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G Siest

Publications and source records attributed to G Siest.

At least 127 records · Page 7Linked to original sources
PubMed1995

Characterization and regulatory effect of gamma-glutamyltransferase messenger RNA untranslated regions in human leukemia.

In this paper we report the presence and function of the 5' untranslated region (5'UTR) from the mRNA encoding human gamma-glutamyltransferase (GGT) in three different hematopoietic cell lines (HL-60, U-937 and K-562) as well as in the RNA of the leukocyte fraction from six acute lymphoblastic leukemias (ALL). Results obtained by RNase protection analysis demonstrate the presence of a unique form of 5'UTR expressed in most human tissues. In order to investigate the possible role of this type of sequence on regulation of GGT in hematopoietic cells, plasmid constructs carrying human hepatoma GGT 5'UTR and a luciferase reporter gene were transfected into the three blood cell lines. Compared to control untransfected cells, transfected HL-60 and K-562 showed a decrease in reporter gene activity of 51 and 73%, respectively. In contrast, transfected U-937 showed a 139% increase of reporter gene activity. Results were compared to GGT activity in the relevant cells and we concluded that the 5'UTR appears to have a regulatory role in GGT expression as a tissue-specific modulator of translation.

Base Sequence↗
PubMed1995

Developmental- and tissue-specific DNA methylation patterns and expression of rat gamma-glutamyltransferase.

In order to investigate the mechanism controlling gamma-glutamyltransferase (GGT) gene expression in adult and fetal rat tissues, as well as in H5-6 and Fao hepatoma cells, the relationship between DNA methylation and GGT expression was studied. Southern blot analysis of genomic DNA digested with MspI and HpaII showed that highly methylated DNA patterns observed especially in adult liver, heart and Fao cells as well as in fetal kidney, pancreas, lung and heart correlate well with low GGT activity determined in these tissues. In contrast, hypomethylated GGT DNA was found in fetal liver, adult kidney and lung, the tissues highly expressing GGT activity. So, observed developmental- and tissue-specific changes in the GGT gene methylation seem to play an important role in the control of GGT gene transcription.

Animals↗
PubMed1995

Stereoselective esterase activity of human serum albumin toward ketoprofen glucuronide.

Many carboxylic acid-containing drugs undergo conjugation with D-glucuronic acid in humans, leading to the formation of acyl glucuronides, which are excreted into urine. However, these metabolites can be hydrolyzed back to the parent aglycon; this reaction can be accelerated by human serum albumin (HSA). Although this phenomenon of interaction between the acyl glucuronide and HSA has been described for various drugs, the kinetics of the protein have not been characterized. The aim of this study was to investigate the HSA-mediated mechanism involved in the in vitro hydrolysis by albumin of the acyl glucuronides of (R)- and (S)-ketoprofen (a nonsteroidal anti-inflammatory drug), as model compounds. The conjugates of both ketoprofen enantiomers were incubated, separately or together, with increasing concentrations of albumin (14.5-145 microM) at pH 7.4 and 37 degrees. The reaction followed Michaelis-Menten kinetics and was stereoselective; the (R)-ketoprofen glucuronide was a better substrate than the S-conjugate. To identify the HSA domain involved in the hydrolysis reaction, specific probes of HSA binding sites were used as potential inhibitors. These probes, added at an equimolar probe/glucuronide ratio (145 microM), slightly decreased the hydrolysis (by up to 30%). They affected the reversible binding of (R)-ketoprofen glucuronide to HSA, as shown by CD studies. Because iodoacetic acid did not modify the single free cysteine residue on HSA, this amino acid residue cannot be the reactive one. In addition, the chemical modification of a single tyrosine residue (probably Tyr-411) on HSA by diisopropyl fluorophosphate significantly but weakly affected the hydrolysis of (R)-ketoprofen glucuronide, suggesting that this residue also is not involved in the catalysis. In contrast, the R-conjugate was not bound to modified albumin, as revealed in CD experiments. These results support the existence of distinct sites on HSA for reversible binding and hydrolysis of (R)-ketoprofen glucuronide.

