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Biomedical subjects

J W Newman

Publications and source records attributed to J W Newman.

At least 19 recordsLinked to original sources
PubMed2004

Cytochrome p450-dependent lipid metabolism in preovulatory follicles.

Estrogen biosynthesis and proteolysis are both important processes involved in ovarian follicular development, which may be influenced by cytochrome P450 (CYP)-dependent fatty acid metabolites. However, CYP-dependent lipid metabolism has not been characterized with respect to follicular maturation in vivo. Therefore, follicular fluid was collected in the hours before and after the LH surge in pigs, and concentrations of epoxy, hydroxy, and dihydroxy lipids were measured by liquid chromatography tandem mass spectrometry. Arachidonate oxidation and epoxyeicosatrienoic acid hydrolysis to dihydroxyeicosatrienoic acids (DHETs) were also assessed in thecal and granulosa tissue fractions, and the expression of CYP epoxygenases was evaluated by immunoblots using available antisera. To evaluate soluble epoxide hydrolase (sEH) expression, the porcine sEH was cloned from ovarian tissue, expressed and purified for antibody generation. The follicular fluid oxylipin concentrations ranged from 1-150 nm depending on the compound and estrous stage. The follicular fluid concentrations of CYP-dependent oxylipins increased at estrus, as did sEH expression; however, significant changes in epoxides were not observed, and the 11,12-DHET peak was delayed. The ratio of 14,15-DHET:11,12-DHET across all samples correlated with the log of follicular fluid estradiol concentrations (P < 0.01). Epoxygenase activities were similar in theca and granulosa, varying little with follicular development, whereas the decline of a single CYP2J isoform at ovulation was observed by immunoblots. The sEH activity was higher in granulosa than in theca. Finally, the dynamic changes in follicular CYP-dependent arachidonic acid metabolites and their modulatory function in vascular models suggest roles for these metabolites in follicular maturation, which may include regulation of estradiol biosynthesis and preovulatory remodeling of the follicular wall that should be fully explored in future studies.

Animals↗
PubMed2002

Effect of nutritional state on Hsp60 levels in the rotifer Brachionus plicatilis following toxicant exposure.

The nutritional state of an organism can affect the results of toxicity testing. Here we exemplified this fact by examining the effect of nutritional deprivation on heat shock protein 60 (hsp60) production in the rotifer Brachionus plicatilis following exposure to two proven inducers of hsp60, a water-accommodated fraction of crude oil (WAF) and a dispersed oil preparation (DO). Both DO and WAF exposures of unfed rotifers resulted in significantly greater hsp60 levels than that of fed DO and WAF exposed rotifers at 8 h: 870 and 3100% of control, respectively. Results clearly demonstrate that a poor nutritional state potentiates stress protein induction upon exposure to water-soluble petroleum products. It is therefore critical to define the organismal nutritional status when reporting toxic responses.

Animals↗
PubMed2001

Optimized thiol derivatizing reagent for the mass spectral analysis of disubstituted epoxy fatty acids.

A novel procedure is described for the derivatization of fatty acid epoxides in the presence of their corresponding diols. The acidic character of 2,3,5,6-tetrafluorobenzenethiol promotes favorable mass fragmentation of linoleate and arachidonate derived epoxide derivatives and reduces alkene isomerization to a manageable side reaction, eliminated through the addition of a thiol scavenger. After silylation, regioisomeric mixtures of epoxy- and dihydroxylipids are simultaneously detected and discriminated using gas chromatography with electron impact mass spectral detection. Silylated hydroxysulfanyloctadecanoids yielded instrumental detection limits of 5 pg/microl, sufficient sensitivity for the quantification of endogenous epoxylipids.

Epoxy Compounds↗
PubMed2001

The role of methyl-linoleic acid epoxide and diol metabolites in the amplified toxicity of linoleic acid and polychlorinated biphenyls to vascular endothelial cells.

