Biomedical subjects

K B Tomer

Publications and source records attributed to K B Tomer.

At least 55 records · Page 3Linked to original sources

PubMed1996

Identification of five hemoglobins in B6C3F1 mice by mass spectrometry and sequence analysis.

The aim of the work is to identify and characterize the hemoglobins found in B6C3F1 mice using mass spectrometry. The primary structures are compared to those reported for BALB/c mice. Individual hemoglobin chains were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the globins were determined using electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The purified globin chains were enzymatically cleaved and the resulting peptides were separated by RP-HPLC. The chains were identified by N-terminal sequencing and mass spectrometry (MALDI). Selected peptides were analysed by Edman degradation. ESI analysis indicates that B6C3F1 mice have two alpha-globin chains (alpha-1 and alpha-2) and at least three beta-globin chains, beta-1, beta-2 and beta-3. This is one additional alpha- and one additional beta-globin chain than reported in the literature for BALB/c mice. Mass and sequence analysis of enzymatically generated peptides showed variations in the amino acid sequence in the alpha-1, alpha-2, beta-2 and beta-3 chains compared to the BALB/c mouse hemoglobins (alpha, beta (minor) and beta (major)). The study showed that mass spectrometry in combination with traditional protein chemistry is able to identify and locate minor protein sequence variations.

Amino Acid Sequence↗

PubMed1996

Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.

RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways.

Animals↗

PubMed1996

Identification of radical adducts formed in the reactions of unsaturated fatty acids with soybean lipoxygenase using continuous flow fast atom bombardment with tandem mass spectrometry.

Structures of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts formed in the reactions of soybean lipoxygenase with linoleic acid, linolenic acid, and arachidonic acid were determined using continuous flow fast atom bombardment (CF-FAB) combined with tandem mass spectrometry. The radical adducts of these lipoxygenase-dependent reactions were: n-octanoic acid radical, 12,13-dihydroxylinoleic acid radical, 12,13-epoxylinoleic acid radical, and n-pentyl radical from linoleic acid; n-octanoic acid radical, ethyl radical, and cis/trans and/or positional isomers (1- and 3-pentenyl) of pentenyl radical from linolenic acid; and 14,15-epoxyarachidonic acid radical and n-pentyl radical from arachidonic acid. Of these radical adducts, the n-octanoic acid radical from linoleic and linolenic acid, the ethyl radical from linolenic acid, and the 12,13-dihydroxylinoleic acid radical are identified for the first time in the reactions of soybean lipoxygenase. Thus the CF-FAB combined with tandem mass spectrometry employed here, by which both radical adducts and their fragment ions can be detected, is shown to be a powerful tool in the structural identification of free radicals.

Arachidonic Acid↗

PubMed1996

Regulation of 13(S)-hydroxyoctadecadienoic acid biosynthesis in Syrian hamster embryo fibroblasts by the epidermal growth factor receptor tyrosine kinase.

Metabolism of arachidonic and linoleic acid can be regulated by polypeptide growth factors in a variety of cell types. In Syrian hamster embryo (SHE) fibroblasts, epidermal growth factor (EGF) stimulates the conversion of exogenous linoleic acid to 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of 13-HODE biosynthesis blocks the EGF-mitogenic response in SHE cells, and 13-HODE and its hydroperoxy precursor are potent and highly specific enhancers of EGF-dependent DNA synthesis. We demonstrated that EGF stimulates a biphasic production and release of endogenous 13-HODE. Through development of a stable isotope-dilution GC/MS assay for 13-HODE, we observed 13-HODE production as early as 5 min after EGF stimulation, and this initial phase peaked at 1 hr. A second rise in 13-HODE formation was seen at 2-4 hr, and this phase plateaued at 4-6 hr at a level of 30-40 ng/10(6) cells. EGF stimulation of 13-HODE biosynthesis is not mediated by transcriptional or translational regulation of the inducible form of prostaglandin H synthase. Based on enzyme inhibitor studies and structural characterization of products, the linoleate metabolite is apparently formed by an n-6 lipoxygenase that remains to be characterized. EGF stimulation of 13-HODE formation is linked with activation of the EGF receptor tyrosine kinase. Inhibition of EGF receptor tyrosine kinase activity with methyl-2,5-dihydroxycinnamate blocked EGF-dependent linoleic acid metabolism and EGF-regulated DNA synthesis. Potentiation of the EGF receptor tyrosine phosphorylation cascade through treatment of SHE cells with the tyrosine phosphatase inhibitor vanadate resulted in a 3-fold increase in EGF-stimulated 13-HODE production and a corresponding enhancement of the EGF mitogenic response. The coupling of EGF-regulated linoleic acid metabolism with the EGF receptor tyrosine kinase activity suggests the importance of specific linoleate compounds in mediating mitogenic signal transduction.