Binding Sites↗
PubMed1994

Localization of a regulatory region on the 5'-untranslated region of human hepatoma HepG2 gamma-glutamyltransferase mRNA and response to dexamethasone and antisense oligonucleotide treatment.

We are reporting the functional analysis of the 5'-untranslated region (5'UTR) of human hepatoma HepG2 gamma-glutamyltransferase (GGT) mRNA. Transient expression of hybrid GGT-luciferase reporter gene mutants in HepG2 shows that this 5'UTR acts as a tissue-specific translational enhancer. A domain of 173 bases containing a steroid hormone response element (HRE) is responsible for the enhancing effect, which can be amplified by addition of dexamethasone at 10(-6) M. Furthermore, the regulatory role of the 5'UTR is demonstrated by interaction with sense and antisense oligonucleotides.

Base Sequence↗
PubMed1994

[Reference values of lipoprotein(a) in a French population].

OBJECTIVES: The aim of this work is to study the effect of different biological factors that could affect Lp(a) level in a presumably healthy population and to establish reference limits. METHODS: We selected 723 subjects (367 men and 356 women) for the age interval 4 to 64 years for evaluation. RESULTS: The distribution of Lp(a) is not Gaussian; 50.5% of subjects had Lp(a) concentrations under 0.10 g/l and the value for the 75th percentile was 0.27 g/l and 0.57 g/l for the 90th percentile. No relationship was observed between Lp(a) concentration and cholesterolaemia, triglyceridaemia, glycaemia, inflammatory proteins (orosomucoide and CRP), overweight, tobacco consumption and oral contraceptive use. The menopause state in women was a factor correlated with increased Lp(a) but this increase was not significant. Moreover, alcohol consumption (more than 44 g per day in men and more than 22 g per day in women) was associated with lower Lp(a) values. Among familial cardiovascular risks, only paternal listing of hypertension was associated with Lp(a) concentration in men. CONCLUSION: The measurement of Lp(a) in a young subject could be used as a genetic marker of cardiovascular risk associated with abnormal lipid metabolism and thrombosis phenomena.

Adolescent↗
PubMed1994

Induction and immunological characterization of the uridine diphosphate-glucuronosyltransferase conjugating 1-naphthol in the rat choroid plexus.

The uridine diphosphate-glucuronosyltransferase isoform conjugating 1-naphthol has a very high activity in the rat choroid plexus. We showed that antibodies raised against the main liver isoenzyme cross-reacted with a choroidal protein exhibiting the same molecular weight as the hepatic form. Both enzymes had a similar affinity for 1-naphthol. After an in vivo treatment by 3-methylcholanthrene, a 3-fold increase of the activity in both the liver and choroid plexuses was observed. These results suggest that the choroidal and hepatic enzymes conjugating 1-naphthol are identical. This high metabolic activity suggests a metabolic protection of the brain by the choroid plexus.

Animals↗
PubMed1994

Molecular and functional characterization of recombinant human gamma-glutamyltransferase. Coupling of its activity to glutathione levels in V79 cells.

We previously described the establishment of a transfected cell line (V79HGGT) that stably produces the highest recombinant human gamma-glutamyltransferase (GGT) activity. We now report the utilization of V79HGGT as a model system for studying human GGT. The papain-solubilized recombinant enzyme has been highly purified from cultured cells by a new procedure. Studies on the purified enzyme, either by N-terminal sequencing or by characterization of its enzymic activities, confirmed that recombinant GGT shares structural and catalytic identity with native human enzymes. The circular dichroism analysis indicated an alpha-helical content of 19%. Based on these data, we have undertaken a study on the functional consequences of elevated GGT activity on the reduced glutathione (GSH) content. GSH status was followed in V79 and V79HGGT cells throughout growth. A particular pattern was observed for each cell line, depending on, but differentially affected by, alteration of the culture medium. Elevated GGT activity was associated with a 2.5-fold reduced GSH content, clearly suggesting a negative influence of the highly expressed enzyme on the GSH level under normal growth conditions. Possible mechanisms involved are proposed. Our findings pointed out that, among the GSH-related enzymes, GGT could constitute an important factor determining the steady-state content of GSH.