Selected dietary lipids may increase the atherogenic effects of environmental chemicals, such as polychlorinated biphenyls (PCBs), by cross-amplifying mechanisms leading to dysfunction of the vascular endothelium. We have shown previously that the omega-6 parent fatty acid, linoleic acid, or 3,3',4,4'-tetrachlorobiphenyl (PCB 77), an aryl hydrocarbon (Ah) receptor agonist, independently can cause disruption of endothelial barrier function. Furthermore, cellular enrichment with linoleic acid can amplify PCB-induced endothelial cell dysfunction. We hypothesize that the amplified toxicity of linoleic acid and PCBs to endothelial cells could be mediated in part by cytotoxic epoxide metabolites of linoleic acid called leukotoxins (LTX) or their diol derivatives (LTXD). Exposure to LTXD resulted in a dose-dependent increase in albumin transfer across endothelial cell monolayers, whereas this disruption of endothelial barrier function was observed only at a high concentration of LTX. Pretreatment with the cytosolic epoxide hydrolase inhibitor 1-cyclohexyl-3-dodecyl urea partially protected against the observed LTX-induced endothelial dysfunction. Endothelial cell activation mediated by LTX and/or LTXD also enhanced nuclear translocation of the transcription factor NF-kappa B and gene expression of the inflammatory cytokine IL-6. Inhibiting cytosolic epoxide hydrolase decreased the LTX-mediated induction of both NF-kappa B and the IL-6 gene, whereas the antioxidant vitamin E did not block LTX-induced endothelial cell activation. Most importantly, inhibition of cytosolic epoxide hydrolase blocked both linoleic acid-induced cytotoxicity, as well as the additive toxicity of linoleic acid plus PCB 77 to endothelial cells. Interestingly, cellular uptake and accumulation of linoleic acid was markedly enhanced in the presence of PCB 77. These data suggest that cytotoxic epoxide metabolites of linoleic acid play a critical role in linoleic acid-induced endothelial cell dysfunction. Furthermore, the severe toxicity of PCBs in the presence of linoleic acid may be due in part to the generation of epoxide and diol metabolites. These findings have implications in understanding interactive mechanisms of how dietary fats can modulate dysfunction of the vascular endothelium mediated by certain environmental contaminants.

Alcohols↗
PubMed2001

Inhibition of microsomal epoxide hydrolases by ureas, amides, and amines.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to several diseases, such as emphysema, spontaneous abortion, and several forms of cancer. To provide new tools for studying the function of mEH, inhibition of this enzyme was investigated. Inhibition of recombinant rat and human mEH was achieved using primary ureas, amides, and amines. Several of these compounds are more potent than previously published inhibitors. Elaidamide, the most potent inhibitor that is obtained, has a K(i) of 70 nM for recombinant rat mEH. This compound interacts with the enzyme forming a noncovalent complex, and blocks substrate turnover through an apparent mix of competitive and noncompetitive inhibition kinetics. Furthermore, in insect cell cultures expressing rat mEH, elaidamide enhances the toxicity effects of epoxide-containing xenobiotics. These inhibitors could be valuable tools for investigating the physiological and toxicological roles of mEH.

Amides↗
PubMed2001

Individual metabolism should guide agriculture toward foods for improved health and nutrition.

Genomics and bioinformatics have the vast potential to identify genes that cause disease by investigating whole-genome databases. Comparison of an individual's geno-type with a genomic database will allow the prescription of drugs to be tailored to an individual's genotype. This same bioinformatic approach, applied to the study of human metabolites, has the potential to identify and validate targets to improve personalized nutritional health and thus serve to define the added value for the next generation of foods and crops. Advances in high-throughput analytic chemistry and computing technologies make the creation of a vast database of metabolites possible for several subsets of metabolites, including lipids and organic acids. In creating integrative databases of metabolites for bioinformatic investigation, the current concept of measuring single biomarkers must be expanded to 3 dimensions to 1) include a highly comprehensive set of metabolite measurements (a profile) by multiparallel analyses, 2) measure the metabolic profile of individuals over time rather than simply in the fasted state, and 3) integrate these metabolic profiles with genomic, expression, and proteomic databases. Application of the knowledge of individual metabolism will revolutionize the ability of nutrition to deliver health benefits through food in the same way that knowledge of genomics will revolutionize individual treatment of dis-ease with pharmaceuticals.