Animals↗

PubMed1996

CYP2J subfamily P450s in the lung: expression, localization, and potential functional significance.

Cytochrome P450 (P450) monooxygenases catalyze the epoxidation of arachidonic acid to form epoxyeicosatrienoic acids, which modulate bronchial smooth muscle tone and airway transepithelial ion transport. We recently described a new human P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homologue (CYP2J3). Northern analysis of lung RNA using CYP2J cDNA probes demonstrated that CYP2J2 and CYP2J3 mRNAs were expressed in the lung. Immunoblotting of microsomal fractions prepared from human and rat lungs using a polyclonal antibody raised against recombinant human CYP2J2 revealed a single 56-kDa band confirming abundant pulmonary CYP2J2 and CYP2J3 protein expression. Immunohistochemical analysis of formalin-fixed paraffin-embedded human and rat lung sections using the anti-human CYP2J2 IgG and avidin/biotin/peroxidase detection showed that CYP2J proteins were primarily expressed in ciliated epithelial cells lining the airway. Prominent staining was also noted in nonciliated airway epithelial cells, bronchial and pulmonary vascular smooth muscle cells, pulmonary vascular endothelium, and alveolar macrophages, whereas less intense staining was noted in alveolar epithelial cells. Endogenous epoxyeicosatrienoic acids were detected in both human and rat lung using gas chromatography/mass spectrometry, thus providing direct evidence for the in vivo human and rat pulmonary P450 metabolism of arachidonic acid. Based on these data, we conclude that CYP2J2 and CYP2J3 are abundant pulmonary arachidonic acid epoxygenases and that CYP2J products, the epoxyeicosatrienoic acids, are endogenous constituents of human and rat lung. In addition to known effects on airway smooth muscle tone and transepithelial electrolyte transport, the localization of CYP2J proteins to vascular smooth muscle and endothelium suggests that epoxyeicosatrienoic acids may also be involved in the modulation of pulmonary vascular tone.

8,11,14-Eicosatrienoic Acid↗

PubMed1995

Characterization of the lower-molecular-mass fraction of venoms from Dendroaspis jamesoni kaimosae and Micrurus fulvius using capillary-electrophoresis electrospray mass spectrometry.

Capillary electrophoresis (CE) with electrospray ionization (ESI) and selected ion-monitoring mass-spectrometric (SIM-MS) detection has been used to provide as much information as possible about the lower molecular-mass fraction (peptides of molecular masses up to 8500 Da) of the venoms of Dendroaspis jamesoni kaimosae (Jameson's Mamba) and Micrurus fulvius (Eastern Coral Snake). Method development was based on the venom of D. jamesoni kaimosae, which contains some previously described peptides, with subsequent application to the completely unknown venom of M. fulvius. CE-ESI-SIM-MS provides a rapid and extremely sensitive method for the detection and molecular-mass determination of peptides present in venoms. It has been utilized to provide molecular masses and thus, by inference, confirmation of the peptide compositions for those toxins which have been previously described in the literature. Our methodology indicates the presence of 83 peptides in the venom of D. jamesoni kaimosae and 49 peptides in the venom of M. fulvius in the molecular-mass range 6000-8500 Da.