Amino Acid Sequence↗
PubMed1994

The chemical modification of human liver UDP-glucuronosyltransferase UGT1*6 reveals the involvement of a carboxyl group in catalysis.

The treatment of UDP-glucuronosyltransferase UGT1*6 stably expressed in V79 cells with three carboxyl-specific reagents, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K), resulted in a fast, dose-dependent decrease of the 4-methylumbelliferone glucuronidation. The inactivation reactions followed pseudo-first order kinetics. The pKa of the modified residue was close to 5.0. A partial protection against inactivation by Woodward's reagent was observed at pH 7.4 in the presence of UDP-glucuronic acid, UDP, and, to a lesser extent, in the presence of 4-methylumbelliferone. Dicyclohexylcarbodiimide significantly decreased the Vmax, without affecting the apparent Km towards UDP-glucuronic acid and 4-methylumbelliferone. The results support the involvement of a carboxyl group in the catalytic process.

Catalysis↗
PubMed1994

Expression of the human UDP-glucuronosyltransferase UGT1*6 in Escherichia coli. Influence of bacterial signal peptides on the production and localization of the recombinant protein.

The membrane-bound human liver UDP-glucuronosyltransferase UGT1*6 was expressed in Escherichia coli. Exchange of the natural signal peptide by the bacterial signal peptides of pclB or OmpT proteins considerably increased the level of expression and, as the natural signal peptide, targeted the protein to the membranes. The extent of maturation of SpelB-UGT1*6 precursor was about 30%. No processing of sOmpT-UGT1*6 occurred but the processing rate of this precursor could be significantly increased by mutagenesis of the first two amino acid residues of the mature sequence. These expression vectors allowed us to produce high levels of recombinant mature UGT1*6 required for further structural studies.

Amino Acid Sequence↗
PubMed1994

Expression of multiple gamma-glutamyltransferase genes in man.

In clinical and pharmacological laboratories, the assay for gamma-glutamyltransferase (GGT) activity is an important diagnostic test, but one with high biological variability. Although the human genome contains multiple GGT genomic sequences, the diagnostic tests generally assume that only a single GGT gene is active. In the current study, segments encompassing parts of seven different potential human GGT genes have been molecularly cloned. Based on sequence determination of exons within these distinct genomic clones, oligonucleotide primers were designed which would prime and PCR-amplify putative mRNA of all seven potential GGT genes, if expressed. Gene-specific oligonucleotide probes were then utilized to assay the transcriptional status of the seven possible GGT genes in a wide variety of human RNAs. Our results show that a single GGT gene exhibits ubiquitous expression in all RNAs tested, including those from fetal and adult liver. A surprisingly large number of four additional GGT genes is expressed in man. Interestingly, these novel GGT genes are expressed in a tissue-restricted manner, which suggests that their corresponding gene products exhibit distinct functions in these specific tissues.

Amino Acid Sequence↗
PubMed1994

Sources of variability of human plasma apolipoprotein A-IV levels and relationships with lipid metabolism.

