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Biomedical subjects

R Drew

Publications and source records attributed to R Drew.

At least 19 recordsLinked to original sources
PubMed1992

The effects of ibuprofen enantiomers on hepatocyte intermediary metabolism and mitochondrial respiration.

In vivo and in vitro (-)R-ibuprofen is inverted to the (+)S antipode via stereoselective formation of an R-ibuprofenyl-CoA intermediate. In this study the effects of (-)R- and (+)S-ibuprofen on metabolism and respiration were studied using isolated rat hepatocytes and mitochondria. R-Ibuprofen significantly increased the lactate to pyruvate ratio, perturbed mitochondrial ketogenesis as evidenced by alterations in the beta-hydroxybutyrate to acetoacetate ratio and uncoupled mitochondrial oxidative phosphorylation. In addition, substantial dose- and time-dependent sequestration of reduced CoA (CoASH) occurred in the presence of the R enantiomer. Similarly, S-ibuprofen altered both the cytosolic and mitochondrial redox states although the magnitude of the effect was substantially less than that observed with the R enantiomer. In contrast to R-ibuprofen, S-ibuprofen did not uncouple oxidative phosphorylation or sequester hepatocyte CoASH. It is proposed that the perturbations observed in hepatocyte intermediary metabolism and mitochondrial function are attributable to a combination of the direct effects of R-ibuprofen per se and the sequestration of CoASH as R-ibuprofenyl-CoA during the process of chiral inversion. On the basis of these results, R-ibuprofen should be considered more in terms of metabolism to a reactive acyl-CoA intermediate rather than as a pro-drug for the pharmacologically active S-enantiomer.

3-Hydroxybutyric Acid↗
PubMed1991

Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product.

Using in vitro-constructed deletions and subcloned DNA fragments, we have identified a new gene, amiC, which regulates expression of the inducible Pseudomonas aeruginosa aliphatic amidase activity. The DNA sequence of the gene has been determined, and an open reading frame encoding a polypeptide of 385 amino acids (molecular mass, 42,834 Da) has been identified. A search of sequence libraries has failed to find homologies with other published sequences. The amiC translation termination codon (A)TGA overlaps the initiation codon for the downstream amiR transcription antitermination factor gene, implying that the amiCR operon is coordinately regulated. Disruption of the amiC open reading frame by insertion and deletion leads to constitutive amidase synthesis, suggesting that AmiC is a negative regulator. This is confirmed by the finding that a broad-host-range expression vector carrying amiC (pSW41) represses amidase expression in a series of previously characterized P. aeruginosa amidase-constitutive mutants. The AmiC polypeptide has been purified from PAC452(pSW41), and N-terminal amino acid sequencing has confirmed the gene identification.

Amidohydrolases↗
PubMed1990

Ocular injury induced by methyl ethyl ketone peroxide.

Methyl ethyl ketone peroxide is a commonly used catalyst in various industries. We studied 19 eyes with a single exposure to methyl ethyl ketone peroxide that developed clinical patterns of mild injury, moderate injury, severe injury, or delayed keratitis. Delayed methyl ethyl ketone peroxide keratitis may cause exacerbations and remissions of corneal and limbal disease lasting more than 20 years with palpebral and bulbar hyperemia equal to the initial chemical exposure. With repeat exacerbation, further pannus may occur, which can be associated with a poorer outcome. Based on the capability of methyl ethyl ketone peroxide to change DNA to a new weak antigen, we suggest possible methods of therapy to prevent or limit delayed methyl ethyl ketone peroxide keratitis. This proposed type of injury has important implications in studying various limbal and corneal diseases. A major factor in the severity of ocular injury was the length of time from exposure to methyl ethyl ketone peroxide to obtaining a topical ocular local anesthetic to perform adequate lavage.

Adult↗
PubMed1989

Positive control of Pseudomonas aeruginosa amidase synthesis is mediated by a transcription anti-termination mechanism.

The DNA sequence of the region upstream from the amidase structural gene (amiE) of Pseudomonas aeruginosa indicates that amidase (EC 3.5.1.4) is transcribed from an Escherichia coli-like promoter located 150 bp before the amiE translation initiation codon. The sequence between the promoter and the coding sequence includes a single open reading frame followed by an E. coli-like rho-independent transcription terminator. A deletion within the presumed terminator region which disrupts the potential stem/loop formation leads to high constitutive amidase expression which is independent of the product of the regulator gene (amiR). It is proposed that the catabolic aliphatic amidase of P. aeruginosa is regulated by a transcription anti-termination mechanism. The magnoconstitutive mutant PAC433 has promoter and terminator sequences identical to the wild-type PAC1 but contains a single base pair change in the amiE gene ribosome-binding site.