Animals↗

PubMed1995

Phenyl N-tert-butyl nitrone forms nitric oxide as a result of its FE(III)-catalyzed hydrolysis or hydroxyl radical adduct formation.

Phenyl N-tert-butyl nitrone (PBN) is commonly employed in spin-trapping studies. We report here evidence that PBN in aqueous solutions is decomposed by two pathways leading to the generation of nitric oxide (.NO). The first pathway is by hydrolysis of PBN, which is strongly catalyzed by ferric iron. The second pathway is via PBN-hydroxyl radical adduct formation. .NO was trapped in the presence of cysteine and ferrous iron to form a [(cys)2Fe(NO)2]-3 complex, which was measured by use of electron paramagnetic resonance (EPR) spectroscopy. A concomitant metabolite, benzaldehyde, was detected from both reaction mixtures. We propose that PBN is hydrolyzed by Fe3+ or attacked by hydroxyl radical, leading eventually to a common transient species, tert-butyl hydronitroxide [t-BuN(O.)H], which is further oxidized to a .NO source, t-BuNO. Our data imply that PBN may decompose to .NO when used in biological models with oxidative stress conditions.

Catalysis↗

PubMed1994

Isolation of hydrophobic lipoproteins in organic solvents by pressure-assisted capillary electrophoresis for subsequent mass spectrometric characterization.

Two capillary electrophoretic (CE) separation techniques with either simultaneous solvent flow induced by hydrostatic pressure or CE followed by low pressurization with helium were developed for the analysis of extremely hydrophobic proteins, such as the lung surfactant protein SP-C. For both related procedures, buffer solutions containing up to 70% of 2-propanol were used for the capillary electrophoretic separation. This high concentration of organic co-solvent, needed to solubilize the protein, dramatically reduces the electroosmotic flow (EOF) in aminopropyltrimethoxysilane-treated fused-silica capillaries. Because the EOF was insufficient to elute the separated analytes from the capillary, two "pressure-assisted" CE techniques were developed. An additional flow to elute the separated analytes was produced either by raising the inlet of the capillary or by helium pressure. Using the pressurization procedure a baseline separation of the SP-C protein and its dimeric complex was obtained in a 55-minute electrophoretic run, followed by pressure elution of the analyte to the detector. The present combination of pressurization and capillary electrophoresis does not require any detergents or involatile buffer additives, which are usually needed to solubilize extremely hydrophobic lipoproteins. It is therefore applicable to on-line coupling with electrospray mass spectrometry for the direct structural characterization of hydrophobic proteins.

Amino Acid Sequence↗

PubMed1994

Inherent versatility of P-450 oxygenase. Conferring dehydroepiandrosterone hydroxylase activity to P-450 2a-4 by a single amino acid mutation at position 117.

Mouse steroid 15 alpha-hydroxylase P-450 2a-4 is restricted in its substrate specificity to the delta 4, 3-ketone steroids such as androstenedione. As a result, the P-450 exhibits little hydroxylase activity toward delta 5, 3-hydroxysteroids including dehydroepiandrosterone (DHEA). A single amino acid mutation of Ala at position 117 to Val, however, is enough to confer a high DHEA hydroxylase activity to P-450 2a-4 with 7 alpha-OH DHEA as one of the two major hydroxylated metabolites. Mouse coumarin 7-hydroxylase P-450 2a-5 contains Val at position 117, but it exhibits very low DHEA hydroxylase activity. P-450 2a-5 acquires high DHEA hydroxylase activity, however, by a mutation of Phe-209 to Asn. Moreover, the mutant P-450 2a-5 loses its activity when Val is replaced by Ala at position 117. The residue at position 117, therefore, plays the principal role in the determination of the DHEA hydroxylase activity of the P-450s. Conversely, mutations at residue 117 have little effect on the androstenedione hydroxylase activities of the P-450s. Further modeling of the DHEA binding orientation in the substrate-heme pocket of bacterial P-450cam (Iwasaki, M., Darden, T., Pedersen, L., Davis, D. G., Juvonen, R. O., Sueyoshi, T., and Negishi, M. (1993) J. Biol. Chem. 268, 759-762) provides support for the hypothesis that the type of residue at position 117 determines the steroid-substrate specificity of the P-450 depending on the substituent at the C3 position of steroid molecule.