Plasma apolipoprotein (apo) A-IV concentration was determined by immunoelectrophoretic assay (EIA) in 119 nuclear families. No significant effect of concomitants such as age, weight, height, body mass index, tobacco, and alcohol consumption was observed on apo A-IV levels in men and in boys. In women, contraceptive use and hormonal status affected apo A-IV levels. In girls, only age influenced the quantitative phenotype. After adjusting by specific concomitants significant correlations were observed between apo A-IV levels and triglycerides, apolipoprotein A-I and apo B levels, suggesting a role of apolipoprotein A-IV in the hepatic lipid metabolism. Intrafamilial correlations were estimated to investigate the plausibility of a common family factor. The results obtained in this study showed a significant correlation between family members with the exception of mother-daughter pairs. Using a variance components model, the contribution of genetic and environmental factors was then investigated. Different statistical models were used and two major hypotheses were statistically acceptable: the first hypothesis supports that shared and specific environmental factors explain 35 and 65%, respectively, of the total adjusted plasma apo A-IV variation. The fraction of apo A-IV variability attributable to genetic factors was null. The second hypothesis supports that the fraction of variability attributable to apo A-IV genetic variation is 67% and the common spouse environmental factors are responsible for 33% of the total variability and no specific environmental effect was found. Among the two hypotheses, taking account of the metabolism function, we support the first one without excluding gene-environment interactions which could mask the genetic influence.

Adult↗
PubMed1994

Segregation analyses of four urinary caffeine metabolite ratios implicated in the determination of human acetylation phenotypes.

Human acetylation phenotypes were determined with caffeine (137X) as the test substance, improved by measuring urinary caffeine metabolites with a previously described HPLC method. Caffeine, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine (IX), 1-methyluric acid (IU), 1,7-dimethylxanthine (17X), and 1,7-dimethyluric acid (17U) were quantified. This study tested the hypothesis, suggested by previous studies, that the acetylation polymorphism is strongly influenced by a major gene. Phenotypes were assessed by using four urinary caffeine metabolite ratios: AFMU/1X, AFMU/[1X + 1U + 17U], AFMU/[AFMU + 1X + 1U], and AFMU/[1X + 1U + 17X + 17U] in a population included 281 nuclear family members who were healthy volunteer subjects. Each urinary ratio revealed strong familial aggregation with correlations between parents and offspring varying from 0.340 to 0.486 as a function of the ratio considered, and between sibs from 0.410 to 0.512 whereas correlations among spouses were not significant, excluding an effect of environmental factors. Segregation analyses were conducted upon these four ratios testing a series of specific models of inheritance and gave evidence for single locus control of N-acetyltransferase (NAT) activity, with Mendelian codominant transmission using the AFMU/1X, AFMU/[1X + 1U + 17U], and AFMU/[1X + 1U + 17X + 17U] ratios. The slow allelic frequencies were 0.739, 0.753, and 0.724, respectively, and the phenotypic concordance was 90 to 92% with the AFMU/1X ratio. The familial aggregation observed in using the AFMU/[AFMU + 1X + 1U] ratio was consistent with a recessive transmission for the allele controlling the homozygous slow phenotype. This last ratio is not convenient to differentiate rapid heterozygous and homozygous phenotype. Considering the number of misclassified subjects, this study would be completed by genotyping the same families as demonstrated by some authors who predicted 97.5% of acetylation phenotype when using PCR-based DNA amplification test.

Acetylation↗
PubMed1994

Heterogeneity of hepatic microsomal UDP-glucuronosyltransferase(s) activities: a new kinetic approach for the study of induction and specificity.

UDP-glucuronosyltransferase (UGT) activities have been described as heterogeneous, i.e. supported by a family of isoenzymes, each of them being capable of conjugating a given chemical family of aglycons, including steroids, coumarines, and phenols. Some of these isoenzymes are specifically induced by xenobiotics. In order to discriminate between the different isoenzymes, we propose a new in situ approach that combines induction (gene regulation) and catalytic activities (specificity). The characterization of one isoenzyme is obtained by (i) increasing its amount by specific inductive stimulation and (ii) studying simultaneously the glucuronidation kinetics of a series of alternative substrates. Provided the substrates are of similar structure, a linear relationship can be established between their glucuronidation rates before versus after induction. We developed a simple mathematical model to analyze the kinetic behaviors observed. With this method, it is possible to know (i) the exact extent of induction of a given isoenzyme by a given inducer (induction factor, n) and (ii) its strict specificity. This in situ methodology is complementary to isolated protein or cDNAs, for the characterization of the real in situ substrate specificity of differentially regulated UGT isoenzymes.

