Biomedical subjects

G Siest

Publications and source records attributed to G Siest.

At least 163 records · Page 9Linked to original sources

PubMed1993

Induction of recombinant human gamma-glutamyl transferase by sodium butyrate in transfected V79 and CHO Chinese hamster cells.

Sodium butyrate was used to enhance biosynthesis rates of recombinant human gamma-glutamyl transferase (GGT) expressed under the control of the SV40 or the cytomegalovirus immediate early promoter, respectively, in transfected V79 and CHO Chinese hamster cell lines. Maximal induction of GGT specific activity in butyrate-treated cells ranged from 3 to 5-fold and resulted from a strong increase in the GGT mRNA ratio. We also observed that maximal transcription level in V79 cells occurred within 12 hr of treatment, whilst the cell proliferation was transiently arrested. Despite its processing requirements, induced GGT exhibited unchanged catalytic and physico-chemical features relative to human serum or hepatoma enzyme, thus appearing as an excellent model for further studies on human GGT.

Animals↗

PubMed1993

Subcellular localization of cytochrome P450, and activities of several enzymes responsible for drug metabolism in the human brain.

We studied the subcellular distribution of cytochrome P450 and related monooxygenase activities in six regions of human brains removed at autopsy. The content of total cytochrome P450 was found to be at least nine times higher in the mitochondrial fraction than in the microsomes in all the regions studied. However, cytochrome P450-dependent enzymatic activities which are representative of different isoforms metabolizing exogenous molecules exhibited a microsomal prevalence, a situation previously observed in rat brain. The other drug-metabolizing enzymes catalysing functionalization and conjugation reactions, presented the following characteristics in human brain: (i) a low activity of NADPH-cytochrome P450 reductase, which also catalyses the reduction of some xenobiotics; (ii) a high specific activity of the membrane-bound epoxide hydrolase; (iii) among the enzymes catalysing conjugation reactions, 1-naphthol-UDP-glucuronosyltransferase activity was barely or not detectable, whereas the mean glutathione-S-transferase activity was 15 times higher than the activity measured in rat brain. The presence of several drug-metabolizing enzyme activities in human brain microvessels, and particularly the high activity of epoxide hydrolase, suggests a participation of these enzymes in the metabolic blood-brain barrier.

Aged↗

PubMed1993

Apolipoprotein B signal peptide polymorphism in patients with myocardial infarction and controls. "The ECTIM study".

We report the allele frequencies of the apolipoprotein B (Apo B) signal peptide polymorphism in patients with myocardial infarction and compare them with controls. The first sample consists of 197 myocardial infarction patients and 168 controls from Belfast (UK). The second sample consists of 167 myocardial infarction patients and 205 controls from Strasbourg (France), and the third consists of 71 patients and 146 controls from Haute-Garonne (Toulouse, France). No significant differences were observed in the frequency distribution of genotypes among cases and controls or between populations. However, there were more rare homozygotes in the Belfast cases. Significant associations were observed between the Apo B signal peptide polymorphism and mean levels of total cholesterol, low density lipoprotein cholesterol, Apo B and lipoprotein particles containing Apo (a) [Lp(a)] in the Strasbourg control population. Individuals homozygous for the rare allele had higher levels of these lipid parameters. In Belfast, although not statistically significant, the Apo B signal peptide polymorphism had a similar effect on Apo-B-related parameters as seen in Strasbourg. No significant associations were observed in the Haute-Garonne population where the risk of myocardial infarction is three times lower than in Belfast. In all three populations, the average Lp(a) levels were consistently different among Apo B signal peptide genotypes. These data implicate the Apo B signal peptide in determining some of the risks of myocardial infarction in these populations. Regardless of the exact mechanism, the Apo B signal peptide is an important candidate locus for the study of potentially atherogenic lipid variants.

Adult↗

PubMed1993

Heterogeneity of hepatic UDP-glucuronosyltransferase activities: investigations of isoenzymes involved in p-nitrophenol glucuronidation.

1. UDP-Glucuronosyltransferase (UGT, EC 2.1.4.17) has been measured routinely with para-nitrophenol (pNP) because the UGT assay using this substrate is easy to assess and run. 2. This compound has been used in several studies as a substrate for purification of the enzyme. 3. In the present work, we characterize the para-nitrophenol-conjugating activity. 4. An analysis of kinetics of para-nitrophenol conjugation obtained from various biological sources (various tissues and various species) leads us to the conclusion that at least three isoenzymes are responsible for this activity in the rat. 5. Both UGT-(testosterone) and the 3-methylcholanthrene-inducible form previously described in the literature, may be responsible for the activity, whilst a highly specific form (UGT-phenol) is reported here for the first time. 6. This work is intended to lay down the basis of further investigations, including purification of the highly specific isoform.

