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Biomedical subjects

J A Gordon

Publications and source records attributed to J A Gordon.

At least 55 records · Page 3Linked to original sources
PubMed1991

Discrimination of mutagenic intermediates derived from alkylating agents by mutational patterns generated in Escherichia coli.

Reactive intermediates (ultimate mutagens/carcinogens) generated by alkylating agents are unstable and difficult to characterize by chemical means. We have used a genetic system to distinguish the in vivo interactions of eight carcinogenic methylating agents and five ethylating agents by the patterns of induced mutations at different target sites in Escherichia coli WU3610. For this multiple locus assay, target sites were an amber (TAG) and an ochre (TAA) triplet, DNA encoding five suppressor tRNA anticodons, and one unidentified locus. Most of the mutations could be classified as specific sequence changes at the target loci by suppressor analysis using T4 bacteriophage. Ratios of the slopes of dose-response curves for induced mutations were used to generate a profile of preferred sites for mutagenesis independent of mutagen potency. 'Mutational fingerprints' derived from different methylating and ethylating agents were compared, as evidence for the existence of common intermediates responsible for their biological effects. Six methylating agents thought to act via SN1 mechanisms were found to generate similar mutational patterns, indicative of a common mechanism, while two methylating agents reacting via SN2 mechanisms gave different patterns. The mutational fingerprints of SN1- and SN-type ethylating agents were also distinct. Mutational fingerprints may be useful in distinguishing the interactions of different ultimate mutagens.

Alkylating Agents↗
PubMed1991

HETEs and coronary artery endothelial cells: metabolic and functional interactions.

Porcine coronary artery endothelial cells have been established in culture. These cells produce prostaglandin (PG) I2, PGF2 alpha, and PGE2 when exposed to either arachidonic acid or ionophore A23187. PG formation was reduced when the cells were exposed to monohydroxy and dihydroxy unsaturated fatty acids. Although all of the hydroxyeicosatetraenoic acids (HETEs) produced reductions, 5-HETE caused the largest decrease in PGI2 formation. Therefore, these lipoxygenase products, especially 5-HETE, may impair the nonthrombogenic surface and some vasodilator responses of coronary endothelium. The cells took up each of the HETEs and incorporated them into phospholipids. Uptake was not affected by equimolar amounts of oleic or linoleic acids; even arachidonic acid reduced 12- and 15-HETE uptake by only 50-60%. Like other cells, the coronary endothelium converted 12- and 15-HETE to polar metabolites. As opposed to other cells, however, these cultures also converted 5-HETE to a more polar metabolite. Thus coronary artery endothelium can take up and metabolize all of the major HETEs, including 5-HETE, and thereby reduce their potentially injurious effects in the coronary circulation.

Animals↗
PubMed1990

Hydroxyeicosatetraenoic acid metabolism in cultured human skin fibroblasts. Evidence for peroxisomal beta-oxidation.

To determine whether the peroxisome is responsible for hydroxyeicosatetraenoic acid (HETE) oxidation, 12- and 15-HETE oxidation was measured in normal and peroxisomal deficient skin fibroblasts from patients with Zellweger's (cerebrohepatorenal) syndrome. When incubated for 1 h with normal fibroblasts, reverse phase HPLC indicated that 24% of the 12-HETE radioactivity was converted to one major polar metabolite. Chemical derivatization followed by reverse phase HPLC and TLC indicated that this metabolite is 8-hydroxyhexadecatrienoic acid [16:3(8-OH)]. Similarly, 33% of the added 15-HETE was also converted to a more polar metabolite. Neither 12- nor 15-HETE were converted to any metabolites by the peroxisomal deficient (Zellweger) cells. No defect in HETE oxidation was found in other human fibroblast cell lines with diverse metabolic abnormalities. Zellweger fibroblasts accumulated increased amounts of 12-HETE, compared with normal fibroblasts. As in the normal cells, most of the 12-HETE incorporated into Zellweger fibroblasts was present in the choline and ethanolamine phosphoglycerides. Protein synthesis, lysosomal acid lipase activity, and mitochondrial butyrate oxidation were not impaired in the Zellweger fibroblasts. Since the Zellweger cells do not convert 12- and 15-HETE to oxidative metabolites, peroxisomes appear to be the cellular organelle responsible for HETE oxidation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

5-HETE: uptake, distribution, and metabolism in MDCK cells.

