Biomedical subjects

Sheldon W May

Publications and source records attributed to Sheldon W May.

9 recordsLinked to original sources

PubMed2006

Analysis of organoselenium and organic acid metabolites by laser desorption single photon ionization mass spectrometry.

A method for analyzing organoselenium and organic acid metabolites using laser desorption from graphite surfaces coupled to vacuum ultraviolet single photon ionization mass spectrometry (LD/SPI MS) is described. The 1-10-fmol sensitivity and linear dynamic range allows quantitative detection of selenomethionine, trimethylselenonium ion, methylselenogalactosamine, and 1beta-methylseleno-N-acetyl-D-galactosamine in complex biological samples such as human urine. In addition, common urinary metabolites such as tartronic, glutaric, orotic, uric, suberic, and hydroxyhippuric acids, are readily detected. Screening and quantitative detection of these organoselenium and organic acid metabolites is achieved within minutes. The results are also consistent with those obtained using high-performance liquid chromatography tandem mass spectrometry techniques. The study demonstrates the viability of matrix-free LD/SPI MS for molecular characterization and quantitative analysis of biological metabolites in the m/z 10-500 range that are present in complex biological fluids.

Journal Article↗

PubMed2006

Anti-inflammatory effects of 4-phenyl-3-butenoic acid and 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester, potential inhibitors of neuropeptide bioactivation.

Substance P (SP) and calcitonin gene-related peptide (CGRP) are well established mediators of inflammation. Therefore, inhibition of the biosynthesis of these neuropeptides is an attractive potential strategy for pharmacological intervention against a number of inflammatory diseases. The final step in the biosynthesis of SP and CGRP is the conversion of their glycine-extended precursors to the active amidated peptide, and this process is catalyzed by sequential action of the enzymes peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase. We have demonstrated previously that 4-phenyl-3-butenoic acid (PBA) is a PAM inhibitor, and we have also shown that in vivo inhibition of serum PAM by PBA correlates with this compound's ability to inhibit carrageenan-induced edema in the rat. Here we demonstrate the ability of PBA to inhibit all three phases of adjuvant-induced polyarthritis (AIP) in rats; this represents the first time that an amidation inhibitor has been shown to be active in a model of chronic inflammation. We recently introduced 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid (AOPHA) as one of a new series of mechanism-based amidation inhibitors. We now report for the first time that AOPHA and its methyl ester (AOPHA-Me) are active inhibitors of serum PAM in vivo, and we show that AOPHA-Me correspondingly inhibits carrageenan-induced edema in rats in a dose-dependent manner. Neither PBA nor AOPHA-Me exhibits significant cyclooxygenase (COX) inhibition in vitro; thus, the anti-inflammatory activities of PBA and AOPHA-Me are apparently not a consequence of COX inhibition. We discuss possible pharmacological mechanisms that may account for the activities of these new anti-inflammatory compounds.

Adjuvants, Immunologic↗

PubMed2005

Iso-coenzyme A.

Iso-coenzyme A is an isomer of coenzyme A in which the monophosphate is attached to the 2'-carbon of the ribose ring. Although iso-CoA was first reported in 1959 (Moffatt, J. G., and Khorana, H. G. (1959) J. Am. Chem. Soc. 81, 1265-1265) to be a by-product of the chemical synthesis of CoA, relatively little attention has been focused on iso-CoA or on acyl-iso-CoA compounds in the literature. We now report structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and beta-hydroxybutyryl-iso-CoA using mass spectrometry (MS), tandem MS, and homonuclear and heteronuclear NMR analyses. Although the 2'-phosphate isomer of malonyl-CoA was recently identified in commercial samples, previous characterizations of iso-CoA itself have been based on chromatographic analyses, which ultimately rest on comparisons with the degradation products of CoA and NADPH or have been based on assumptions regarding enzyme specificity. We describe a high performance liquid chromatography methodology for separating the isomers of several CoA-containing compounds. We also report here the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl transfer reactions. Finally, we describe a simple synthesis of iso-CoA from CoA, which utilizes beta-cyclodextrin to produce iso-CoA with high regioselectivity, and we demonstrate a plausible mechanism that accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. We anticipate that these results will provide methodology and impetus for investigating iso-CoA compounds as potential pseudo-substrates or inhibitors of the >350 known CoA-utilizing enzymes.

Acetyl Coenzyme A↗

PubMed2004

Analysis of organoselenium compounds in human urine using active carbon and chemically modified silica sol-gel surface-assisted laser desorption/ionization high-resolution time-of-flight mass spectrometry.