Animals↗

PubMed1993

Peroxisome proliferators as inducers and substrates of UDP-glucuronosyltransferases.

Peroxisome proliferators, despite their chemically unrelated structures, share the common property of being able to stimulate the glucuronidation of bilirubin in rodents and, probably, also in man. The aryloxycarboxylic acids (clofibric acid, fenofibrate, bezafibrate, ciprofibrate), tiadenol and probucol, all of which have hypolipidemic properties, as well as the fatty acid-like perfluorodecanoic acid all enhanced the expression of the UDP-glucuronosyltransferase (UGT) form involved in the conjugation of the pigment. This induction is manifested by an increase in the mRNA species encoding the protein with a subsequent increase in the neosynthesis of the corresponding protein in the endoplasmic reticulum. The induction process is concomitant with that of cytochrome P-450-IVA1 and cytosolic epoxide hydrolase, which, like bilirubin UGT, are mainly involved in the metabolism of endogenous substrates. With a series of carboxylic acids related to clofibric acid, it was possible to demonstrate that induction was mediated via specific interactions based on the physicochemical properties of the inducers. Until now, the molecular basis of induction of bilirubin UGT is not known. The peroxisome poliferators that possess a carboxyl group are good substrates of UGT, especially in man. The acylglucuronides formed are known for their instability and reactivity which could contribute to the toxicity encountered in some patients treated with the drugs. There is convincing evidence that UGT bilirubin does not catalyze the glucuronidation of these substances even if the two types of substrate form acylglucuronides.

Animals↗

PubMed1993

Acetylation phenotypes and biological variation in a French Caucasian population.

Factors affecting the caffeine acetylation phenotype were investigated in a French Caucasian population of 150 unrelated supposedly healthy subjects, aged 18 to 63 years. This population, including 75 men and 75 women, was used to determine whether the acetylation polymorphism is related to environmental influences such as smoking habits, intake of alcohol, use of oral contraceptives, use of certain drugs. The acetylation phenotype was assessed from the molar ratio of two caffeine metabolites: 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine. For values less than 0.85, the subjects were classified as poor acetylators (frequency, mean +/- SD: 61.3 +/- 7.9%) in this study. Dose recoveries of 5-acetylamino-6-formylamino-3-methyluracil (mean +/- SD) were 1.26 +/- 0.85% and 3.58 +/- 1.64% in slow and rapid acetylators, respectively. The recovery (mean +/- SD) of 1-methylxanthine in the 3 hour-urine was 2.86 +/- 1.51% in slow acetylators and 2.36 +/- 1.27% in rapid acetylators. The mean value (and SD) of the molar ratio was 0.437 (0.177) and 1.669 (0.651) for slow and rapid acetylators. Three other metabolite ratios can also provide an acetylation index: 5-acetylamino-6-formylamino-3-methyluracil/5-acetylamino-6-formylamino-3 - methyluracil + 1-methylxanthine + 1-methyluric acid; 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine + 1-methyluric acid + 1,7-dimethyluric acid; and 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine + 1-methyluric acid + 1,7-dimethyluric acid + 1,7-dimethylxanthine with a bimodal distribution for the former and a trimodal distribution for the two latter ratios, both showing about 95% concordance with the 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine ratio. Age did not influence the excretion of caffeine and its five major metabolites. A marked influence of sex was observed only on the unchanged caffeine excretion, and the effect was greater in slow acetylators than in rapid acetylators. The 5-acetylamino-6-formylamino-3-methyluracil excretion was about three times higher in rapid acetylators than in slow acetylators in both sexes.

Acetylation↗

PubMed1993

Blood activities of antioxidant enzymes in alcoholics before and after withdrawal.

Since free radicals and peroxides seem to be involved in the toxicity of alcohol, several authors have examined the variations of blood activities of antioxidant enzymes in alcoholics, but published results are somewhat conflicting. In this study, erythrocyte (E) activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT), and plasma (P) activities of SOD and GPX were measured in 58 male alcoholics without evidence of severe liver disease before and after a 21-day weaning period, and in a control group of 78 healthy men. Before abstinence, E-SOD and E-GPX activities were, respectively, 6.8% and 13.0% lower in alcoholics than in controls (p < or = .05 and p < or = .01, respectively), whereas the slight increases of E-CAT, P-SOD and P-GPX were not statistically significant. After 21 days of abstinence, no change in activities of the erythrocyte enzymes was noticed; conversely, P-SOD activity was reduced by 8.3% (p < or = .01) and P-GPX by 23.3% (p < or = .001). Variations of blood antioxidant enzymes observed in patients were of limited amplitude and do not allow the use of either of them as markers of alcohol abuse.

