Search PubMed⌕ Search

Biomedical subjects

S L Kelly

Publications and source records attributed to S L Kelly.

At least 91 records · Page 5Linked to original sources
PubMed1995

Metabolism of the herbicide chlortoluron by human cytochrome P450 3A4.

Studies were carried out to investigate the metabolism of herbicide chlortoluron in the microsomal fractions and whole cells of Saccharomyces cerevisiae expressing human cytochrome P450 3A4. Both whole cells and microsomal fractions of yeast expressing human cytochrome P450 3A4 exhibited a typical dithionite-reduced, CO-difference absorbance spectrum with maximum absorbance at 448 nm. Chlortoluron produced a type I binding spectrum with cytochrome P450 3A4 with a Ks value of 200 microM. Chlortoluron was metabolised into four metabolites; hydroxylated-N-monodemethylated, hydroxylated ring methylated, N-didemethylated and N-monodemethylated products. Chlortoluron metabolism was absolutely dependent on NADPH and no metabolism was observed in control transformants.

Autoradiography↗
PubMed1995

Role of neutrophil membrane proteases in fibrin degradation.

The cellular components of blood play a significant role in the breakdown of fibrin, with specific cellular adhesive processes allowing for accumulation of neutrophils within the fibrin clot. Fibrinolysis by elastase and cathepsin G, enzymes present within the azurophilic granules of the neutrophil, has previously been shown. Recent studies have demonstrated neutrophil-mediated fibrinogenolysis by a membrane-associated protease which suggests that proteases connected with the neutrophil membrane might also be capable of clot dissolution. Intact neutrophils were found to solubilize fibrin clots with the rate of fibrin solubilization being greater when the cells were incorporated into the clot than when the cells were added to preformed clots. Stimulation of intact neutrophils with phorbol ester upregulated this neutrophil-mediated fibrinolysis. Solubilization was detectable within 2 min of incubating the cells with fibrin clot, was always faster than that by neutrophil conditioned medium and lacked inhibition with lysosomal enzyme inhibitors. Neutrophil-mediated clot lysis was effected by membrane-associated serine proteases that migrated to apparent molecular weights of 501 kD, 398 kD, 316 kD, 245 kD and 209 kD on 3-13% SDS-PAGE. This degradation was distinct from that produced by plasmin, neutrophil lysosomal enzymes and purified human neutrophil elastase. The neutrophil membrane proteolytic systems were found to enhance the action of plasmin in clot solubilization. These results suggest neutrophil membrane proteolytic activity could assist thrombus dissolution and may be of particular value in assisting early clot dissolution by plasmin and when clot stabilization occurs through plasminogen activator inhibitor-1 (PAI-1) inhibition of plasminogen activation.

Cell Membrane↗
PubMed1995

Movement hyperacuity threshold deficit in juvenile onset diabetes mellitus.

Oscillatory movement displacement thresholds from a group of 18 normal children (mean age of 9.2 +/- 3.6 years) were compared with those obtained from a group of 26 age-matched children (mean age of 9.9 +/- 2.9 years) having insulin-dependent diabetes mellitus. Disease durations ranged from 6 months to 12 years, although no ophthalmoscopic evidence of retinopathy was found in any subject. Up to about 8 years of age there is no significant difference in the performance of diabetics and controls (t = 1.00, p = n.s.), oscillatory movement displacement thresholds correlating with age in both groups (r= -0.59, p < 0.02). Over about 8 years of age oscillatory movement displacement thresholds no longer improve and the two groups perform significantly differently, thresholds being degraded in the insulin-dependent diabetes mellitus group compared to normals (t = 5.25, p <<0.0001). It is suggested that the measurement of oscillatory movement displacement thresholds in children with insulin-dependent diabetes mellitus can reveal its effects in the absence of retinopathy.

Adolescent↗
PubMed1995

Cross-resistance to polyene and azole drugs in Cryptococcus neoformans.

