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B Clement

Publications and source records attributed to B Clement.

At least 19 recordsLinked to original sources
PubMed1992

The reduction of 6-N-hydroxylaminopurine to adenine by xanthine oxidase.

The genotoxic and mutagenic compound 6-N-hydroxylaminopurine (HAP) can be detoxified in vitro by enzymatic N-reduction to adenine. This reaction is catalysed by both rat and rabbit liver cytosolic fractions. The formation of adenine was monitored using HPLC. Subcellular distribution of the activity, kinetic parameters and the influence of various cofactors and inhibitors were determined. The N-reduction required NADH or hypoxanthine or xanthine and was strongly inhibited by allopurinol. These observations suggested that the N-reductase activity is due to xanthine oxidase (EC 1.2.3.2). Moreover, the involvement of xanthine oxidase is supported by the observation that purified cow milk xanthine oxidase also catalysed this reaction. The N-reduction of HAP was inhibited only weakly by oxygen. In addition, the formation of adenine is catalysed by either the oxidase or dehydrogenase form of xanthine oxidase. Thus, this reaction should be significant for the in vivo detoxification of HAP.

Adenine↗
PubMed1992

Genes encoding a histone H3.3-like variant in Arabidopsis contain intervening sequences.

Two genes encoding a particular H3 histone variant were isolated from Arabidopsis thaliana. These genes differ from the H3 genes previously cloned from Arabidopsis and other plants by several interesting properties: (1) the two genes are located close to each other; (2) their coding regions are interrupted by two or three small introns, the two closest to the initiation codon being located at the same place in the two genes; (3) another, long intron is located in the 5'-untranslated region just before the initiation codon of gene I as deduced from the sequence of several corresponding cDNAs, and very likely also of gene II; (4) these genes do not show preferential expression in organs containing meristematic tissues contrary to the classical intronless replication-dependent histone genes, thus suggesting that their expression is not replication-dependent; (5) the protein encoded by both genes is the same and corresponds to a minor H3 variant highly conserved among all the plant species studied up to now. All these characteristics are common with the animal replication-independent H3.3 histone genes and it is assumed that the genes described here are the first example of the equivalent H3.3 gene family in plants. Interestingly, the promoter regions of the two genes have the same general structure as the Arabidopsis intronless genes. Possible implications on the regulation of H3 genes expression are discussed.

Amino Acid Sequence↗
PubMed1992

Metabolic N-hydroxylation of diminazene in vitro.

The two N-hydroxylated derivatives (amide oximes) and the corresponding amides of the trypanocidal diamidine diminazene (Berenil, CAS 536-71-0) have been synthesized. These reference compounds made it possible to investigate the in vitro metabolism of the amidine functionalities of diminazene. Diminazene metabolites were detected for the first time, in the form of the corresponding mono- and di(amide oxime), after incubation with 12000 g supernatants from rabbit liver homogenates and careful workup (freeze drying). The identification was based on the behavior in thin-layer chromatography and on comparison of the mass spectral data for the metabolites with those for synthetic material. The N-hydroxylation of diminazene showed the properties typical of a reaction catalyzed by a microsomal monoxygenase. Diminazene amides did not occur as metabolites. The di(amide oxime) of diminazene was tested for its trypanocidal and leishmanicidal activity on various laboratory strains of trypanosomes in mice and on Leishmania donovani in hamsters. The studies showed that the di(amide oxime) has a trypanocidal effect on various strains of trypanosomes (T. brucei, T. vivax, T. congolense and T. evansi), but this is distinctly weaker than that of diminazene. A diminazene-resistant strain of T. rhodesiense is also unaffected by the di(amide oxime). The di(amide oxime) also has a leishmanicidal effect, but this again is distinctly less than that of diminazene.

Animals↗
PubMed1991

Identification of a 110-kDa nonintegrin cell surface laminin-binding protein which recognizes an A chain neurite-promoting peptide.

Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identified a 110-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the 110-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact sequence identity with nucleolin, a major 110-kDa nucleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-peptide-binding 110-kDa protein. The 110-kDa protein appears to be a ligand for a specific site on laminin.

Amino Acid Sequence↗
PubMed1991

Differential expression of laminin chains in hepatic lipocytes.