Biomarkers↗
PubMed2001

Evaluation of fish models of soluble epoxide hydrolase inhibition.

Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrolysis and inhibitor susceptibility. Low lethality was observed in either larval or embryonic fish exposed to diuron [N-(3,4-dichlorophenyl), N'-dimethyl urea], desmethyl diuron [N-(3,4-dichlorophenyl), N'-methyl urea], or siduron [N-(1-methylcyclohexyl), N'-phenyl urea]. Dose-dependent inhibition of sEH was a sublethal effect of substituted urea exposure with the potency of siduron < desmethyl diuron = diuron, differing from the observed in vitro sEH inhibition potency of siduron > desmethyl diuron > diuron. Further, siduron exposure synergized the toxicity of trans-stilbene oxide in fathead minnows. Medaka embryos exposed to diuron, desmethyl diuron, or siduron displayed dose-dependent delays in hatch, and elevated concentrations of diuron and desmethyl diuron produced developmental toxicity. The dose-dependent toxicity and in vivo sEH inhibition correlated, suggesting a potential, albeit undefined, relationship between these factors. Additionally, the observed inversion of in vitro to in vivo potency suggests that these fish models may provide tools for investigating the in vivo stability of in vitro inhibitors while screening for untoward effects.

Animals↗
PubMed2000

Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids.

The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14, 15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22+/-4 mm Hg (P:<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N:, N:'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.

8,11,14-Eicosatrienoic Acid↗
PubMed2000

Cress and potato soluble epoxide hydrolases: purification, biochemical characterization, and comparison to mammalian enzymes.

Affinity chromatographic methods were developed for the one-step purification to homogeneity of recombinant soluble epoxide hydrolases (sEHs) from cress and potato. The enzymes are monomeric, with masses of 36 and 39 kDa and pI values of 4.5 and 5.0, respectively. In spite of a large difference in sequence, the two plant enzymes have properties of inhibition and substrate selectivity which differ only slightly from mammalian sEHs. Whereas mammalian sEHs are highly selective for trans- versus cis-substituted stilbene oxide and 1,3-diphenylpropene oxide (DPPO), plant sEHs exhibit far greater selectivity for trans- versus cis-stilbene oxide, but little to no selectivity for DPPO isomers. The isolation of a covalently linked plant sEH-substrate complex indicated that the plant and mammalian sEHs have a similar mechanism of action. We hypothesize an in vivo role for plant sEH in cutin biosynthesis, based on relatively high plant sEH activity on epoxystearate to form a cutin precursor, 9,10-dihydroxystearate. Plant sEHs display a high thermal stability relative to mammalian sEHs. This stability and their high enantioselectivity for a single substrate suggest that their potential as biocatalysts for the preparation of enantiopure epoxides should be evaluated.

Animals↗
PubMed2000

Metabolism of monoepoxides of methyl linoleate: bioactivation and detoxification.

Leukotoxin (ltx) and isoleukotoxin (iltx) methyl esters, are metabolites of methyl linoleic acid, an essential fatty acid. They have been associated with acute respiratory distress syndrome. The observed toxicity of ltx and iltx is, in fact, due to the metabolism of the epoxides to their corresponding diols by soluble epoxide hydrolase (sEH). Herein, we demonstrate that ltx/iltx are toxic in a time-dependent manner to human sEH expressing cells with a LT(50) of 10.6 +/- 0.8 h and that ltx and iltx have K(M) of 6.15 +/- 1.0 and 5. 17 +/- 0.56 microM, respectively, and V(max) of 2.67 +/- 0.04 and 1. 86 +/- 0.06 micromol/min/mg, respectively, which can be inhibited by sEH inhibitors. We show that four major metabolites of ltx/iltx are formed in our system, including ltx/iltx free acid, ltxd/iltxd, free acid, and phosphotidylcholine and phosphotidylethanolamine containing the carboxylic acid forms of both ltx/iltx and ltxd/iltxd, but that the only metabolite associated with toxicity is the carboxylic acid form of ltxd/iltxd, suggesting the involvement of cellular esterases. We demonstrate that a serine esterase inhibitor provides some protection from the toxicity of epoxy fatty esters to sEH expressing cells as do intercellular free sulfhydryls, but that this protection is not due to glutathione conjugation. With these data, we have proposed an extension of the metabolic pathway for ltx/iltx in eukaryotic cells.