Fluconazole was observed to inhibit sterol 14 alpha-demethylase in the human pathogen Cryptococcus neoformans, and accumulation of a ketosteroid product was associated with growth arrest. A novel mechanism(s) of azole and amphotericin B cross-resistance was identified, unrelated to changes in sterol biosynthesis, as previously identified in Saccharomyces cerevisiae. Reduced cellular content of drug could account for the resistance phenotype, indicating the possible involvement of a mechanism similar to multidrug resistance observed in higher eukaryotes.

Amphotericin B↗
PubMed1994

Investigation of the role of sterol delta 8-->7-isomerase in the sensitivity of Saccharomyces cerevisiae to fenpropimorph.

Treatment of Saccharomyces cerevisiae with the morpholine fungicide fenpropimorph was examined using both a wild-type and a mutant strain (erg2) defective in sterol delta 8-->7-isomerase. No resistance to fenpropimorph was observed in the mutant strain after 3 days, although after 7 days the mutant and the wild-type strains had grown in concentrations of fenpropimorph close to the saturating dose. Re-inoculation of both strains into fresh medium containing fenpropimorph resulted in continued growth and this adaptation to fungicide tolerance was lost on subculture in the absence of fenpropimorph. Analysis of the sterols present in the cells indicated that fenpropimorph treatment resulted in the accumulation of delta 8,14-sterols. This accumulation and the corresponding depletion of ergosterol were correlated with growth inhibition rather than the presence of delta 8-sterols. Together with an absence of gene dosage effect for ERG2 on fenpropimorph sensitivity, this supports the hypothesis that sterol delta 8-->7-isomerase inhibition does not contribute to the fungicidal activity of fenpropimorph.

Base Sequence↗
PubMed1994

Resistance to amphotericin B associated with defective sterol delta 8-->7 isomerase in a Cryptococcus neoformans strain from an AIDS patient.

Two Cryptococcus neoformans strains isolated from an AIDS patient were investigated, a pretreatment isolate (CN1) and a second isolate (CN3) following failure of fluconazole and amphotericin B treatment. No difference in fluconazole sensitivity, but relative resistance to amphotericin B was observed for CN3. The sterol composition of CN3 indicated a defect in sterol delta 8-->7 isomerase in this strain and depletion of ergosterol, the major sterol of the CN1.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Arylamine N-acetyltransferase in Balb/c mice: identification of a novel mouse isoenzyme by cloning and expression in vitro.

Three genes encoding arylamine N-acetyltransferase were identified in Balb/c mice. All three genes were cloned from genomic DNA, sequenced and expressed in a bacterial expression system. Two of the genes corresponded to Nat-1 and Nat-2 which have been previously identified in A/J and C57B1/6 strains of mice (Martell et al., 1991). The new gene, designated Nat-3, can be distinguished from the other mouse Nat genes both by specific amplification using PCR and by restriction-endonuclease digestion. The products of all three genes are demonstrated to catalyse acetylation of aminofluorene and anisidine following expression in Escherichia coli.

Acetylation↗
PubMed1994

Fibrinogenolysis by a neutrophil membrane protease generates an A alpha 1-21 fragment.

The 600 kDa neutrophil membrane neutral protease, which had been shown to generate bioactive peptides from the acute-phase reactant C-reactive protein, has now been shown to have fibrinogenolytic activity that is distinct from fibrinogenolysis by plasmin and neutrophil lysosomal enzymes. This protease gradually reduces the apparent molecular mass of fibrinogen (340 kDa) to non-clottable products and generates terminal products with apparent molecular mass values of 270 kDa, 200 kDa, 100 kDa and less than 40 kDa through cleavage of all three of the constituent chains. Characteristics of fibrinogenolysis by this neutrophil protease are cleavage of the bond between amino acids valine and glutamic acid at positions 21 and 22 respectively from the N-terminus of the A alpha chain to release an A alpha 1-21 peptide, digestion of the B beta chain at positions within the C-terminus, and proteolysis of the bond between amino acids isoleucine and glycine at positions 394 and 395 respectively from the N-terminus of the gamma chain. This generates products that lack anticoagulant activity. The thrombin clotting time of the product with an apparent molecular mass of 330 kDa was prolonged, although clot formation was still observed. Loss of coagulability and inability to clot was found with further degradation of fibrinogen to an apparent molecular mass of 290 kDa. Activity of this neutrophil membrane protease in vivo could be important for the regulation of fibrin deposition at sites of inflammation, and may contribute to the reported plasma levels of the A alpha 1-21 peptide.