The lipocyte is an important source of laminin in the normal liver. We have investigated the expression of the 3 chains of laminin in isolated rat lipocytes. Both B1 and B2 chains, but not A, were found in medium from 5-day-old lipocyte primary cultures by immunoblotting and immunoprecipitation of 35S-labeled proteins after reducing SDS-polyacrylamide gel electrophoresis. An additional polypeptide of Mr = 380,000 was identified by immunoprecipitation. Under non-reducing conditions only one Mr = 900,000 band was revealed. High levels of B1 and B2 mRNAs were also demonstrated in 5-day-old cultured lipocytes while at the time of seeding, only B2 chain mRNAs were clearly detectable. A chain mRNA was constantly absent. These results suggest that lipocytes produce a variant form of laminin in primary culture and that the Mr = 380,000 polypeptide could be unrelated to the A chain of laminin.

Adipose Tissue↗
PubMed1991

Cellular sources of matrix proteins in experimentally induced cholestatic rat liver.

Collagens (I, III, and IV), fibronectin, and laminin were localized using the indirect immunoperoxidase technique 14 days after bile duct ligation, i.e., when extensive fibrosis and numerous neoformed bile ducts were observed. Extensive fibrous septa in enlarged portal spaces were stained for collagens I, III and IV, fibronectin, and laminin. Collagen IV and laminin were abundant around proliferative bile ducts. In addition, collagen IV was nearly continuous in the sinusoids. At the ultrastructural level, antigens were localized in the endoplasmic reticulum of several liver cell types. In portal spaces, bile duct cells and cells that form the transitional canal of Hering were strongly labelled for basement membrane components, particularly laminin, but not for collagens I and III and fibronectin, which were abundant in fibroblast-like cells. Inside the lobule, only Ito cells and, to a lesser extent, endothelial cells contained collagens, fibronectin, and laminin. Ito cells were found to be heavily stained for collagens III and IV, and laminin. Except for fibronectin, which was always abundant, precursors of extracellular matrix proteins were only slightly detectable in the endoplasmic reticulum of some hepatocytes, particularly those located close to altered areas. This study demonstrates that experimental extrahepatic cholestasis in the rat induces periportal fibrosis and continuous deposition of collagen IV in the sinusoids. Several cell types participate in the formation of extracellular matrix components, particularly bile duct cells and Ito cells, with a possible involvement of hepatocytes, thus suggesting that cholestasis provokes changes in the pattern of matrix protein production in liver cells.

Animals↗
PubMed1991

Expression of laminin and its receptor LBP-32 in human and rat hepatoma cells.

Dramatic cellular changes that occur during hepatocarcinogenesis are associated with major alterations in extracellular matrix formation and in the relationships between cells and their microenvironment. We have studied the expression of laminin, the major noncollagenous glycoprotein of basement membrane, and the laminin receptor 32 kD laminin-binding protein in two rat (Faza 967 and HTC) and two human (HepG2 and HBGC2) hepatoma cell lines that express a variety of liver-specific functions. Laminin was found in the rough endoplasmic reticulum of these cells when the indirect immunoperoxidase method and electron microscopic examination were used. Radiolabeled laminin, immunoprecipitated from both media and cell extracts, was resolved by electrophoresis on sodium dodecyl sulfate gel in two major polypeptides that comigrated with the A and B subunits from Engelbreth-Holm-Swarm tumor laminin. Immunoblot analysis showed that the Mr = 400,000 polypeptide did not correspond to the A subunit of laminin. Northern blot analyses demonstrated large amounts of B1 and B2 mRNAs but no A chain mRNA. We conclude that the tumor cells produce the laminin B chains only. In contrast, normal adult hepatocytes from either man or rat lacked laminin mRNAs, whereas in 1-day primary culture, B chain mRNAs became detectable. The steady-state level of 32 kD laminin-binding protein mRNA was 10-fold and threefold higher in rat hepatoma cells than in freshly isolated and 1-day cultured normal rat hepatocytes, respectively. In human hepatocytes, the steady-state levels of 32 kD laminin-binding protein mRNAs varied depending on the donor and never reached the level of the human hepatoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Hepatic microsomal N-hydroxylation of adenine to 6-N-hydroxylaminopurine.

