties of HNSCC as a result of miR-375 expression. In conclusion, increased miR-375 expression may suppress the invasive properties of HNSCC through diminished invadopodia activity. Citation Format: Lizandra Jimenez, Ved P. Sharma, Jihyeon Lim, Ruth Angeletti, John Condeelis, Thomas Harris, Michael B. Prystowsky, Geoffrey Childs, Jeffrey E. Segall. MicroRNA-375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1452. doi:10.1158/1538-7445.AM2014-1452
- Published
- 2014
194. Interchain cross-linking of DNA mediated by the principal adduct of acrolein
- Author
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Lloyd Rs, Thomas M. Harris, Lubomir V. Nechev, Constance M. Harris, Ivan D. Kozekov, and Ana M. Sanchez
- Subjects
Magnetic Resonance Spectroscopy ,Stereochemistry ,Acrolein ,Deoxyguanosine ,General Medicine ,Toxicology ,Photochemistry ,Mass spectrometry ,High-performance liquid chromatography ,Adduct ,chemistry.chemical_compound ,DNA Adducts ,Cross-Linking Reagents ,chemistry ,Duplex (building) ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Humans ,A-DNA ,DNA ,Chromatography, High Pressure Liquid - Abstract
A DNA duplex containing the primary acrolein adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purin-10(3H)-one (2), of deoxyguanosine in a 5'-CpG sequence context spontaneously but reversibly formed an interchain cross-link with the exocyclic amino group of deoxyguanosine in the opposing chain. The linkage was sufficiently stable that the cross-linked duplex could be isolated by HPLC and characterized by MALDI-TOF mass spectrometry. Enzymatic degradation gave bis-nucleoside 6, which was independently prepared by direct reaction of 2 with dGuo.
- Published
- 2001
195. Intercalation of the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in the N-ras codon 61 sequence: DNA sequence effects
- Author
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Amanda S. Wilkinson, Zhijun Li, Thomas M. Harris, Constance M. Harris, Pamela J. Tamura, and Michael P. Stone
- Subjects
Stereochemistry ,Base pair ,Stereoisomerism ,Biochemistry ,Adduct ,chemistry.chemical_compound ,DNA Adducts ,Benz(a)Anthracenes ,Carbohydrate Conformation ,Moiety ,Humans ,Nucleotide ,Codon ,Base Pairing ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Bay-Region, Polycyclic Aromatic Hydrocarbon ,Deoxyribose ,Chemical shift ,Adenine ,Intercalating Agents ,Solutions ,Genes, ras ,chemistry ,Oligodeoxyribonucleotides ,Mutagenesis, Site-Directed ,Pseudorotation ,Thermodynamics ,Protons - Abstract
The structure of the bay region (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X(7) of 5'-d(CGGACAXGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined by NMR. This was the bay region benz[a]anthracene RSRS (61,3) adduct. The BA moiety intercalated above the 5'-face of the modified base pair. NOE connectivities between imino protons were disrupted at T16 and T17. Large chemical shifts at the lesion site were consistent with ring current shielding arising from the BA moiety. A large chemical shift dispersion was observed for the BA aromatic protons. An increased rise of 8.17 A was observed between base pairs A6 x T17 and X7 x T(16). The PAH moiety stacked with the purine ring of A6, the 5'-neighbor nucleotide. This resulted in buckling of the 5'-neighbor A6 x T17 base pair, evidenced by exchange broadening for the T17 imino resonance. It also interrupted sequential NOE connectivities between nucleotides C5 and A6. The A6 deoxyribose ring showed an increased percentage of the C3'-endo conformation. This differed from the bay region BA RSRS (61,2) adduct, in which the lesion was located at position X6 [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981], but was similar to the benzo[a]pyrene BP SRSR (61,3) adduct [Zegar I. S., Chary, P., Jabil, R. J., Tamura, P. J., Johansen, T. N., Lloyd, R. S., Harris, C. M., Harris, T. M., and Stone, M. P. (1998) Biochemistry 37, 16516-16528]. The altered sugar pseudorotation at A6 appears to be common to both bay region BA RSRS (61,3) and BP SRSR (61,3) adducts. It could not be discerned if the C3'-endo conformation at A6 in the BA RSRS (61,3) adduct altered base pairing geometry at X7 x T16, as compared to the C2'-endo conformation. The structural studies suggest that the mutational spectrum of this adduct may be more complex than that of the BA RSRS (61,2) adduct.