Amino Acid Sequence↗
PubMed1994

Studies on azole-induced cell death in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae strain XL16-5B exhibited a fungicidal response to treatment with ketoconazole. Cell death became apparent during prolonged treatment over 72 h following an initial period over 24 h where viable cells were found and limited cell division occurred. Sterol analysis showed some differences between XL16-5B and the strain XY729-5a, which had a fungistatic response to ketoconazole. In particular, the level of ergosterol was higher in XL16-5B and remained high during treatment.

Cytochrome P-450 Enzyme System↗
PubMed1994

Microbial transformations of steroids--VIII. Transformation of progesterone by whole cells and microsomes of Aspergillus fumigatus.

The filamentous fungus, Aspergillus fumigatus, efficiently hydroxylated exogenous progesterone producing, after 3 h of incubation, 11 alpha- and 15 beta-hydroxyprogesterone as major products, 7 beta-hydroxyprogesterone as a minor product and trace amounts of 7 beta, 15 beta- and 11 alpha, 15 beta-dihydroxyprogesterone. After 72 h the dihydroxyprogesterones were the sole metabolites in the culture medium. Microsomes, prepared by Ca2+ precipitation, catalysed only monohydroxylation of progesterone at the same sites as whole cells. Hydroxylation was dependent on NADPH (but not NADH) which was replaceable by NaIO4. Hydroxylation was inhibited by carbon monoxide and by the azole fungicide, ketoconazole. Microsomes gave a dithionite-reduced, carbon monoxide difference absorbance spectrum with a peak at 448 nm and a Type-I progesterone-binding spectrum typical of cytochrome P450 interaction with substrate. Ketoconazole inhibition studies suggest the presence of two non-inducible cytochrome P450 progesterone hydroxylases, one possessing 7 beta site-selectivity, the other 11 alpha/15 beta site-selectivity.

Aspergillus fumigatus↗
PubMed1993

Benzo(a)pyrene hydroxylase activity in yeast is mediated by P450 other than sterol 14 alpha-demethylase.

Benzo(a)pyrene hydroxylation has been observed in Saccharomyces cerevisiae and the role of sterol 14 alpha-demethylase (CYP51A1) in this activity has been examined by using a strain which contains a gene disruption of CYP51A1. This strain still contained P450 protein(s) with a Soret absorption maximum at 448nm in reduced carbon monoxide difference spectra of the microsomal fraction. On addition of benzo(a)pyrene to this microsomal extract a typical Type I substrate-binding spectrum was obtained and was also observed for the isogenic sister strain containing no CYP51A1 gene disruption. Microsomal extracts of both strains had equivalent activity in the aryl hydrocarbon hydroxylase assay. These results indicate a yeast benzo(a)pyrene hydroxylase activity distinct from sterol 14 alpha-demethylase P450.

Aryl Hydrocarbon Hydroxylases↗
PubMed1992

Characterization of neutrophil-mediated degradation of human C-reactive protein and identification of the protease.

