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K B Tomer

Publications and source records attributed to K B Tomer.

At least 19 recordsLinked to original sources
PubMed2005

Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2005

Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning.

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.

Animals↗
PubMed2005

Moderate alcohol consumption and levels of antioxidant vitamins and isoprostanes in postmenopausal women.

BACKGROUND: Although alcohol intake has been positively associated with breast cancer risk in epidemiologic studies, the mechanisms mediating this association are speculative. OBJECTIVE: The Postmenopausal Women's Alcohol Study was designed to explore the effects of moderate alcohol consumption on potential risk factors for breast cancer. In the present analysis, we evaluated the relationship of alcohol consumption with antioxidant nutrients and a biomarker of oxidative stress. DESIGN: Participants (n=53) consumed a controlled diet plus each of three treatments (15 or 30 g alcohol/day or a no-alcohol placebo beverage), during three 8-week periods in random order. We measured the antioxidants, vitamin E (alpha (alpha)- and gamma (gamma)-tocopherols), selenium, and vitamin C in fasting blood samples which were collected at the end of diet periods, treated and frozen for assay at the end of the study. We also measured 15-F(2t)-IsoP isoprostane, produced by lipid peroxidation, which serves as an indicator of oxidative stress and may serve as a biomarker for conditions favorable to carcinogenesis. RESULTS: After adjusting for BMI (all models) and total serum cholesterol (tocopherol and isoprostane models) we observed a significant 4.6% decrease (P=0.02) in alpha-tocopherol and a marginally significant 4.9% increase (P=0.07) in isoprostane levels when women consumed 30 g alcohol/day (P=0.06 and 0.05 for overall effect of alcohol on alpha-tocopherol and isoprostanes, respectively). The other antioxidants were not significantly modified by the alcohol treatment. CONCLUSIONS: These results suggest that moderate alcohol consumption increases some biomarkers of oxidative stress in postmenopausal women.

Alcohol Drinking↗
PubMed2001

A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking.

The combination of limited proteolysis and MALDI-TOF mass spectrometry has become an important tool for the determination of epitopes but works best with highly purified antibodies. Here we report the use of capture antibodies to reduce the need for purification of the antibody in the mass spectrometric determination of the epitope. In this new method, a secondary Fc-specific antibody, covalently bound to Sepharose beads, is used to capture the primary antibody (the antibody of interest). After capture, the two antibodies are cross-linked. The antigen is then bound to the immobilized antibodies and subjected to proteolysis using several successive proteinases. In this study, this strategy is demonstrated with a crude mouse anti-ACTH IgG solution and adrenocorticotropin (ACTH). Comparing this strategy with previous methods where the antibody is bound directly to activated beads, the new method (1) results in a higher binding capacity of the bound antibody to ACTH, (2) does not require purification of the antibody of interest, and (3) dramatically reduces the chemical background in the MALDI mass spectra.

Adrenocorticotropic Hormone↗
PubMed2001

Hormone-mediated dephosphorylation of specific histone H1 isoforms.

We have previously shown a connection between histone H1 phosphorylation and the transcriptional competence of the hormone inducible mouse mammary tumor virus (MMTV) promoter. Prolonged exposure of mouse cells to dexamethasone concurrently dephosphorylated histone H1 and rendered the MMTV promoter refractory to hormonal stimulation and, therefore, transcriptionally unresponsive. Using electrospray mass spectrometry, we demonstrate here that prolonged dexamethasone treatment differentially effects a subset of the six somatic H1 isoforms in mouse cells. H1 isoforms H1.0, H1.1, and H1.2 are non-responsive to hormone whereas prolonged dexamethasone treatment effectively dephosphorylated the H1.3, H1.4, and H1.5 isoforms. The protein kinase inhibitor staurosporine, shown to dephosphorylate histone H1 and down-regulate MMTV in cultured cells, appears only to completely dephosphorylate the H1.3 isoform. These results suggest that dephosphorylation of specific histone H1 isoforms may contribute to the previously observed decrease in transcriptional competence of the MMTV promoter through the modulation of chromatin structure. In a broader sense, this work advances the hypothesis that post-translational modifications of individual histone H1 isoforms directly influence the transcriptional activation/repression of specific genes.

Animals↗
PubMed2001

Site-specific phosphorylation of human p53 protein determined by mass spectrometry.