Animals↗
PubMed2000

Toxicity of epoxy fatty acids and related compounds to cells expressing human soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) is suggested to alter the mode of action and increase the toxic potency of fatty acid epoxides. To characterize the structural features necessary for sEH-dependent epoxy fatty acid toxicity, 75 aliphatic compounds were assayed for cytotoxicity in the presence and absence of sEH. Three groups of aliphatic epoxide-diol pairs were described by their observed differential toxicity. Group I compounds were typified by terminal epoxides whose toxicity was reduced in the presence of sEH. Group II compounds were toxic in either their epoxide or diol form, but toxicity was unaffected by sEH. Group III compounds exhibited sEH-dependent toxicity and were therefore used to investigate the structural elements required for cytotoxicity in this study. The optimal structure for group III compounds appeared to be a fatty acid 18-20 atoms long (e.g., a carbon backbone plus a terminal heteroatom) with an epoxide positioned between C-7 and C-12. In the absence of sEH, replacement of epoxides with a vicinal diol was required for toxicity. While diol stereochemistry was unimportant, vicinal diol-induced toxicity exhibited fewer positional constraints to toxicity than sEH-dependent epoxide toxicity. Tested fatty acids and esters with neither an epoxide nor a vicinal diol were not toxic. These data support the hypothesis that long-chain epoxy fatty acid methyl esters are potential pro-toxins metabolized by sEH to more toxic diols. Furthermore, our results suggest that the endogenous compounds, leukotoxin methyl ester, 9,10(Z)-epoxyoctadec-12(Z)-enoic acid methyl ester, and isoleukotoxin methyl ester, 12, 13(Z)-epoxyoctadec-9(Z)-enoic acid methyl ester, are structurally optimized to elicit the observed effect.

Epoxide Hydrolases↗
PubMed2000

Foot-and-mouth disease virus is a ligand for the high-affinity binding conformation of integrin alpha5beta1: influence of the leucine residue within the RGDL motif on selectivity of integrin binding.

Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors for internalization, whereas strains that are adapted for growth in cultured cell lines appear to be able to use alternative receptors like heparan sulphate proteoglycans (HSPG). The ligand-binding potential of integrins is regulated by changes in the conformation of their ectodomains and the ligand-binding state would be expected to be an important determinant of tropism for viruses that use integrins as cellular receptors. Currently, alphavbeta3 is the only integrin that has been shown to act as a receptor for FMDV. In this study, a solid-phase receptor-binding assay has been used to characterize the binding of FMDV to purified preparations of the human integrin alpha5beta1, in the absence of HSPG and other RGD-binding integrins. In this assay, binding of FMDV resembled authentic ligand binding to alpha5beta1 in its dependence on divalent cations and specific inhibition by RGD peptides. Most importantly, binding was found to be critically dependent on the conformation of the integrin, as virus bound only after induction of the high-affinity ligand-binding state. In addition, the identity of the amino acid residue immediately following the RGD motif is shown to influence differentially the ability of FMDV to bind integrins alpha5beta1 and alphavbeta3 and evidence is provided that alpha5beta1 might be an important FMDV receptor in vivo.

Amino Acid Motifs↗
PubMed1999

The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex.

Heparan sulfate has an important role in cell entry by foot-and-mouth disease virus (FMDV). We find that subtype O1 FMDV binds this glycosaminoglycan with a high affinity by immobilizing a specific highly abundant motif of sulfated sugars. The binding site is a shallow depression on the virion surface, located at the junction of the three major capsid proteins, VP1, VP2 and VP3. Two pre-formed sulfate-binding sites control receptor specificity. Residue 56 of VP3, an arginine in this virus, is critical to this recognition, forming a key component of both sites. This residue is a histidine in field isolates of the virus, switching to an arginine in adaptation to tissue culture, forming the high affinity heparan sulfate-binding site. We postulate that this site is a conserved feature of FMDVs, such that in the infected animal there is a biological advantage to low affinity, or more selective, interactions with glycosaminoglycan receptors.