- Published
- 2001
196. Rapamycin blocks IL-2-driven T cell cycle progression while preserving T cell survival
- Author
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Michael B. Prystowsky, Thomas M. Harris, Geoffrey Childs, and Juana Gonzalez
- Subjects
Programmed cell death ,Cell Survival ,T cell ,T-Lymphocytes ,Cell Culture Techniques ,Apoptosis ,Biology ,Lymphocyte Activation ,Cell Line ,Mice ,Gene expression ,medicine ,Animals ,Drug Interactions ,Molecular Biology ,Oligonucleotide Array Sequence Analysis ,Regulation of gene expression ,Sirolimus ,Gene Expression Profiling ,Cell Cycle ,Cell Biology ,Hematology ,Cell cycle ,Cell biology ,Gene expression profiling ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Gene Expression Regulation ,Cell culture ,Protein Biosynthesis ,Molecular Medicine ,Interleukin-2 ,Signal transduction ,Immunosuppressive Agents ,Spleen ,Signal Transduction - Abstract
Effective cellular immune responses require increases in antigen-specific T lymphocytes; IL-2 drives antigen-stimulated T cell proliferation and is largely responsible for the increases observed. We used microarrays containing approximately 9000 mouse cDNAs to study IL-2-induced gene expression. IL-2 induces the expression of genes that regulate cell cycle progression, control cell survival, and increase synthetic and metabolic processes during proliferation. IL-2 also suppresses expression of genes that block cell cycle progression and promote cell death. Rapamycin inhibits IL-2-driven proliferation by downregulating the expression of genes required for key processes required for cell cycle progression. Rapamycin also preserves cell survival by keeping intact the IL-2-induced cell survival programs. These complex multifaceted programs of gene expression permit a dynamic regulation of cellular proliferation and cellular survival.
- Published
- 2001
197. Synthesis and characterization of nucleosides and oligonucleotides bearing adducts of butadiene epoxides on adenine n(6) and guanine n(2)
- Author
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Amanda S. Wilkinson, Mingzhu Zhang, Pamela J. Tamura, Lubomir V. Nechev, Thomas M. Harris, Dimitrios Tsarouhtsis, and Constance M. Harris
- Subjects
Purine ,Circular dichroism ,Guanine ,Base Sequence ,Oligonucleotide ,Stereochemistry ,Adenine ,Circular Dichroism ,Oligonucleotides ,Nucleosides ,General Medicine ,Toxicology ,Butene ,Mass Spectrometry ,Adduct ,chemistry.chemical_compound ,chemistry ,Epoxy Compounds ,Nucleic acid analogue ,Nuclear Magnetic Resonance, Biomolecular ,DNA - Abstract
Butadiene is a major industrial chemical whose genotoxic effects are attributed to the reaction of its oxidized metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), with DNA. Nucleosides and oligonucleotides containing regio- and stereochemically specific adducts of BDO and the BDO2-related compound, butene 3,4-diol 1,2-epoxide (BDE), on guanine [(2R)- and (2S)-N(2)-(1-hydroxy-3-buten-2-yl) and (2R,3R)- and (2S,3S)-N(2)-(2,3,4-trihydroxybut-1-yl), respectively] and on adenine [(2R)- and (2S)-N(6)-(1-hydroxy-3-buten-2-yl) and (2R,3R)- and (2S,3S)-N(6)-(2,3,4-trihydroxybut-1-yl), respectively] have been prepared by nonbiomimetic routes. For guanine adducts, 2-fluoro-O(6)-(trimethylsilylethyl)-2'-deoxyinosine was treated with (2R)- and (2S)-2-amino-3-buten-1-ol to give the BDO adducts and with (2R,3R)- and (2S,3S)-1-amino-2,3,4-butanetriol to produce the BDE adducts; the adducted oligonucleotides were prepared from 11-mer oligonucleotides containing the halopurine. Adenine adducts were prepared in a similar fashion using 6-chloropurine 2'-deoxyriboside as the reactive purine component.
