Adipose tissue secretes a variety of proteins into the bloodstream. We have previously reported a novel cDNA, apM1 (adipose most abundant gene transcript 1), which is specifically and abundantly expressed in adipose tissue [1]. Primary structure analysis predicted that the apM1 gene product possesses significant homology to collagens VIII, X and complement factor C1q, and we named it adiponectin. In the current study, we analyzed characteristics of adiponectin in vitro and in vivo. Adiponectin protein was proved to be secreted into the medium when the cDNA was transfected to COS cells. Anti-adiponectin cross-reactivities were abundantly detected in the human plasma. In solid-phase binding assays, adiponectin specifically bound to collagen types I, III and V, which are present in vascular intima. Immunohistochemical analysis revealed that adiponectin was detected in the walls of the catheter-injured vessels but not in the intact vascular walls. These data suggest that adiponectin is a plasma protein produced by adipose tissue and accumulates in vascular walls when the endothelial barrier is injured.
An acetyltransferase-overexpressing strain of Salmonella typhimurium (NM2009) has been used to investigate roles of human liver microsomal cytochrome P450 (P450) enzymes in the activation of carcinogenic nitrosamine derivatives, including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines, to genotoxic products. Studies employing correlation of activities with several P450-dependent monooxygenase reactions in different human liver samples, inhibition of microsomal activities by antibodies raised against human P450 enzymes and by specific P450 inhibitors, and reconstitution of activities with purified P450 enzymes suggest that the tobacco-smoke-related nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N-nitrosonornicotine (NNN) as well as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are oxidized to genotoxic products by different P450 enzymes, particularly P450 2E1 and 2A6. The activation of NDMA and NNN by liver microsomes was suggested to be catalyzed more actively by P450 2E1 than by other P450 enzymes because the activities were well correlated with NDMA N-demethylation and aniline p-hydroxylation in different human samples, and purified P450 2E1 had the highest activities in reconstituted monooxygenase systems. The relatively high contribution of P450 2A6 to the activation of NDEA and NNK was supported by the correlation seen with coumarin 7-hydroxylation in human liver microsomes, and antibodies raised against P450 2A6 inhibited both activities by approximately 50%. P450 3A4, 2D6 and 2C enzymes appear not to be extensively involved in the activation of these nitrosamines as judged by several criteria examined. Thus, this work indicates that several P450 enzymes, particularly P450 2E1 and 2A6, catalyze metabolic activation of nitrosamine derivatives including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines in human liver microsomes.
The possible roles of cytochrome P450 (P450) enzymes in the metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) by rat liver microsomes have been examined in a system containing the bacterial tester strain Salmonella typhimurium NM2009, a newly developed strain showing high O-acetyltransfer activities. The DNA-damaging activity could be determined by measuring expression of the umu gene in a plasmid containing the fused umuC-lacZ gene construct in the bacteria. The following lines of evidence support the view that both NDMA and NDEA are principally oxidized to reactive products by P450 2E1 in rat liver microsomes. First, NDMA and NDEA were activated by rat liver microsomes in a protein- and substrate-dependent manner and the former chemical was more active than the latter; both activities were induced in rats treated with P450 2E1 inducers such as ethanol, acetone and isoniazid and by starvation. Second, activation of NDMA and NDEA were both inhibited significantly by antibodies raised against rat P450 2E1 and by P450 2E1 inhibitors such as diethyldithiocarbamate and 4-methylpyrazole in rat liver microsomes. Finally, in reconstituted monooxygenase systems containing purified rat P450 enzymes, P450 2E1 gave the highest rates of the activation of both NDMA and NDEA; the addition of rabbit cytochrome b5 to the system caused about a 1.5-fold increase in both reactions. In separate experiments we also found that N-nitrosomethylacethoxymethylamine, a compound that reacts with DNA after ester cleavage, is more genotoxic in S.typhimurium NM2009 than in S.typhimurium NM2000, a strain that is defective in O-acetyltransferase activity. Part of the pathway involved in the activation of nitrosamines is suggested to be acetylation of alkyldiazohydroxides formed by P450 or acetylesterase, because the genotoxic activity of N-nitrosomethylacethoxymethylamine in S.typhimurium NM2009 could be inhibited by the O-acetyltransferase inhibitor pentachlorophenol. These results indicate that NDMA and NDEA are oxidized to gentoxoic products by rat liver microsomes and that a P450 2E1 enzyme plays a major role in the activation of these two potent carcinogens. The activation pathway of N-nitrosodialkylamines through acetylation by O-acetyltransferase has been proposed. This simple bacterial system for measuring genotoxicity should facilitate studies on the activation of N-nitroso alkylamines.
Uptake of methionine, a-aminoisobutyric acid, and a-(methylamino)-isobutyric acid has been shown to occur by at least two transport systems, one sensitive and the other insensitive to the Na+ concentration. For a-aminoisobutyric acid and its N-methyl derivative, the Na+-insensitive uptake is not concentrative and its rate increases almost linearly with concentration within the range examined. In contrast, the Na+-insensitive uptake of methionine is concentrative and subject to inhibition by such amino acids as phenylalanine, leucine, and valine, although not in a manner to indicate that the uptake is mediated by a single agency. This component is not produced by a residual operation of the Na+-requiring transport system, handicapped by the absence of Na+ or by its having combined with a-aminoisobutyric acid. The increase in the rate of methionine uptake is linear with concentration only above about 16 mM methionine. The Na+-sensitive uptakes of methionine, a-aminoisobutyric, and a-(methylamino)-isobutyric acid appear to occur by the same population of transport-mediating sites. Both Km and Vm,, of the Na+ -sensitive uptake of these three amino acids change with changes in the concentration of Na+, an effect which is shown to have a theoretical basis. A similarity in the values of Vm, for ten amino acids entering principally by the Na + -sensitive agency indicates that differences in their Km values probably measure differences in their affinities for that transport-mediating system.
Recent studies indicate that cytochrome P450 (P450) 3A4 plays important roles in the activation of procarcinogens such as aflatoxin B1 and sterigmatocystin, as well as in the oxidation of a number of structurally diverse chemicals and endogenous compounds. Since P450 3A5 has been reported to be present at significant levels in liver microsomes in approximately 25% of human adults, we examined and compared the role of P450 3A4 and 3A5 in procarcinogen activation in humans. Immunoblot experiments with liver microsomes from 60 human samples suggested that 4/30 Japanese and 4/30 Caucasians contained considerable levels of P450 3A5, although P450 3A4 could be determined at relatively high levels in all of the human samples examined. Good correlation was observed between P450 3A4, but not P450 3A5, levels versus activation of aflatoxin B1 and stergmatocystin in these human samples. Comparisons of the activation of procarcinogens in reconstituted monooxygenase systems containing modified P450 3A4 and 3A5 enzymes expressed in Escherichia coli were carried out in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strain for genotoxicity assay, and it was found that P450 3A4 had similar activities to or higher rates than P450 3A5 for the 24 procarcinogens tested.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.