Human C-reactive protein (CRP) is shown to mediate a release of enzymatic activity from neutrophils which promotes its own degradation in the extracellular medium. This egress of proteolytic activity, which was upregulated by phorbol 12-myristate 13-acetate (PMA), was found to occur from both the cytoskeleton and membrane fractions of neutrophils and was dependent on the time of incubation of CRP with the cells and the concentration of CRP. Neutrophil kinases activated by PMA are found to be involved in upregulating the activity of the CRP-degrading protease. The apparent molecular weight of the CRP-degrading protease associated with the conditioned medium from PMA-stimulated neutrophils and neutrophil membrane and cytoskeleton preparations, was found by size exclusion chromatography to be 600 kD and migrated on 3-13% SDS-PAGE as four discrete bands to positions corresponding to apparent molecular weights of 209 kD, 316 kD, 398 kD and 501 kD.

C-Reactive Protein↗
PubMed1991

The expression of human cytochrome P450IA1 in the yeast Saccharomyces cerevisiae.

Data from animal studies suggest that cytochrome P450IA1 catalyses the metabolic activation of several procarcinogenic compounds. In the present study, we have expressed human cytochrome P450IA1 in yeast cells. A 1.70 kb BclI/BamHI fragment containing a full-length human cytochrome P450IA1 cDNA was inserted into the BglII expression site of the yeast expression plasmid pMA91 thereby allowing the ATG initiation codon to be located adjacent to the PGK (phosphoglycerate kinase) promoter. The resulting recombinant plasmid, pCK-1, was introduced into Saccharomyces cerevisiae strains ATCC 44773 and AH22. Microsomes prepared from yeast transformatants of strain ATCC 44773 contained undetectable levels of cytochrome P450. In contrast, microsomes from strain AH22 contained cytochrome P450 with a specific content of 33.3 +/- 10.8 pmol/mg of microsomal protein and showed a reduced carbon monoxide difference spectrum with a peak at 448 nm. Control yeast cells transformed with pMA91 showed no cytochrome P450. Western blots were carried out using an antibody that reacts against rat cytochrome P450IA1 and an antibody that reacts against a synthetic peptide representing a short sequence of human cytochrome P450IA1. A band with a molecular weight of 54 kD was observed in microsomes of yeast transformed with pCK-1, but not with pMA91. When microsomes from yeast transformed with pCK-1 were incubated with benzo(a)pyrene (10 min, 10-160 microM), an estimated Km value of 7 microM was obtained. The availability of yeast cells with functionally active human cytochrome P450IA1 will facilitate molecular structure-activity studies of procarcinogen and drug metabolism by this enzyme in man.

Cell Line, Transformed↗
PubMed1991

Drug metabolising N-acetyltransferase activity in human cell lines.

Many arylamine and hydrazine drugs and xenobiotics are acetylated by N-acetyltransferase (NAT), a cytosolic enzymic activity which has a wide tissue distribution. Humans can be classified as either fast or slow acetylators on the basis of their ability to metabolise isoniazid or sulphamethazine. These are termed polymorphic substrates. The acetylation of other compounds does not vary amongst individuals, e.g., p-aminobenzoic acid, and are termed monomorphic substrates. NAT from human hepatic and non-hepatic tissues, viz., (i) liver, (ii) the hepatoma cell line HepG2, (iii) tonsil lymphocytes and (iv) the monocytic cell line U937 have been compared with respect to substrate specificity towards polymorphic and monomorphic substrates. The chromatographic and centrifugation behaviour of NAT from these sources has also been investigated. NAT from liver shows 2-fold greater activity towards sulphamethazine than towards p-aminobenzoic acid as substrate. All other cell types tested show at least 70-fold greater activity with p-aminobenzoic as substrate compared to sulphamethazine. NAT from HepG2 cells, U937 cells and tonsil lymphocytes migrates as a single peak during ion-exchange chromatography, whereas the liver NAT activity is separated into two peaks. NAT in HepG2 cells resembles extra-hepatic tissue NAT rather than NAT in liver. HepG2 cells do not therefore represent a good in vitro model for investigation of human metabolism of arylamines or hydrazines. The molecular weight of NAT from U937 cells has been determined by a combination of sucrose density gradient centrifugation and gel filtration to be 31,600 +/- 1200 daltons.

Arylamine N-Acetyltransferase↗