Amidohydrolases↗
PubMed1988

Studies on the expression of Cu,Zn superoxide dismutase in human tissues during development.

The developmental expression of Cu,Zn superoxide dismutase in human lung and erythrocytes has been studied using activity measurements, immunoblotting and immunohistochemistry. Enzyme activity in erythrocytes increased significantly during gestation but no developmental trend was seen in lung. Immunoblotting identified a single enzyme form that was present in a variety of tissues and immunohistochemistry showed the enzyme to have widespread distribution in lung tissue. These data indicate that Cu,Zn superoxide dismutase is consistently expressed during human development and that, unlike in other species, no late-fetal surge in expression occurs.

Cytosol↗
PubMed1988

A radioisotopic assay of picomolar concentrations of coenzyme A in liver tissue.

A single-step enzyme assay using [14C]palmitic acid and bacterial acyl-coenzyme A synthetase (EC 6.2.1.3) is described for the determination of reduced coenzyme A (CoASH) levels in liver samples. Use of this technique provides a rapid and accurate determination of CoASH in the range 1-250 pmol. Application of the method to the quantitation of CoASH in samples of human liver tissue and rat liver homogenate, isolated hepatocytes, and mitochondria is described.

Animals↗
PubMed1987

Studies on the developmental expression of glutathione S-transferase isoenzymes in human heart and diaphragm.

The developmental expression of the basic, near-neutral and acidic isoenzymes of glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) has been studied in heart and diaphragm. Neither these enzymes nor the putative muscle-specific GST4 isoenzyme demonstrated any developmental trends in expression. In vitro hybridisation and SDS-discontinuous polyacrylamide gel electrophoresis were used to show that the GST4 isoenzyme is a homodimer composed of monomers that have a slightly larger molecular weight than the near-neutral isoenzyme. The sensitivity of GST4 to inhibitors also appeared similar to that of the GST1 2 isoenzyme. Immunodiffusion and immunoblotting techniques were used to show that the acidic enzyme in muscle is immunologically identical to that in other tissues.

Chromatography↗
PubMed1987

The amidase regulatory gene (amiR) of Pseudomonas aeruginosa.

Recombinant plasmids carrying the amidase genes of Pseudomonas aeruginosa were used to study the genetic control of amidase synthesis in Escherichia coli and Pseudomonas aeruginosa. The amidase regulator gene, amiR, was found to lie about 2 kbp downstream from the structural gene, amiE. Using plasmids with in vitro-constructed deletions, and plasmids containing subcloned DNA fragments, the amiR gene was located within a 1 kbp ClaI-XhoI DNA fragment. The structural and regulator genes were shown to be transcribed in the same direction. Deletion of DNA sequences between the two genes resulted in increased synthesis of amidase in both E. coli and P. aeruginosa. The intervening sequences showed no repressing effect when tested in trans. The results suggested that the amiR gene could be transcribed from more than one promoter.

Amidohydrolases↗
PubMed1987

Haemoglobin A/F ratio in neonates at 7 days correlated with birth weight and estimated gestational age.

Haemoglobin (Hb) A and Hb F has been determined in neonates of Afro-Caribbean and North European origin with gestational ages varying from 32 to 42 weeks. There was no difference in the distribution of Hb A/F ratios between the two groups. Only weak correlations could be established between the Hb A/F ratio and the estimated gestational age or birth weight. This would indicate that there is a considerable interindividual variation in the timing of the switching of haemoglobin synthesis from Hb F to Hb A and erythrocyte production from liver to bone marrow and of oxygen affinity of fetal blood. Thus, intra-uterine adjustments of the oxygen release capacity of haemoglobin would have to rest on biochemical mechanisms during the third trimester.

Birth Weight↗
PubMed1986

Benoxaprofen induced toxicity in isolated rat hepatocytes.

The toxicity of benoxaprofen, a non-steroidal anti-inflammatory compound was investigated using rat hepatic microsomal and isolated hepatocyte suspensions. In microsomes, benoxaprofen produced a Type I binding spectra and competitively inhibited (ki 380 microM) the oxidative metabolism of aminopyrine. Marked toxicity was observed following incubation of benoxaprofen with isolated hepatocytes from either untreated, phenobarbitone (PB) or 3-methylcholanthrene (3-MC) pretreated male rats. In untreated hepatocytes increases in the intracellular lactate/pyruvate (L/P) ratio and alanine aminotransferase (ALT) release were related to the benoxaprofen concentration and duration of incubation. Alterations in L/P ratio preceded the release of cytosolic ALT and at 4 h a well defined dose-response relationship existed between the benoxaprofen concentration and the observed increases in the L/P ratio and ALT release. Pretreatment of animals with either PB or 3-MC did not affect the temporal nature nor the magnitude of the hepatocyte response to benoxaprofen. In addition, inhibitors of cytochrome P-450 isozymes (SKF-525A, metyrapone and alpha-napthoflavone) were ineffective with regard to modifying the observed toxicity. The results of this study suggest that hepatic cytochrome P-450 mediated metabolism may not be implicated in the toxicity of benoxaprofen in isolated hepatocytes. However, alterations in the cellular redox state and evidence of plasma membrane bleb formation suggest that benoxaprofen may uncouple oxidative phosphorylation and disturb intracellular calcium ion homeostasis.