Androstenedione↗

PubMed1994

Capillary electrophoresis combined with 252Cf plasma desorption and electrospray mass spectrometry for the structural characterization of hydrophobic polypeptides using organic solvents.

Capillary electrophoresis (CE) conditions have been developed for the separation of hydrophobic polypeptides, such as fatty acid-acylated peptides, and their subsequent structural identification by 252Cf plasma desorption (PDMS) and electrospray mass spectrometry (ESMS). Salt- and detergent-free aqueous acetic acid buffers containing up to 20% 2-propanol or 25% acetonitrile were employed for CE separations of hydrophobic peptides with (i) untreated, and (ii) 3-aminopropyltrimethoxysilane-derived fused silica capillaries. For both capillary types, electroosmotic flow rates suitable for sample isolation and transfer were determined, and CE separations of polypeptide mixtures were compared for aqueous buffers containing 2-propanol or acetonitrile. For the mass spectrometric identification of CE-separated peptides, a sheath flow sample isolation method was developed for subsequent transfer to PDMS. This procedure enabled the efficient isolation of peptide fractions for PDMS analysis, or alternative microanalytical techniques.

Amino Acid Sequence↗

PubMed1994

Tris(2-chloroethyl) phosphate pharmacokinetics in the Fischer 344 rat: a comparison of conventional methods and in vivo microdialysis coupled with tandem mass spectrometry.

The pharmacokinetics of tris(2-chloroethyl) phosphate (TRCP, 20 mg/kg, iv) were investigated in awake male and female and anesthetized male Fischer 344 (F344) rats by conventional (CONV) sampling/detection methods (blood withdrawal with sample workup and analysis for TRCP). TRCP pharmacokinetics were also investigated in anesthetized male F344 rats using a new sampling/detection technique, in vivo microdialysis coupled with tandem mass spectrometry (MD/MS/MS). The concentration of free TRCP in plasma versus time profiles were analyzed using noncompartmental methods to estimate pharmacokinetic parameters. Comparisons of mean parameter estimates were made for (1) awake males versus females in CONV studies (t test, no significant differences, p < or = 0.05) and (2) awake and anesthetized males in CONV studies and anesthetized males in MD/MS/MS studies. There were significant differences (Scheffe's test) for the three groups of male rats, most notably the free TRCP concentration in plasma at early time points in CONV versus MD/MS/MS studies. The contributions of an indwelling jugular cannula, the blood sampling regimen, and the in vitro MD/MS/MS standard calibration curve were investigated. It appears that quantitation of TRCP by mass spectrometry using an in vitro standard calibration is responsible for the difference.

Anesthesia↗

PubMed1994

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

Amino Acid Sequence↗

PubMed1993

Nanoscale packed capillary liquid chromatography-electrospray ionization mass spectrometry: analysis of penicillins and cephems.

A series of seventeen penicillins and cephems (cephalosporins and cephamycins) was examined by electrospray ionization. Separations by nanoscale packed-capillary liquid chromatography, with sub-microliter flow-rates, were performed using methanol-water and acetonitrile-water both containing trifluoroacetic acid gradients. In the on-column analyses, the protonated species usually predominate, and the fragment ions are often present which can be used for confirmation of compound identity. With combined nanoscale packed-capillary liquid chromatography-electrospray ionization mass spectrometry, separations and full-scan mass spectra can be obtained on *q12-15 ng of analyte, allowing the analysis of therapeutic levels of these antibiotics from only a few microliters of serum.

Cefotaxime↗

PubMed1992

On-line coupling of in vivo microdialysis with tandem mass spectrometry.