The enzymatic N-hydroxylation of the purine base adenine to the genotoxic and mutagenic compound 6-N-hydroxylaminopurine is reported for the first time. Adenine was N-oxygenated in vitro by aerobic incubations with 3-methylcholanthrene or isosafrole induced microsomal fractions of rat liver homogenates and NADPH. The formation of 6-N-hydroxylaminopurine in the incubation mixtures under widely differing conditions was assayed using newly-developed, high-performance liquid- and thin-layer chromatographic methods. Optimal reaction conditions and kinetic parameters were determined. Neither superoxide anion nor hydrogen peroxide was directly involved in the N-hydroxylation reaction. Oxidases like xanthine oxidase and peroxidase (in the presence of hydrogen peroxide) did not catalyse this N-hydroxylation. The involvement of cytochrome P-450 isoenzymes in this reaction is supported by the observation that the N-hydroxylation is only observed after pretreatment of the rats with 3-methylcholanthrene or isosafrole. Other inducers (phenobarbital, ethanol, 5-pregnen-3 beta ol-20-one-16 alpha-carbonitrile) were without effect. This is the first example of the microsomal transformation of an endogenous substance to a toxic derivative by usually foreign substances (xenobiotics) metabolizing cytochrome P-450 isoenzymes. The significance for the in vivo situation is discussed on the basis of the data obtained in this study.

Adenine↗
PubMed1989

[Biotransformation of benzamidine and benzamidoxime by microsomal enzymes of the rabbit].

At pH 7.4 neither benzamidine (1) is ring-hydroxylated nor benzamidoxime (2) is N-hydroxylated, reduced or ring-hydroxylated by aerobic incubations with microsomal fractions (12000 g supernatant, microsomes) of rabbit liver homogenates and NADPH. Products of hydrolytic processes are also not detected. A very long incubation period and a pH 6.3 are necessary for the detection of a slight reduction of benzamidoxime (2) to benzamidine (1). Results are obtained by use of synthetic reference material and by newly developed HPLC methods. Thus, kinetic studies of the microsomal N-hydroxylation of benzamidine (1) to benzamidoxime (2) in the presence of N-methylbenzamidine (3) performed at pH 7.4 are not influenced by other transformations and provide evidence for the involvement of the same isoenzyme of cytochrome P-450 for both the N-hydroxylation of 1 and the N-dealkylation of 3.

Amidines↗
PubMed1989

Structural requirements of microsomal N-oxygenations derived from studies on amidines.

A short description of the chemical and pharmacological properties of amidines is followed by a comprehensive discussion of investigations on the N-oxidative biotransformations of amidines. The results of these investigations have confirmed the author's hypothesis, based on mechanisms, that N-oxygenation by the cytochrome P-450 enzyme system is observed particularly when N-dealkylation is not possible because of the absence of hydrogen atoms on the carbon atoms adjacent to the nitrogen atom, alpha-H-atoms. The results obtained with amidines are discussed in their relationship to other microsomal N-oxygenations both by cytochrome P-450 and flavine-containing monooxygenase. Attempts are made to deduce a scheme for predicting N-oxygenations.

Amidines↗
PubMed1988

Mechanism of the microsomal N-hydroxylation of para-substituted benzamidines.

With the aid of HPLC analyses and simple Michaelis-Menten kinetics, the maximum rates of the microsomal N-oxygenation of various para-substituted benzamidines 1 to benzamidoximes 2 were determined. The presence of electron-donating substituents increased the rates whereas the presence of electron-accepting substituents decreased them. A significant correlation between the logarithm of the maximum rates with the Hammett sigma p constants was found for a reaction constant of rho = -0.88. These results support the postulated radical mechanism for the N-oxygenation by the cytochrome P-450 enzyme system.

Amidines↗
PubMed1988

Polyadenylation of histone H3 and H4 mRNAs in dicotyledonous plants.

The histone H3 and H4 genes are shown to be expressed in both Arabidopsis plantlets and transitory multicellular suspension. The 5'- and 3'-ends of the H4 mRNAs have been localized on two H4 genes previously sequenced, H4A748 and H4A777. S1-nuclease mapping and reverse-transcriptase-primer-elongation experiments revealed the existence of two start points for transcription, located 31 and 37 nucleotides downstream from the TATA-box. The 3'-end of the mRNA corresponding to H4A748 was localized at 177 nt after the stop codon. The other gene, H4A777, most probably is not expressed. In addition to a long 3'-untranslated region, the H4 mRNA was shown to be polyadenylated in both plantlets and cell-suspension. This observation was extended to the H3 mRNAs of Arabidopsis and of two other dicots, tobacco and sunflower. Previous results on maize H3 and H4 mRNAs suggest that polyadenylation is a common feature for histone mRNAs in higher plants.