Adult↗

PubMed1993

Application of gene transfer technologies to the production of enzyme reference materials: example of gamma-glutamyltransferase.

Protein reference materials are traditionally prepared by purification from mammalian or human tissues. The supply of these tissues is limited; consequently, there is a growing need for applied molecular and cellular biology technologies for the production of human recombinant proteins. This is especially true when only small amounts of the proteins are available in the tissues. We review the current knowledge necessary for high-level production of such proteins in different heterologous expression systems, using our data on gamma-glutamyltransferase (EC 2.3.2.2) as an example. We describe the steps required to achieve the expression of enzymes and other proteins in Escherichia coli, yeast, or mammalian cells. We list many of the problems investigators may face in preparing recombinant proteins, and provide information on selecting the most appropriate system as well as the most favorable experimental conditions. Depending on the expression system, recombinant proteins can potentially be obtained for most, if not all, enzymes of interest in clinical chemistry, and such proteins should possess characteristics very similar to those of the corresponding human native proteins. Studies suggest that these products can be used as reference materials in clinical chemistry laboratories.

Animals↗

PubMed1993

[Viral theory of atherosclerosis. Role of cytomegalovirus].

The multifactorial aetiology of atherosclerosis is nowadays well established. In parallel with confirmation of the lipidic hypothesis, a tumoral theory for this pathology was built up during the past decade. This theory considers atheroma a benign tumor. Among agents that can induce cell proliferation, oncogenic viruses seem to be the most efficient. In this review, we present several works suggesting viral involvement in atherosclerosis. The viral theory is based not only on clinical and epidemiologic data, but also on cell and molecular biology research. These three complementary approaches have established a relationship between herpes viruses and the atherogenic process. Cytomegalovirus (CMV) seems to be the suspect in human atherosclerosis. Several studies of the effects of CMV on cells involved in atheroma, especially smooth muscle cells and endothelial cells, are consistent with a possible role of this virus in atherosclerosis. This paper presents not only recent research developments in this increasingly explored field, but also questions that remain to be elucidated and the consequences of the viral theory of atherosclerosis.

Animals↗

PubMed1993

The role of genetics in defining reference values and health status.

Since its establishment, the Center for Preventive Medicine in Vandoeuvre-les-Nancy, France, performed specific studies on healthy humans, and its approach was very useful for defining reference values. Prevention should extend its interest to chronic diseases. The majority of important adult disorders are partially genetically determined. Genetic markers are also useful as exclusion or as partition criteria in the production of reference values. Results are presented that were obtained for apolipoproteins E, B and AIV, frequencies of these polymorphisms in the Lorraine population, and relationships between these polymorphisms and lipid metabolism-related parameters. Health checkup centers, in particular those involved in family screening, are well suited for resembling many data concerning environmental factors: tobacco consumption, alimentation habits, or alcohol and drug consumption. Simultaneous determination of genetic markers could allow the determination of an individual's susceptibility or resistance to developing a disease and to prepare a preventive action.

Environmental Exposure↗

PubMed1992

Selective and potent inhibition of different hepatic UDP-glucuronosyltransferase activities by omega,omega,omega-triphenylalcohols and UDP derivatives.

A homologous series of omega,omega,omega-triphenylalcohols and corresponding omega,omega,omega-triphenylalkyl-UDP derivatives was synthesized and tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes, with 1-naphthol, testosterone and bilirubin as substrates. Introduction of the UDP moiety in the triphenylalcohols increased their inhibition potency markedly toward the isoforms which glucuronidate 1-naphthol and testosterone, but strongly decreased that toward bilirubin. The inhibiting potency of the UDP-derivatives increased as a function of the length of the hydrocarbon chain. The best inhibitor 7,7,7-triphenylheptyl-UDP showed an I50 of 30 and 10 microM for 1-naphthol and testosterone glucuronidation, respectively; even a 1 mM concentration of the compound had little, if any, effect on bilirubin glucuronidation. The inhibition by 7,7,7-triphenylheptyl-UDP was mixed-type toward 1-naphthol, and non competitive toward testosterone (apparent K(i) 30 microM and 1.7 microM, respectively); on the other hand, the inhibition was competitive toward the common substrate UDP-glucuronic acid (apparent K(i) 1.9-1.2 microM). In addition, 7,7,7-triphenylheptyl-UDP (0.25-0.50 mM) almost inhibited glucuronidation of 1-naphthol and testosterone catalyzed by the recombinant rat liver UGT-2B1 and human liver UGT-1A1, whose cDNA has been expressed in V79 cells. In conclusion, the data indicate that 7,7,7-triphenyheptyl-UDP interacted competitively with the UDP binding site of UGT. The results also indicate that it is possible to design transition state analogue inhibitors with specificity for different UGT forms.