Alanine Transaminase↗
PubMed1986

Interaction of benoxaprofen with rat erythrocytes: effects on oxidative metabolism and membrane ATPase activities.

Incubation of rat erythrocytes with benoxaprofen resulted in increased glucose utilization, lactate production, depletion of cellular ATP and significant haemolysis. In isolated red cell membranes, benoxaprofen produced a dose related stimulation of both Ca2+ dependent and Ca2+ independent ATPase activities. Coincubation of either erythrocytes or red cell membranes with cysteine ameliorated the effects of benoxaprofen. A possible association exists between the mechanism of the reported anti-inflammatory activity of benoxaprofen and its cellular toxicity.

Adenosine Triphosphatases↗
PubMed1984

The effects of buthionine sulphoximine (BSO) on glutathione depletion and xenobiotic biotransformation.

Buthionine sulphoximine (BSO) is an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS) and, consequently lowers tissue glutathione (GSH) concentrations. In fed male C3H mice, liver and kidney GSH levels were depleted by BSO in a dose dependent manner with maximum effect (35% of initial levels) occurring with doses between 0.8 and 1.6 g/kg, i.p. At these doses maximum effects on gamma-GCS and GSH were observed 2-4 hr after BSO administration; initial gamma-GCS activity and GSH content were restored approximately 16 hr post BSO. BSO, either in vivo or in vitro, had no effect on hepatic microsomal cytochrome P-450 levels, a range of cytochrome P-450 dependent enzyme activities or p-nitrophenol glucuronyl transferase activity. Similarly, BSO had no effect on phenol sulphotransferase and two GSH-transferase activities in the 105,000 g supernatant fraction. BSO had no effect on the duration of hexobarbitone induced narcosis in mice. Consistent with specific inhibition of GSH synthesis, BSO pretreatment of mice decreased the proportion of a 50 mg/kg dose of paracetamol excreted in the urine as GSH-derived conjugates but did not affect paracetamol clearance through the glucuronidation or sulphation pathways. Since BSO does not affect cytochrome P-450 or conjugating enzyme activity, its use as a specific depletor of tissue GSH in the investigation of mechanisms of xenobiotic-induced toxicities is preferable to the standard GSH-depleting agents as these have other enzymic effects.

Acetaminophen↗
PubMed1984

Mechanism of action of paracetamol protective agents in mice in vivo.

The mechanism of action of cysteine, methionine, N-acetylcysteine (NAC) and cysteamine in protecting against paracetamol (APAP) induced hepatotoxicity in male C3H mice in vivo has been investigated by, characterising the effect of the individual protective agents on the metabolism of an hepatotoxic dose of APAP, and determining the efficacy of the protective agents in animals treated with buthionine sulphoximine (BSO), a specific inhibitor of glutathione (GSH) synthesis. Co-administration of cysteine, methionine or NAC increased, while co-administration of cysteamine decreased, the proportion of GSH-derived conjugates of APAP excreted in the urine of mice administered APAP, 300 mg/kg. Pretreatment of animals with BSO abolished the protective effect of cysteine, methionine and NAC, whereas cysteamine still afforded protection against APAP after BSO treatment. In conjunction with other data, these results suggest the most likely mechanism for the protective effect of cysteine, methionine and NAC is by facilitating GSH synthesis, while the most likely mechanism for the protective effect of cysteamine is inhibition of cytochrome P-450 mediated formation of the reactive metabolite of APAP.

Acetaminophen↗
PubMed1984

Complementation analysis of the aliphatic amidase genes of Pseudomonas aeruginosa.

A plasmid, pCL34, capable of autonomous replication in Escherichia coli and Pseudomonas aeruginosa has been constructed which carries the promoter and structural gene (amiE) for P. aeruginosa amidase, but not the regulator gene (amiR). Plasmid pCL34 has been mobilized from E. coli to P. aeruginosa using the broad host range plasmid RP4. Complementation studies were performed in P. aeruginosa strains carrying various amidase mutations. Measurements of amidase activity in the recipients under inducing, non-inducing and repressing conditions showed trans-complementation by the chromosomally located regulator gene product. These results confirmed the positive control model for amidase gene expression. Levels of amidase expression seen during these studies were approximately threefold higher than in the parental, amidase-positive strains.

Amidohydrolases↗