Base Sequence↗
PubMed1988

Clinical and angiographic correlates and prognostic significance of the coronary extent score.

The clinical and angiographic correlates and the prognostic significance of the "coronary extent score" in a consecutive series of 313 patients who were catheterized twice were studied. The extent score was defined as the number of 5 to 75% stenosed segments in a 15-segment coding system. The extent score was higher in subgroups of patients with new onset angina at the time of the first angiogram (4.3 +/- 2.4 vs 3.3 +/- 1.9, p less than 0.01), unstable angina at the time of the second angiogram (4.0 +/- 2.0 vs 3.3 +/- 1.9, p less than 0.05) or multifocal progression from the first to the second angiogram (4.0 +/- 2.1 vs 3.3 +/- 1.9, p less than 0.01), suggesting that it is an index of active coronary artery disease. The extent score did not correlate with the number of diseased vessels (r = 0.03), the ejection fraction (r = 0.03), the Friesinger score (r = 0.04) and the Gensini score (r = -0.07) (difference not significant for each). Cox's model was fit to the survival data recorded on a prospective basis after the second angiogram. Independent predictors of survival were ejection fraction (p less than 0.001), extent score (p = 0.001), number of diseased vessels (p = 0.01) and percent of left main luminal stenosis (p less than 0.05). The extent score was also an independent predictor of myocardial infarction and unstable angina. Thus, the extent score, an index of active progressive disease, is an independent predictor of mortality and cardiac events in patients with coronary artery disease.

Angina Pectoris↗
PubMed1988

Genotoxic activities of benzamidine and its N-hydroxylated metabolite benzamidoxime in Salmonella typhimurium and mammalian cells.

The genotoxic potentials of benzamidine and benzamidoxime were determined to study the toxicological relevance of the metabolic N-oxygenation (N-hydroxylation) of benzamidines to benzamidoximes. Benzamidoxime induced DNA single-strand breaks (in rat hepatocytes) and DNA amplification in SV40-transformed hamster cells. In the experiments performed, benzamidine itself was only marginally positive in the hepatocyte/DNA single-strand break assay. Since these cells possess an intact metabolization apparatus, the biological activities may be attributed to toxic and genotoxic metabolites formed by biotransformation. In the Salmonella typhimurium mutagenicity test (TA 98 and TA 100) benzamidoxime alone exhibited a low mutagenicity in the TA 98 strain in the presence of rabbit liver S-9 fractions. These results permit recognition of the metabolic N-hydroxylation of benzamidines to benzamidoximes as a process to toxication. Indirect evidence for the formation of a glucuronide of benzamidoxime has been obtained from in vitro experiments, but it could not be established that this process was a decisive factor in the genotoxicity of benzamidoxime.

Amidines↗
PubMed1988

Types I and IV procollagen gene expression in cultured rat hepatocytes.

The molecular mechanism involved in the expression of collagens by hepatocytes were investigated in both pure and co-culture with another rat liver epithelial cell type (RLEC). We measured the steady-state levels of mRNAs coding for pro alpha 1(I) and pro alpha 1(IV) chains by Northern analysis and by dot blotting, using specific recombinant cDNA probes. In freshly isolated hepatocytes, only small amounts of pro alpha 1(I) and pro alpha 1(IV) mRNAs were detected by dot-blot analysis. After 3 days in culture, the pro alpha 1(I) and pro alpha 1(IV) mRNA levels increased 2 to 5 times. The amount of pro alpha 1(IV) mRNAs was identical in hepatocyte cultured with RLECs while the pro alpha 1(I) mRNA level was 5 times that in pure hepatocyte culture. Hydrocortisone reduced pro alpha 1(I) mRNA in hepatocyte cultures, but had no effect on co-cultured cells. In both culture systems, this glucocorticoid did not act on the steady-state pro alpha 1(IV) mRNA level. Whatever the age and the type of culture (pure or mixed) RLECs exhibited the highest levels of pro alpha 1(I) and pro alpha 1(IV) mRNAs, which were reduced by hydrocortisone. These results show that procollagen gene expression by hepatocytes is not directly correlated with their functional state and that corticosteroids differently affect the expression of different collagen genes and collagen deposition.

Animals↗