Animals↗
PubMed1994

Chemical modification of human UDP-glucuronosyltransferase UGT1*6 by diethyl pyrocarbonate: possible involvement of a histidine residue in the catalytic process.

Chemical modification with diethyl pyrocarbonate (DEPC) of the recombinant human liver UDP-glucuronosyltransferase UGT1*6 in enriched membrane fractions from a V79 cell line resulted in a rapid inactivation of the glucuronidation reaction, measured with 4-methyl-umbelliferone as aglycone substrate, with a second-order rate constant of 3110 M-1.min-1 at pH 6.0 and 25 degrees C. The enzymatic activity was restored by hydroxylamine. Chemical modification with 0.2 mM DEPC for 60 s decreased the apparent Vmax 2.4-fold without significantly affecting the apparent Km toward 4-methylumbelliferone and UDP-glucuronic acid. Similarly, the binding of the photoactivatable cosubstrate analog [beta-32P]5-azido-UDP-glucuronic acid to the active site was not affected by the chemical modification. The enzyme was protected against this inactivation by 4-methylumbelliferone, suggesting that the modified residue was located in or near the aglycone binding site. In contrast, the cosubstrate UDP-glucuronic acid potentiated the irreversible inhibition, indicating a conformational change in the protein upon binding. The pH-dependence of the inactivation was in agreement with the modification of an amino acid residue with a pKa of 6.1. On the other hand, analysis of the variation of Vmax and Vmax/Km values of the glucuronidation reaction as a function of the pH revealed the presence of two essential residues with a pKa within the range 5.7-6.0. The data of the chemical modification of the recombinant enzyme together with that of the pH dependence of the activity strongly suggest the involvement of a histidine residue, highly reactive toward DEPC, which could be the base catalyst of the glucuronidation reaction supported by human UGT1*6.

Affinity Labels↗
PubMed1994

Expression and role of the human liver UDP-glucuronosyltransferase UGT1*6 analyzed by specific antibodies raised against a hybrid protein produced in Escherichia coli.

Characterization of human UDP-glucuronyltransferases (UGTs) has been limited by the unavailability of probes selective for each of several highly related isoforms. To better understand the role of this superfamily in the metabolism of drugs and xenobiotics, we describe a molecular/immunological strategy for discriminating the implication of each human isoenzyme in this process. Specific polyclonal antibodies were generated against the divergent amino-terminal domain of the UGT isoform UGT1*6 which is involved in the detoxification of nucleophilic compounds related to phenols and naphthols in human liver. The novel approach consists of the expression of a N-terminal UGT polypeptide fused to Staphylococcus aureus protein A in Escherichia coli and a single step purification of the fusion protein by immunoaffinity chromatography. Immunoblot and immunoinhibition analysis showed that the antibodies raised against the fusion protein selectively recognized both the denaturated and the native forms of UGT1*6, when expressed in V79 cell lines, but not three other recombinant UGT isoenzymes. In human liver microsomes, specific immunoinhibition analysis demonstrated that glucuronidation by UGT1*6 represented 20 to 50% of the total 1-naphthol UGT activity with a good correlation with the amount of protein selectively quantified on immunoblot. The specific expression of UGT1*6 was found to be significantly reduced in tumoral tissues but enhanced in cholestatic livers, when compared with healthy hepatic tissues. Interestingly, in human kidney microsomes, antibodies revealed a high level of UGT1*6 expression on immunoblot and inhibited 1-naphthol glucuronidation up to 55%, indicating that this isoform is also expressed in kidney and extensively contributes to phenol glucuronidation in this tissue.

Adult↗