Laser desorption/ionization time-of-flight mass spectra of three thermally labile low molecular weight organoselenium compounds (selenomethionine, selenoethionine, trimethylselenonium iodide) in human urine matrix have been obtained by using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). Four active layers, active carbon, silica sol-gel, and silica sol-gel impregnated with crown ether and with active carbon, were deposited on three different support substrates: (i) microscope slide coverslips; (ii) Al foil; and (iii) Cu tapes. Primarily protonated molecular ions and alkali metal adducts were observed in the mass spectra. A mixed-silica sol-gel and active carbon substrate active layer gave the best signal response for all compounds. Sol-gel substrates offered very clean backgrounds compared to the pure active carbon layers; however, the mass spectral signal intensities acquired were substantially lower for the sol-gel surfaces than those of the pure active carbon under the same conditions. Cu tape and Al foil support materials gave strong mass spectra of molecular ions and alkali metal molecular ion adducts, with lower Na and K adducts found with the Cu tape than with Al foil. Glass coverslips gave no response under all experimental conditions examined. Aerosol deposition was used to prepare SALDI substrates to avoid the localized "sweet spot" phenomenon encountered in conventional SALDI substrate preparation. Various nebulizers were examined and found to be effective in producing SALDI films with controlled and reproducible thicknesses. We were able to obtain exact mass identification of all three selenium species by high-resolution TOF-MS. To the best of our knowledge, this is the first time low molecular weight organoselenium compounds have been identified by SALDI-high-resolution TOF-MS.

Carbon↗

PubMed2004

Selenium redox cycling in the protective effects of organoselenides against oxidant-induced DNA damage.

The biological role of selenium is a subject of intense current interest, and the antioxidant activity of selenoenzymes is now known to be dependent upon redox cycling of selenium within their active sites. Exogenously supplied or metabolically generated organoselenium compounds, capable of propagating a selenium redox cycle, might therefore supplement natural cellular defenses against the oxidizing agents generated during metabolism. We now report evidence that selenium redox cycling can enhance the protective effects of organoselenium compounds against oxidant-induced DNA damage. Phenylaminoethyl selenides were found to protect plasmid DNA from peroxynitrite-mediated damage by scavenging this powerful cellular oxidant and forming phenylaminoethyl selenoxides as the sole selenium-containing products. The redox properties of these organoselenoxide compounds were investigated, and the first redox potentials of selenoxides in the literature are reported here. Rate constants were determined for the reactions of the selenoxides with cellular reductants such as glutathione (GSH). These kinetic data were then used in a MatLab simulation, which showed the feasibility of selenium redox cycling by GSH in the presence of the cellular oxidant, peroxynitrite. Experiments were then carried out in which peroxynitrite-mediated plasmid DNA nick formation in the presence or absence of organoselenium compounds and GSH was monitored. The results demonstrate that GSH-mediated redox cycling of selenium enhances the protective effects of phenylaminoethyl selenides against peroxynitrite-induced DNA damage.

Antioxidants↗

PubMed2004

Reversal of the transformed phenotype and inhibition of peptidylglycine alpha-monooxygenase in Ras-transformed cells by 4-phenyl-3-butenoic acid.

Recent studies have shown that the proliferation of some tumor cells is dependent on autocrine growth loops that require amidated autocrine growth factors. Peptidylglycine alpha-monooxygenase (PAM) is required for amidation of these growth factors and, therefore, this enzyme is an attractive target for anti-tumor compounds. 4-Phenyl-3-butenoic acid (PBA) is an irreversible turnover-dependent inhibitor of PAM in vitro and has been shown to decrease lung cancer cell proliferation by inhibiting the synthesis of amidated growth factors. We show here that PBA (0.1 mg/mL) inhibits the growth of Ras-transformed epithelial cells (WB-Ras) but has little effect on the proliferation of normal epithelial cells (WB-Neo). The methyl ester derivative of PBA (PBA-Me) at 10-fold lower concentration also exhibits a selective inhibition of Ras-transformed cell growth compared to normal epithelial cell growth. In addition, PBA produces a significant upregulation of gap junctional communication between WB-Ras cells following 2-5 day treatments, with a corresponding increase in the degree of connexin 43 phosphorylation and an increase in the number of connexin 43-containing plasma membrane gap junction plaques. Western blot analyses indicate no effect of PBA on the proportion of p21 Ras in the membrane versus cytosolic fractions or on p44/42 MAP kinase phosphorylation. Furthermore, the cell morphology of PBA-treated WB-Ras cells is altered, so as to more closely resemble that of non-transformed WB-Neo cells. PAM activity was assayed in both WB-Ras and WB-Neo cells, and we demonstrate that PBA at long treatment times (4 days) inhibits PAM activity in both cell types at concentrations that produce selective growth inhibition of WB-Ras cells. Shorter PBA treatment times (24 h), however, inhibit PAM activity in WB-Ras but not WB-Neo cells, an effect that was mimicked by PBA-Me. Taken together, these results clearly demonstrate that PBA returns Ras-transformed cells to a more normal phenotype, a finding consistent with the known increased dominance of the Ras signaling pathway in transformed epithelial cells.