Human recombinant p53 (r-p53) protein was studied by mass spectrometry (MS) to determine site-specific posttranslational differences between basal and hyperphosphorylated r-p53. Wild-type p53 was basally expressed after baculovirus infection while a parallel preparation was treated with the phosphatase inhibitor okadaic acid during the terminal stages of expression to create a hyperphosphorylated form of p53 known for its higher DNA binding and transcriptional activation. After immunoaffinity and HPLC purification, MALDI/MS measured a higher molecular mass for r-p53 from okadaic acid treatment relative to control, suggesting a higher phosphorylation state. This was supported by an acidic shift of r-p53 isoforms separated by gel isoelectric focusing. Employing a variety of mass spectrometric analyses combined with separation and affinity techniques, six specific phosphorylation sites of p53 were identified. The MS data indicated that hyperphosphorylated p53 showed a higher degree of phosphorylation than basal p53 at specific amino- and carboxy-terminal sites. In particular, ESI-MS demonstrated that Ser(315) was entirely phosphorylated after okadaic acid treatment, as confirmed biochemically by CDK2 kinase assay and by isoelectric focusing. In summary, MS analysis uniquely revealed increased, site-specific phosphorylations on p53 after phosphatase inhibition, particularly at Ser(315), which may be critical molecular events in defining p53 activity.

Amino Acid Sequence↗
PubMed2001

The dimerization motif of cytosolic sulfotransferases.

Cytosolic sulfotransferases sulfate steroids such as estrogens and hydroxysteroids. The enzymes, including human estrogen sulfotransferase (hEST) and hydroxysteroid sulfotransferase (hHST), are generally homodimers in solution with mouse estrogen sulfotransferase (mEST) being one of few exceptions. To identify the amino acid residues responsible for the dimerization, eight residues on the surface of hEST were mutated to their counterparts in mEST and mutated hESTs were then analyzed by gel filtration chromatography. A single mutation of Val(269) to Glu was sufficient to convert hEST to a monomer and the corresponding mutation of Val(260) also altered hHST to a monomer. The hHST crystal structure revealed a short stretch of peptide with the side-chains from two hHST monomers forming a hydrophobic zipper-like structure enforced by ion pairs at both ends. This peptide consisted of 10 residues near the C-terminus that, including the critical Val residue, is conserved as KXXXTVXXXE in nearly all cytosolic sulfotransferases. When mEST underwent the double mutations Pro269Thr/Glu270Val dimerization resulted. Thus, the KXXXTVXXXE sequence appears to be the common protein-protein interaction motif that mediates the homo- as well as heterodimerization of cytosolic sulfotransferases.

Amino Acid Motifs↗
PubMed2001

Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.

AIDS Vaccines↗
PubMed2001

An improved GC/MS-based procedure for the quantitation of the isoprostane 15-F2t-IsoP in rat plasma.

This article describes a procedure for the quantitation of the isoprostane 15-F2t-IsoP (9a,11a,15S-trihydroxy-(8b)-prosta-5Z,13E-dien-1-oic acid [CAS#27415-26-5] formerly known as 8-epi-PGF2a or 8-iso-PGF2a, and also as iPF2a-III). We have combined features from several earlier methods for 15-F2t-IsoP and prostaglandins, and identified and modified those steps that may lead to poor recoveries. The resulting protocol is precise and reliable, and was validated by a blind time-course study of plasma levels in rats treated with 120 and 1200 mg CCl4/kg body weight. Plasma levels of 15-F2t-IsoP, as measured according to the procedure described above, are good indicators of acute oxidative stress as induced by CCl4. The precision of the measurements allows detection of elevated plasma 15-F2t-IsoP levels as long as 16 h after an acute exposure of 120 mg CCl4/kg body weight, and 2 h after an exposure of 1 mg CCl4/kg body weight. The results of this low-dose, pilot study suggest that this method has sufficient analytical precision to allow the detection of the small changes in plasma isoprostane levels, which result from chronic and/or lower-level exposures to agents causing oxidative stress.

Animals↗
PubMed2000

Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells.

An analytical approach is reported for the characterization of the specific glycans found on highly glycosylated proteins based on a combination of specific proteolysis and deglycosylation combined with two different mass spectrometric approaches, matrix-assisted laser desorption/ionization mass spectrometry, and nanoelectrospray mass spectrometry/tandem mass spectrometry using a hybrid quadrupole-time-of-flight tandem mass spectrometer. The high resolution and mass accuracy of the mass spectrometric data obtained on the hybrid instrument combined with the high parent mass capabilities are shown to be extremely useful in the site-specific assignment of heterogeneous glycans. Using this methodology, 25 of 26 consensus glycosylation sites on HIV-1(SF2) gp120, expressed in Chinese hamster ovary cells, could be assigned. Good correlations between the relative abundances of members of heterogeneous series in the matrix-assisted laser desorption/ionization mass spectra and the nanoelectrospray mass spectra were observed, indicating that the mass spectrometric data reflected the actual abundances of the members of the series. These data were incorporated with molecular modeling based on the solved structure of a mutant truncated, highly deglycosylated gp120 to propose a structural model for the completely glycosylated form.

Amino Acid Sequence↗
PubMed2000

Evaluation of the binding between potential anti-HIV DNA-based drugs and viral envelope glycoprotein gp120 by capillary electrophoresis with laser-induced fluorescence detection.