The interaction of (S)-5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HETE) with Madin-Darby canine kidney (MDCK) cells was investigated to determine whether this lipoxygenase product might influence tubular epithelial function. When incubated with arachidonic acid, MDCK cells failed to synthesize any 5-HETE. However, MDCK cells can take up 5-HETE to a much greater extent than either 12- or 15-HETE. 5-HETE uptake occurred from both the apical and basolateral surfaces and was not saturated at concentrations up to 10 microM. Much of the 5-HETE was incorporated into phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine. After a 1-h incubation 5-HETE was found to be localized in either the microsomal and/or plasma membrane of MDCK cells. After pulse labeling for 1 h, MDCK cells released 35% of 5-HETE compared with 10% of the incorporated arachidonate during the next 24 h, indicating a much more rapid turnover of newly incorporated 5-HETE. When MDCK cells were incubated with 5.0 microM 5-HETE, their capacity to produce prostaglandin E2 was reduced greater than 50% in as little as 5.0 min. Since 5-HETE enters epithelial phospholipids and reduces prostaglandin production, it apparently has the capacity to modulate renal function if it is released in the proximity of the tubular epithelium during inflammatory reactions.

Animals↗
PubMed1989

Aortic wraps a modification to aortic grafting.

Aortic wrapping has been attempted on occasion when the standard replacement of a diseased lower third of the aorta by a trouser graft is not feasible. Cellophane has been the most common agent employed, but these wraps have met with minimal success and on occasion has even been passed rectally. Kartchner and Lovett have reviewed the techniques and results of the later procedures replacing the cellophane wraps. In most instances a Dacron sheet was utilized. Six cases are reviewed from a solo private practice in which a standard trouser graft replacement was varied by using an alternative form of wrapping.

Aortic Diseases↗
PubMed1989

Identification of the major metabolite of 12-HETE produced by renal tubular epithelial cells.

The identification and polarity of release of the major metabolite of 12-HETE produced by cultured canine renal tubular epithelial cells was determined. When incubated with 1.0 microM [3H]12-HETE for 1 h, cultured Madin Darby Canine Kidney (MDCK) cells converted 35% of the radiolabeled 12-HETE to a more polar metabolite. Following high performance liquid chromatography isolation and chemical derivatization, gas-liquid chromatography combined with mass spectrometry was used to identify the compound as 8-hydroxyhexadecatrienoic acid [16:3(8-OH)]. The electron impact mass spectrum of the hydrogenated derivative contained major ions at m/z = 215 and 245, corresponding to cleavage on either side of the trimethylsilyl group, and chemical ionization with NH3 yielded a major ion at m/z = 359, corresponding to the protonated molecular weight of the methyl ester. Incubation with 25 mM alpha-naphthoflavone, 20 microM nordihydroguaiaretic acid, and 0.1 mM 4-pentenoic acid failed to inhibit the formation 16:3 (8-OH), suggesting that the formation of 16:3 (8-OH) is not mediated by the cytochrome P-450, lipoxygenase, or mitochondrial beta-oxidation pathways. When grown on fibronectin-treated polycarbonate filters, MDCK cells released the 16:3 (8-OH) in both the apical and basolateral directions, irrespective of which side the 12-HETE was encountered. These results demonstrate the conversion of 12-HETE to a 16-carbon monohydroxy derivative by renal tubular epithelium and suggest that this product can be released to either the potential urinary space or the kidney parenchyma and renal microcirculation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1988

Human progesterone receptor transformation and nuclear down-regulation are independent of phosphorylation.