Adaptation, Physiological↗
PubMed1998

Mechanism of mammalian soluble epoxide hydrolase inhibition by chalcone oxide derivatives.

A series of substituted chalcone oxides (1,3-diphenyl-2-oxiranyl propanones) and structural analogs was synthesized to investigate the mechanism by which they inhibit soluble epoxide hydrolases (sEH). The inhibitor potency and inhibition kinetics were evaluated using both murine and human recombinant sEH. Inhibition kinetics were well described by the kinetic models of A. R. Main (1982, in Introduction to Biochemical Toxicology, pp. 193-223, Elsevier, New York) supporting the formation of a covalent enzyme-inhibitor intermediate with a half-life inversely proportional to inhibitor potency. Structure-activity relationships describe active-site steric constraints and support a mechanism of inhibition consistent with the electronic stabilization of the covalent enzyme-inhibitor intermediate. The electronic effects induced by altering the ketone functionality and the para-substitution of the phenyl attached to the epoxy C1 (i.e., the alpha-carbon) had the greatest influence on inhibitor potency. The direction of the observed influence was reversed for the inhibitory potency of glycidol (1-phenyl-2-oxiranylpropanol) derivatives. Recent insights into the mechanism of epoxide hydrolase activity are combined with these experimental results to support a proposed mechanism of sEH inhibition by chalcone oxides.

Animals↗
PubMed1997

Arginine-glycine-aspartic acid-specific binding by foot-and-mouth disease viruses to the purified integrin alpha(v)beta3 in vitro.

The integrin alpha(v)beta3 has been shown to act as the receptor for internalization of foot-and-mouth disease virus (FMDV) (A12), with attachment being through a highly conserved RGD motif located on the G-H loop of viral capsid protein VP1. In addition, however, we have recently shown that efficient infection of culture-grown cells by FMDV (O1BFS) requires binding to cell surface heparan sulfate. In this study, we have used a solid-phase receptor binding assay to characterize the binding by FMDV to purified alpha(v)beta3 in the absence of heparan sulfate and other cell surface components. In this assay, FMDV (O1BFS) successfully replicated authentic ligand binding by cellular alpha(v)beta3 in terms of its high affinity, dependence on divalent cations, and activation by manganese ions. Virus binding to this preparation of alpha(v)beta3 was exquisitely sensitive to competition by short RGD-containing peptides (50% inhibition at < 10(-8) M peptide), and this inhibition was highly sequence specific, with the equivalent RGE peptide being at least 10(4) fold less effective as a competitor. Representative viruses of the other six serotypes of FMDV bound to alpha(v)beta3 in a similar RGD-specific manner, although significant differences in sensitivity to RGD peptides suggest that the affinity of the different FMDV serotypes for alpha(v)beta3 is influenced, in part, by the variable amino acid residues in the VP1 G-H loop on either side of the RGD.

Aphthovirus↗
PubMed1996

Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.

Animals↗
PubMed1994

A method for the determination of environmental contaminants in living marine mammals using microscale samples of blubber and blood.

As part of a study examining the possible effects of organochlorine compounds on juvenile northern elephant seals (Mirounga angustirostris), blubber and blood samples were taken from animals present on the Año Nuevo (California) rookery, and from animals admitted for rehabilitation at The Marine Mammal Center (Sausalito CA). Blubber samples were collected from immobilized seals. A pre-cleaned 6 mm K-medic biopsy punch was used to extract the blubber from a 1 cm incision near the hip, near the dorsal mid point. Blood samples were taken from the extradural vein; two mL of serum was analyzed for organochlorine compounds. Blubber samples (approximately 0.1g) were ground with Na2SO4 and extracted with 20 mL hexane:methylene chloride (1:1). Sera samples were extracted using commercially available disposable C18 columns. The extracts were separated on a micro-Florisil column, and analyzed by HRGC-ECD. Lipid determination in the serum was obtained by colorimetric analysis with 20 microliters samples. Results from the analysis of replicates and standard reference materials showed good recoveries, precision, and accuracy for both the blubber and blood methods.

Adipose Tissue↗