The capability of interfacing in vivo microdialysis with mass spectrometry has been demonstrated. The goal of this research was to demonstrate the feasibility of real-time analysis in biological systems using microdialysis in combination with tandem mass spectrometry (MS/MS). Microdialysis sampling was accomplished by surgically implanting a small microdialysis probe into a tissue or area of interest. Molecules diffuse through the membrane of the microdialysis probe due to concentration differences. These molecules are collected in a sample loop and analyzed by tandem mass spectrometry. Sequential injections can be made in as little as 2 min. This capability is advantageous in the study of molecules with very rapid elimination rates. Tris(2-chloroethyl)phosphate (TRCP) was used as a model compound in the development of this analytical technique. As an example of an application of the microdialysis/MS/MS technique, plasma concentration vs time curves were obtained and compared with the plasma concentration profiles obtained using conventional studies. For the microdialysis/MS/MS studies, the average slope from three animals was -0.086 min-1. In comparison, the average slope from four animals from the conventional studies was -0.035 min-1.

Animals↗

PubMed1992

Combined liquid chromatography/electron paramagnetic resonance spectrometry/electrospray ionization mass spectrometry for radical identification.

Electron paramagnetic resonance (EPR) spectrometry and mass spectrometry (MS) have been coupled together on-line with liquid chromatography (LC)/UV detection. These combined techniques have been applied to the determination of spin-trapped radical adducts, including phenyl, 2-, 3-, and 4-chlorophenyl, and 2-bromophenyl radicals trapped with alpha-(1-oxo-4-pyridyl)-N-tert-butylnitrone (4-POBN), and phenyl radicals trapped with 2-methyl-2-nitrosopropane (MNP), alpha-phenyl-N-tert-butylnitrone (PBN), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Oxidized and reduced forms of the radical adducts were also detected by the on-line LC/EPR/MS system.

Chromatography, Liquid↗

PubMed1992

Detection of the ethyl- and pentyl-radical adducts of alpha-(4-pyridyl-1- oxide)-N-tert-butylnitrone in rat-liver microsomes treated with ADP, NADPH and ferric chloride.

HPLC-EPR analyses of the reaction mixtures of microsomal suspensions incubated with ADP, ferric chloride, NADPH and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were performed. In the elution pattern of the reaction mixture, three peaks (peaks 1, 2 and 3) were detected. The radical adducts (1 and 3) were identified as being the pentyl- and ethyl-radical adducts of 4-POBN by comparing their retention times with those of the authentic radical adducts.

Adenosine Diphosphate↗

PubMed1991

Nanoscale separations combined with tandem mass spectrometry.

High-efficiency separations of peptide mixtures, tryptic digest and other biological compounds have been achieved using nanoscale packed capillaries and capillary zone electrophoresis (CZE). The coaxial continuous-flow fast atom bombardment design is an excellent interface for coupling these separation techniques with mass spectrometry (MS). In addition, this interface is very useful for the acquisition of MS-MS data from compounds separated by nanoscale packed capillary liquid chromatography and CZE. Structurally informative daughter-ion spectra can be obtained at the low picomole to femtomole level.

Amino Acid Sequence↗

PubMed1991

Nanoscale packed-capillary liquid chromatography coupled with mass spectrometry using a coaxial continuous-flow fast atom bombardment interface.

Nanoscale packed-capillary liquid chromatography (LC) columns have been coupled with mass spectrometry (MS) using a coaxial continuous-flow fast atom bombardment interface. The combined system has been applied to the analysis of mixtures of peptides, including synthetic mixtures of bioactive peptides and tryptic digests of proteins. Nanoscale packed-capillary columns offer two principal advantages for LC/MS analysis--high chromatographic separation efficiencies and low mobile-phase flow rates. The high separation efficiencies facilitate the separation of complex mixtures, and the low mobile-phase flow rates reduce problems with coupling the LC effluent with the high-vacuum, high-voltage environment of sector MS ion sources. The columns used in this work were 50- or 75-micron i.d., 1-2 m long, packed with 10-micron C18 particles, using mobile-phase flow rates of 50-350 nL/min.

Animals↗