We have studied the phosphorylation of progesterone receptors (PR) in T47Dco human breast cancer cells using a monoclonal antibody directed against human PR called AB-52. This antibody recognizes both the A- (Mr approximately 94,000) and B- (Mr approximately 120,000) hormone binding proteins of PR, and was used to immunoprecipitate phosphorylated receptors isolated from cells incubated in vivo with [32P]orthophosphate. The specific activity, or phosphorylation levels, relative to protein levels was quantified by combined immunoblotting and autoradiography followed by densitometry. We find that immunopurified untransformed hormone-free receptors, which have a characteristic triplet B, singlet A structure, are phosphoproteins with similar levels of phosphate incorporation in all protein bands. If PR are first transformed to the nuclear binding form by treatment of cells with progesterone, and then labeled with [32P]orthophosphate, the receptor proteins are additionally phosphorylated. These chromatin-bound hormone occupied receptors incorporate five to 10 times more labeled phosphate per total receptor protein than do PR from untreated cells during the same [32P]incubation time. The second round of phosphorylation may also account for mobility shifts of transformed A- and B-receptors observed in sodium dodecyl sulfate-polyacrylamide gels. Both untransformed and transformed species of A- and B-receptors are phosphorylated only on serine residues, and neither the extent of phosphorylation, nor the phosphoamino acids, are affected by treatment of the cells with epidermal growth factor or insulin. We previously reported that after hormone binding and transformation of receptors to the tight chromatin binding state, PR undergo processing, or nuclear down-regulation. AB-52 was used to compare PR protein and phosphorylation levels when cells were treated for 0.5-48 h with progesterone or the synthetic progestin R5020. Both agonists lead to hyperphosphorylation of nuclear PR before phosphorylation levels decrease, in parallel with the drop in protein levels as receptors down-regulate. Treatment of cells with RU 486, an antiprogestin, leads to PR transformation as determined by immunoblotting, but subsequent down-regulation does not occur. After transformation, chromatin-bound RU 486-occupied receptors become intensely phosphorylated however, with specific activities 15 times greater than those of untransformed PR. Since these receptors are phosphorylated but not processed, the hormone-induced nuclear phosphorylation of PR is unlikely to be a signal for receptor processing.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1987

Determination of the cleavage specificity of Streptomyces griseus protease B in the presence of guanidinium chloride.

Streptomyces griseus protease B (SGPB), a Pronase enzyme, has been shown to be stable and active in the presence of 6.0 M guanidinium chloride (Siegel, S. et al. (1972) J. Biol. Chem. 247, 4155-4159). In order to determine the cleavage specificity of this unusual enzyme under denaturing conditions, 12 peptides of known amino acid sequence were hydrolyzed by SGPB in the absence and presence of 6.0 M guanidinium chloride. The new N-terminal amino acids produced by the action of SGPB were dansylated and quantitatively identified by reverse phase HPLC. The results indicate that SGPB retained its cleavage specificity for phenylalanyl, tyrosyl, tryptophanyl, and leucyl peptide bonds in the presence of 6.0 M guanidinium chloride. Of these peptide bonds, SGPB exhibited a greater cleavage preference for phenylalanyl and tryptophanyl bonds, which was relatively unaffected by the presence of the denaturant. The SGPB-catalyzed cleavages of the leucyl peptide bonds examined (Leu-Met, Leu-Arg, Leu-Val, Leu-Thr, and Leu-Ile) were substantially decreased under denaturing conditions, while Leu-Gly bond cleavage by SGPB was virtually unaffected by denaturant. The demonstrated predictability of the catalytic preference of this unusual protease for phenylalanyl, tyrosyl, tryptophanyl, and leucyl-glycine peptide bonds under denaturing conditions enhances its utility in the site-specific proteolysis of insoluble or otherwise proteolysis-resistant polypeptide substrates.

Amino Acid Sequence↗
PubMed1987

In vivo effect of sodium orthovanadate on pp60c-src kinase.

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.

Amino Acid Sequence↗
PubMed1987

Effects of 12-HETE on renal tubular epithelial cells.

The interaction of 12-HETE with MDCK cells was investigated to determine whether this lipoxygenase product might influence tubular epithelial function. MDCK cells took up 12-HETE from both the luminal and interstitial surfaces. Much of the 12-HETE was incorporated into phospholipids but, unlike arachidonic acid, there was no specificity for the sn-2 position. 12-HETE was incorporated without chemical modification, suggesting that it might perturb tight packing in the lipid bilayer. 12-HETE uptake was reduced by the presence of arachidonic acid, but not by oleic acid. 12-HETE was removed from the cell lipids more rapidly than arachidonic acid and it was released into the medium in the form of both more and less polar metabolites. When MDCK cells were incubated with 12-HETE, their capacity to produce prostaglandin E2 was reduced. Since 12-HETE enters epithelial phospholipids and is converted to metabolic products and reduces prostaglandin production, it apparently has the capacity to modulate renal function if it is released in the proximity of the tubular epithelium.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1986

Calcium-dependent isolation of the 36-kilodalton substrate of pp60src-kinase. Fractionation of the phosphorylated and unphosphorylated species.