Alcohols↗

PubMed1992

Enzyme induction by drugs and toxins.

Enzyme induction by drugs mostly concerns those enzymes involved in drug metabolism: cytochromes P-450, UDP-glucuronosyltransferases, glutathione S-transferases, gamma-glutamyltransferases and epoxide hydrolases. A large variety of molecular forms exists, but not all of them are inducible (e.g. the inducible cytochromes P-450 in man are members of family IA, IIA, IIC, IIE, IIIA). Induction is most common in the liver, but also occurs in other organs (lung, placenta, lymphocytes). Over the past 20 years a relatively small number of drugs and environmental chemicals have been identified as enzyme inducers, perhaps fewer than early studies suggested. Information on inducing properties must be obtained as early as possible during the development of a new drug and made available to clinicians and clinical chemists when the drug is marketed. The main consequences of enzyme induction are changes in pharmacokinetics of the drug itself or of an associated drug. Much progress has been made in methods to identify these inducers.

Animals↗

PubMed1992

Gamma-glutamyltransferase: nucleotide sequence of the human pancreatic cDNA. Evidence for a ubiquitous gamma-glutamyltransferase polypeptide in human tissues.

gamma-Glutamyltransferase (GGT, EC 2.3.2.2) is an enzyme involved in glutathione metabolism and drug and xenobiotic detoxification. Using human hepatoma Hep G2 GGT cDNA as probe, we isolated a cDNA from a human pancreatic cDNA library. Analysis of the nucleotide sequences revealed a 2244-bp insert that includes an open reading frame of 1710 bp, encoding a protein identical to the Hep G2 and human placenta GGTs. Similarly, the 5' untranslated region, though shorter, is highly homologous to that of Hep G2 cDNA. These data suggest strongly that the same gene encodes human GGT in the placenta, Hep G2 and the pancreas. We further studied the distribution of the corresponding mRNA, called type I mRNA, in different human tissues. Using a highly sensitive method associating reverse transcription with specific amplification by polymerase chain reaction, cDNA was synthesized from total RNA isolated from the tissues and GGT specific fragments were amplified. We observed the presence of a specific cDNA fragment corresponding to the type I mRNA in the human tissues and cells tested, providing the evidence for a ubiquitous expression of this GGT mRNA in human tissues.

Amino Acid Sequence↗

PubMed1992

Glucuronidation of 3'-azido-3'-deoxythymidine in human liver microsomes: enzyme inhibition by drugs and steroid hormones.

The molecular form of UDP-glucuronosyltransferase involved in the catalysis of 3'-azido-3'-deoxythymidine (AZT)-5'-O-glucuronide was characterized in human liver microsomes. The specific activity (1.3 nmol/min per mg protein) in transplantable liver was more than 2-times higher than in post-mortem fragments. Liver microsomes from patients suffering Crigler-Najjar syndrome, who are genetically deficient in bilirubin UDP-glucuronosyltransferase, could also glucuronidate AZT to a similar extent, thus indicating that this protein was not involved in that process. A genetically engineered V79 cell line stably expressing a cDNA which encodes a human isozyme active towards 1-naphthol was unable to glucuronidate AZT. Clinically used drugs, most of them being glucuronidated, were tested as potential inhibitors of the glucuronidation of AZT in human liver microsomes. The drugs chemically related to 2-phenylpropionic acid, naproxen and flurbiprofen, and the steroid compounds testosterone, estrone and ethynylestradiol strongly inhibited AZT glucuronidation. Codeine and morphine also decreased the reaction rate although to a lower extent. Except estrone which elicited a partial competitive inhibition, ethynylestradiol, flurbiprofen naproxen and testosterone could competitively inhibit AZT glucuronidation with an apparent Ki of 38, 50, 172 and 250 microM, respectively. The results suggest that these drugs were substrates of the same isozyme(s) involved in AZT glucuronidation. Probenecid was a weak inhibitor of the reaction (Ki 900 microM), only when non-disrupted microsomes were used. This drug may compete with the anion carrier system involved in the microsomal uptake of UDP-glucuronic acid.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1992

Age-related variations of enzymatic defenses against free radicals and peroxides.