Cell Line, Transformed↗

PubMed2003

Separation methods applicable to the evaluation of enzyme-inhibitor and enzyme-substrate interactions.

Enzymes catalyze a rich variety of metabolic transformations, and do so with very high catalytic rates under mild conditions, and with high reaction regioselectivity and stereospecificity. These characteristics make biocatalysis highly attractive from the perspectives of biotechnology, analytical chemistry, and organic synthesis. This review, containing 128 references, focuses on the use of separation techniques in the elucidation of enzyme-inhibitor and enzyme-substrate interactions. While coverage of the literature is selective, a broad perspective is maintained. Topics considered include chromatographic methods with soluble or immobilized enzymes, capillary electrophoresis, biomolecular interaction analysis tandem mass spectrometry (BIA-MS), phage and ribosomal display, and immobilized enzyme reactors (IMERs). Examples were selected to demonstrate the relevance and application of these methods for determining enzyme kinetic parameters, ranking of enzyme inhibitors, and stereoselective synthesis and separation of chiral entities.

Electrophoresis↗

PubMed2003

Catalysis, stereochemistry, and inhibition of ureidoglycolate lyase.

Ureidoglycolate lyase (UGL, EC 4.3.2.3) catalyzes the breakdown of ureidoglycolate to glyoxylate and urea, which is the final step in the catabolic pathway leading from purines to urea. Although the sequence of enzymatic steps was worked out nearly 40 years ago, the stereochemistry of the uric acid degradation pathway and the catalytic properties of UGL have remained very poorly described. We now report the first direct investigation of the absolute stereochemistry of UGL catalysis. Using chiral chromatographic analyses with substrate enantiomers, we demonstrate that UGL catalysis is stereospecific for substrates with the (S)-hydroxyglycine configuration. The first potent competitive inhibitors for UGL are reported here. These inhibitors are compounds which contain a 2,4-dioxocarboxylate moiety, designed to mimic transient species produced during lyase catalysis. The most potent inhibitor, 2,4-dioxo-4-phenylbutanoic acid, exhibits a KI value of 2.2 nM and is therefore among the most potent competitive inhibitors ever reported for a lyase enzyme. New synthetic alternate substrates for UGL, which are acyl-alpha-hydroxyglycine compounds, are described. Based on these alternate substrates, we introduce the first assay method for monitoring UGL activity directly. Finally, we report the first putative primary nucleotide and derived peptide sequence for UGL. This sequence exhibits a high level of similarity to the fumarylacetoacetate hydrolase family of proteins. Close mechanistic similarities can be visualized between the chemistries of ureidoglycolate lyase and fumarylacetoacetate hydrolase catalysis.

Amidine-Lyases↗

PubMed2002

Selenium-based pharmacological agents: an update.

The biochemistry and pharmacology of selenium is a subject of intense current interest, particularly from the viewpoint of public health. Selenium, long recognised as a dietary antioxidant, is now known to be an essential component of the active sites of several enzymes, including glutathione peroxidase and thioredoxin reductase, which catalyse reactions essential to the protection of cellular components against oxidative and free radical damage. A low concentration of selenium in plasma has been identified as a risk factor for several diseases, including cancer, cardiovascular disease, osteoarthritis and AIDS, and several large-scale selenium supplementation human trials are now underway. Evidence is emerging that, at least in the case of cancer, the antitumorigenic effect of selenium supplementation arises at least in part from enhanced production of specific selenium-containing metabolites, not just from maximal expression of selenoenzymes. Therefore a number of novel pharmaceutical agents which are selenium-based or which target specific aspects of selenium metabolism are under development. Among these are orally-active antihypertensive agents, anticancer, antiviral, immunosuppressive and antimicrobial agents, and organoselenium compounds which reduce oxidative tissue damage and edema. It can be anticipated that as our understanding of the basic biology and biochemistry of selenium increases, the coming years will bring further development of new selenium-based pharmaceutical agents with therapeutic potential toward a variety of human diseases.

Animals↗