- Published
- 2001
198. N-Methoxy-N-methylacetamide
- Author
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Thomas M. Harris
- Subjects
chemistry.chemical_compound ,Nucleophile ,Chemistry ,Hydrochloride ,Acetyl chloride ,Reagent ,Pyridine ,Organic chemistry ,Ether ,Carbon-13 NMR ,Solubility ,Medicinal chemistry - Abstract
[78191-00-1] C4H9NO2 (MW 103.14) InChI = 1S/C4H9NO2/c1-4(6)5(2)7-3/h1-3H3 InChIKey = OYVXVLSZQHSNDK-UHFFFAOYSA-N (acetylation of carbon nucleophiles; formation and transient protection of ketones; C-nucleophile in enolate anion condensations; preparation of aldehydes) Physical Data: bp 40–44 °C/20 mmHg.2 Solubility: sol ether and other organic solvents. Analysis of Reagent Purity: 1H NMR δ 2.12, 3.18, 3.70; 13C NMR δ 19.58, 31.98, 60.86, 171.59; IR (neat) 2950, 1670, 1410, 1380 cm−1.2 Preparative Method: reaction of Acetyl Chloride with commercially available N,O-Dimethylhydroxylamine hydrochloride in pyridine, followed by distillation at reduced pressure (65%).2 Handling, Storage, and Precautions: the compound is stable but should be protected from moisture; use in a fume hood.
- Published
- 2001
199. 2,4-Pentanedione
- Author
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Thomas M. Harris
- Subjects
Metal ,chemistry.chemical_compound ,Acetic anhydride ,chemistry ,visual_art ,Acetone ,visual_art.visual_art_medium ,Ethyl acetate ,Organic chemistry ,Chelation ,Solubility ,Enol ,Catalysis - Abstract
(keto) [123-54-6] C5H8O2 (MW 100.13) InChI = 1S/C5H8O2/c1-4(6)3-5(2)7/h3H2,1-2H3 InChIKey = YRKCREAYFQTBPV-UHFFFAOYSA-N (enol) [1522-20-9] InChI = 1S/C5H8O2/c1-4(6)3-5(2)7/h3,6H,1-2H3/b4-3- InChIKey = POILWHVDKZOXJZ-ARJAWSKDSA-N (carbon–carbon condensations involving mono-, di-, and trianions; formation of pyrazoles, isoxazoles, pyrroles, and pyridines; catalysis by metal chelates) Alternate Names: acetylacetone; acac. Physical Data: liquid, bp 45 °C/30 mmHg;26 d 0.975 g cm−3. Keto form: mp −23 °C; bp 134–136 °C.24 Enol form: mp −9 °C.24 Solubility: misc organic solvents; sol dil HCl; slightly sol hot H2O.24 Form Supplied in: commercially available liquid, in which the enol form predominates; the keto form predominates in aq soln. Preparative Methods: conveniently prepared by BF3-catalyzed reaction of acetone and acetic anhydride, or by reaction of ethyl acetate, acetone, and NaOEt.25 Purification: distillation after drying over anhyd K2CO3; several other methods are available.26 Handling, Storage, and Precautions: moderately toxic; reactions should be conducted in a well-ventilated fume hood.
- Published
- 2001
200. Mutagenic potential of guanine N(2) adducts of butadiene mono- and diolepoxide volume 13, number 1, january 2000, pp 18-25
- Author
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J. Russ Carmical, Mingzhu Zhang, Lubomir Nechev, Constance M. Harris, Thomas M. Harris, and R. Stephen Lloyd
- Subjects
General Medicine ,Toxicology - Published
- 2000
Catalog
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