Superoxide dismutase, glutathione peroxidase and catalase are the three main enzymatic systems of defense of the organism against free radicals and peroxides. A survey of the literature shows that no general tendency of evolution of these systems in aging emerges, even if some recent studies in humans demonstrate the existence of a concomitant decrease in most of the antioxidant enzymes in blood of the elderly. The study of the antioxidant systems and their interrelations in the elderly represents a large field of future investigations.

Adult↗

PubMed1992

Multivariate genetic analysis of high density lipoprotein particles.

The aim of this study was to investigate the effect of genetic factors on three components of plasma high density lipoproteins, HDL-cholesterol (HDL-C), apolipoprotein A-I (apo A-I) and lipoprotein particle Lp A-I (Lp A-I), which contains apo A-I but not apo A-II. These analyses were carried out on 106 nuclear families with one or more children (407 subjects) who volunteered for health screening at the Center for Preventive Medicine, Vandoeuvre, France. After adjustment by stepwise multiple linear regression analysis for age, gender, weight, height, ponderosity, alcohol consumption, smoking habits, and hormonal treatment in females, a multifactorial model (considering the effect of polygenes, individual, specific, environmental and common household factors) was fitted to each variable separately. The hypothesis of no common household effects was accepted for each of the traits. The contribution of genetic factors to inter-individual variance was larger than the contribution of environmental factors for apo A-I (h2 = 0.81) and Lp A-I (h2 = 0.63) but not for HDL-C (h2 = 0.44). Bivariate analyses were carried out by parameterizing covariance components between traits. The genetic correlations were always significantly different from zero. They were estimated to be 0.73 between HDL-C and apo A-I, 0.40 between HDL-C and Lp A-I, 0.51 between apo A-I and Lp A-I. These results suggest that HDL-C, apo A-I and Lp A-I are only in part affected by the same genes and that the measurement of lipids as well as the apo A-I and Lp A-I gives complementary and different information on the metabolic and genetic aspects.

Adolescent↗

PubMed1992

Allele frequency distribution of the (TG)n(AG)m microsatellite in the apolipoprotein C-II gene.

The dinucleotide (TG)n interspersed repetitive sequences are the most abundant microsatellites in the human genome. Using the polymerase chain reaction to amplify a (TG)n(AG)m microsatellite in the first intron of the apo C-II gene, we have detected 15 different alleles in 242 unrelated individuals of French ancestry. The heterozygosity index was 0.85 and codominant Mendelian inheritance of the alleles was observed in individuals from 121 nuclear families. We report that polymorphism at this locus is attributable to length variation at both (TG)n and (AG)m motifs, although the (AG)m motif contains only two alleles differing by one repeat unit. A quadrimodal allele frequency distribution was observed at the (TG)n(AG)m locus. Each of the first three modes comprises one frequent allele and one very rare allele adjacent in size. No alleles of intermediate size were found between the three first modes. The fourth mode encompasses nine alleles that span from 27 to 35 repeat units. We suggest that this distribution reflects the molecular mechanisms by which alleles give rise to one another.

Alleles↗

PubMed1992

Drug metabolizing enzymes in the rat pituitary gland.

Brain protection against chemicals is mainly provided by the specific properties of cerebral microvessels forming the blood-brain barrier. In addition, several drug metabolizing enzymes have been evidenced both in brain tissue and in cerebral capillaries, suggesting their participation in the enzymatic protection of this organ. The pituitary gland, like true circumventricular organs, lacks a tight vascular endothelium and therefore is especially sensitive to blood-native toxic or pharmacologically active molecules. We report here the presence of cytochrome P-450 in the pituitary gland and its main mitochondrial localization. The O-dealkylase activity measured towards 7-benzoxyresorufin, a substrate for the main cytochrome P-450 isoforms involved in the metabolism of xenobiotics, was 5 times higher in the pituitary gland than in the brain cortex. Similarly, microsomal epoxide hydrolase, which inactivates reactive epoxides to trans diol molecules, and two conjugating enzymes, 1-naphthol UDP-glucuronosyltransferase and glutathione-S-transferase, display respectively 6, 4 and 7 times higher activities in the pituitary gland. 7-Benzoxyresorufin-O-dealkylase, 1-naphthol UDP-glucuronosyltransferase and membrane-bound epoxide hydrolase activities were significantly increased in the pituitary gland as an adaptive response to an in vivo treatment by an exogenous inducer, 3-methylcholanthrene. These results suggest that these enzymatic systems play a role in the protection of the pituitary gland towards drugs or toxic substances.

Animals↗