In this paper, we present a new and simple purification of the 36-kDa protein, a major substrate of both viral and growth factor-receptor associated tyrosine protein kinases, and its complex from normal and Schmidt-Ruppin strain Rous sarcoma virus-transformed chicken embryo fibroblasts that employs a DEAE-Sephacel column and introduces the calcium-dependent adsorption of 36-kDa protein. The use of EGTA step gradients differentially elutes the 36-kDa molecules from the DEAE-Sephacel column. An average total yield of the 36-kDa protein in all fractions approached 80% and represented 0.78% of the [35S] methionine-labeled cellular protein. A purity of 95-99% was obtained with a yield of 60% in the central elution fractions from normal or Rous sarcoma virus-transformed chicken embryo fibroblasts. Furthermore, 2 mM EGTA elutes poorly phosphorylated molecules while heavily phosphorylated 36-kDa protein requires 4 or 6 M EGTA; a small residual fraction is released at 8-10 mM EGTA. If the EGTA step gradients were neutralized with Ca2+ ion, elution of the 36-kDa protein is inhibited. The complex of the 36-kDa protein and the 6-10-kDa protein may not be dependent on the phosphorylation as the associated 6-10-kDa peptide is seen in all fractions containing the 36-kDa protein. Tyrosine phosphorylation of the 36-kDa protein is increased 2-3-fold following a short term incubation of whole cells with micromolar vanadate. The elution pattern (but not intensity) of the 36-kDa protein obtained from lysates of vanadate-treated cells was identical to untreated cell lysates. The additional phosphorylation appears to result from a recruitment of unphosphorylated 36-kDa protein as the position (but not intensity) of the phosphorylated tryptic peptides is unchanged. We conclude that the function of the 36-kDa protein may be calcium ion-dependent and may be influenced by the phosphorylation state of the protein.

Animals↗
PubMed1986

Unexpected, unsuspected and missed injuries in a paediatric trauma unit.

In a newly established trauma unit 12 282 patients were seen in 1 year, of whom 1 557 were admitted. Out of the total there was 0.3% of missed, unexpected or unsuspected injuries; this was 2.5% of ward admissions. These injuries are classified and analysed, highlighting the need for constant vigilance. Although the majority were orthopaedic injuries, the abdominal injury cases show that these can be more severe than clinically suspected.

Abdominal Injuries↗
PubMed1986

gamma-Glutamyl transpeptidase excretion in cisplatin-induced acute renal failure.

Cisplatin (cisdiamminedichloroplatinum), an important antineoplastic agent, possesses nephrotoxicity as its major side effect. A mild partially reversible nonoliguric form of acute renal failure (ARF) is generally the most common form of nephrotoxicity and occurs in experimental animals following a single dose. gamma-Glutamyl transpeptidase (gamma GT) is an enzyme with maximal activity located in the brush border of proximal renal tubular epithelium. To test the relationship between cisplatin nephrotoxicity and urinary gamma GT excretion, rats received a single 5.5 mg/kg dose of cisplatin and gamma GT excretion was evaluated and compared to anatomic and functional damage. Twenty-four hours following cisplatin administration, there was a marked enhancement of urinary gamma GT excretion, prior to the onset of azotemia. Urinary gamma GT excretion peaked at day 4, then returned to baseline, and decreased to values below baseline on days 8 through 10. By days 11 through 12, renal function and urinary gamma GT excretion had returned to normal. The correlation between nephrotoxicity, changes in urinary gamma GT excretion, and anatomic damage was excellent. Morphologically, increased gamma GT excretion was associated with loss of microvilli, and the return of urinary gamma GT excretion to normal correlated with their regeneration. We conclude that cisplatin administration results in increased urinary gamma GT excretion. This early enhancement, prior to the onset of azotemia, may provide a useful noninvasive marker of early cisplatin nephrotoxicity.

Acute Kidney Injury↗