The fusion of the human immunodeficiency virus (HIV) with the target cell was assisted by the interaction between the viral envelope glycoprotein HIV-1 gp120 and a chemokine receptor. Studies have shown that the efficiency of the binding depends on the presence of the V3 loop of the gp120 which is known to interact with polyanions, such as phosphorothioate oligodeoxynucleotides (Sd, potential anti-HIV drugs). In this study, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to systematically evaluate binding between Sd and HIV-1 gp120. A 25-mer fluorescently tagged phosphorothioate oligodeoxynucleotide (GEM) was employed as a probe to study this interaction. The dissociation constant (K(d)) between GEM and gp120 was determined to be 0.98 nM by Scatchard analysis. The competition constants (K(c)) of a set of Sd that compete with GEM for binding to gp120 were also determined. The results showed that the interaction had a strong dependence on the sulfur phosphorothioate backbone. Chain length and the sequence of Sd also affect the ability of binding to gp120. The ability to study the protein-drug binding in the solution with minimal sample consumption makes CE-LIF very attractive for biological studies.

Anti-HIV Agents↗
PubMed2000

Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical.

Previous studies established that the cyanyl radical ((*)CN), detected as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(*)CN by the electron spin resonance (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H(2)O(2)) and by mitochondrial cytochrome c oxidase (CcO) in the absence of H(2)O(2). To investigate the mechanism of inhibition by cyanyl radical, we isolated and characterized the iron protoporphyrin IX and heme a from the reactions of CN(-) with HRP and CcO, respectively. The purified heme from the reaction mixture of HRP/H(2)O(2)/KCN was unambiguously identified as cyanoheme by the observation of the protonated molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons was missing and had been replaced with a cyanyl group, indicating that the single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a isolated from partially or fully inhibited CcO did not show any change in the structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. However, a protein-centered radical on the CcO can be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in heme structures between HRP and CcO, the available crystallographic data also suggested that the distinct heme environments may contribute to the different inhibition mechanisms of HRP and CcO by cyanyl radical.

Apoenzymes↗
PubMed2000

Mass spectrometric characterization of a discontinuous epitope of the HIV envelope protein HIV-gp120 recognized by the human monoclonal antibody 1331A.

The characterization of a discontinuous epitope in the C5 region of the HIV envelope protein HIV-gp120, recognized by 1331A, a human mAb, is reported. Regions involved in affinity binding in the HIV-gp120 molecule were identified by epitope excision/extraction methods followed by matrix assisted laser desorption-time of flight mass spectrometry. In epitope excision, the protein is bound in its native conformation to an immobilized Ab and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the Ab. A series of proteolytic digestions of the 1331A/HIV-gp120 complex allowed the identification of protected amino acids in two noncontinuous regions of the C5 region of HIV-gp120. Interaction of the Ab with amino acids I487 and E507 of HIV-gp120 is essential for efficient binding. This is the first application of this approach for the identification and characterization of a discontinuous epitope. The results are consistent with molecular modeling results, indicating that these amino acids are located on opposite sides of a hydrophobic pocket. This pocket is thought to be of importance for the interaction of HIV-gp120 with the transmembrane protein HIV-gp41.

Amino Acid Sequence↗
PubMed2000

Mapping of the 5'-2-deoxyribose-5-phosphate lyase active site in DNA polymerase beta by mass spectrometry.

The mechanism of the 5'-2-deoxyribose-5-phosphate lyase reaction catalyzed by mammalian DNA beta-polymerase (beta-pol) was investigated using a cross-linking methodology in combination with mass spectrometric analyses. The approach included proteolysis of the covalently cross-linked protein-DNA complex with trypsin, followed by isolation, peptide mapping, and mass spectrometric and tandem mass spectrometric analyses. The 8-kDa domain of beta-pol was covalently cross-linked to a 5'-2-deoxyribose-5-phosphate-containing DNA substrate by sodium borohydride reduction. Using tandem mass spectrometry, the location of the DNA adduct on the 8-kDa domain was unequivocally determined to be at the Lys(72) residue. No additional amino acid residues were found as minor cross-linked species. These data allow assignment of Lys(72) as the sole Schiff base nucleophile in the 8-kDa domain of beta-pol. These results provide the first direct evidence in support of a catalytic mechanism involving nucleophilic attack by Lys(72) at the abasic site.

Amino Acid Sequence↗
PubMed2000

Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer.

The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction. It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags). Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity. Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search. This uncertainty, however, is overcome by MS/MS analysis of the peptides. Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein. This procedure is thus, highly sensitive, accurate, and rapid. In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling. Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.

Consecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, beta-casein and alpha-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation.

Amino Acid Sequence↗
PubMed2000

Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis.

A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope.

Acetylation↗