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At least 667 records · Page 37Linked to original sources
PubMed2000

Retrospective evaluation of serum ornithine carbamyltransferase activity as an index of hepatotoxicity in toxicological studies with rats.

The sensitivity of elevated serum ornithine carbamyltransferase (OCT) as an index of hepatotoxicity in rats was assessed in different studies conducted over a number of years and originally designed to examine the toxicity or carcinogenicity of a variety of test chemicals and diets. Changes in serum OCT activities were compared with the more widely used clinical endpoints, alanine aminotranserase (ALT) and aspartate aminotransferase (AST). In the first study, rats received a single oral dose of the hepatotoxic and hepatocarcinogenic fungal toxin aflatoxin B(1) (AFB(1)). The increase in enzyme levels between control and AFB(1)-treated rats was greater for serum OCT than for ALT or AST. This response was similar to the changes in serum enzyme levels in studies where rats ingested a hepatotoxic and hepatocarcinogenic choline deficient (CD) diet. When rats were exposed to the hepatotoxic and nephrotoxic fungal toxin fumonisin B(1) (FB(1)) by intraperitoneal injection for 6 days, serum AST and ALT were significantly elevated above control levels while OCT was unaffected. The peroxisome proliferator ciprofibrate caused elevated ALT and AST but not OCT at week 52 of dietary exposure, after the development of liver nodules and tumours. Of the two liver-specific enzymes examined in all of the studies, ALT was more consistently predictive of hepatotoxicity than OCT.

Administration, Oral↗
PubMed2000

Glutamine metabolism in endothelial cells: ornithine synthesis from glutamine via pyrroline-5-carboxylate synthase.

L-Glutamine (the most abundant free amino acid in plasma and the body) is a potent inhibitor of endothelial NO synthesis. However, little is known about glutamine metabolism in endothelial cells (EC). As an initial step toward understanding the role of glutamine in endothelial physiology, the present study was conducted to quantify glutamine catabolism in microvascular, aortic and venous EC. For metabolic studies, EC were incubated for 1 h in Krebs bicarbonate buffer containing 5 mM glucose and 0.5-4 mM L-[U-(14)C]-glutamine. For enzymological studies, cell extracts and mitochondrial fractions were prepared to determine the activities of glutamine-degrading enzymes. Our results reveal extensive hydrolysis of glutamine to glutamate and ammonia in a concentration-dependent manner via phosphate-dependent glutaminase in all EC studied. In addition, both metabolic and enzymological evidence indicate a novel pathway for endothelial synthesis of ornithine from glutamine via pyrroline-5-carboxylate synthase. This new knowledge of glutamine metabolism may pave a new path for understanding the physiological role of glutamine in vascular function.

Alanine↗
PubMed2001

Mechanism for acivicin inactivation of triad glutamine amidotransferases.

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.

Amino Acid Sequence↗
PubMed1998

Effects of cholecystokinin octapeptide on a pancreatic acinar carcinoma in the rat.

PURPOSE: To investigate the effects of increasing concentrations of cholecystokinin octapeptide (CCK-8) on a pancreatic acinar adenocarcinoma. METHODS: Growth of the tumour was estimated in vivo on rats bearing a subcutaneous pancreatic carcinoma, and in vitro on primary cultured tumour cells. CCK receptors were characterized by binding assays. RESULTS: CCK-8, administered for 12 successive days, exerted a biphasic action on tumour growth: a dose-dependent stimulation with low doses (0.1 and 0.5 microg/kg) and inhibition with high doses (2 and 4 microg/ kg) as shown by respective increases and decreases in tumor volume, protein, RNA and amylase contents. In cell cultures, [3H]thymidine incorporation was dose-dependently increased with 10-(10) to 10(-8) M CCK-8 and inhibited with 10(-7) M. Both effects were completely suppressed by the CCK-receptor antagonists CR 1409 and L 364,718 (10(-4) M). Binding studies showed the overexpression of two classes of CCK-A receptors of low and high affinity when compared to the normal pancreas which was less sensitive to CCK-8. CONCLUSIONS: CCK-8 exerts a biphasic growth response on the acinar pancreatic carcinoma, mediated by two classes of CCK-A receptors overexpressed in the tumour.

Animals↗
PubMed1991

Purification and partial characterization of glutamate synthase from Rhodospirillum rubrum grown under nitrogen-fixing conditions.

Glutamate synthase, a key enzyme in ammonia assimilation, has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The purification procedure involves ion-exchange chromatography, affinity chromatography and gel filtration. The recovery in the procedure is high (62%) and the specific activity is 21 mumol of NADPH oxidized/min per mg. The enzyme is specific for its substrates, and no activity was demonstrated with NADH or NH4+ ions substituting for NADPH and glutamine respectively. The enzyme is composed of two dissimilar subunits with molecular masses of 53 and 152 kDa, and it is shown that Cl- ions have an effect on the aggregation of the enzyme. Km values for the substrates are: NADPH, 16 microM; 2-oxoglutarate, 10 microM; and glutamine, 65 microM. The enzyme is inhibited by amidotransferase inhibitors at micromolar concentrations. The role of the enzyme in the metabolic regulation of nitrogenase is discussed.

Amino Acids↗
PubMed2000

Sterol regulatory element-binding protein-1 is regulated by glucose at the transcriptional level.

In vivo studies suggest that sterol regulatory element-binding protein (SREBP)-1 plays a key role in the up-regulation of lipogenic genes in the livers of animals that have consumed excess amounts of carbohydrates. In light of this, we sought to use an established mouse hepatocyte cell line, H2-35, to further define the mechanism by which glucose regulates nuclear SREBP-1 levels. First, we show that these cells transcribe high levels of SREBP-1c that are increased 4-fold upon differentiation from a prehepatocyte to a hepatocyte phenotype, making them an ideal cell culture model for the study of SREBP-1c induction. Second, we demonstrate that the presence of precursor and mature forms of SREBP-1 protein are positively regulated by medium glucose concentrations ranging from 5. 5 to 25 mm and are also regulated by insulin, with the amount of insulin in the fetal bovine serum being sufficient for maximal stimulation of SREBP-1 expression. Third, we show that the increase in SREBP-1 protein is due to an increase in SREBP-1 mRNA. Reporter gene analysis of the SREBP-1c promoter demonstrated a glucose-dependent induction of transcription. In contrast, expression of a fixed amount of the precursor form of SREBP-1c protein showed that glucose does not influence its cleavage. Fourth, we demonstrate that the glucose induction of SREBP could not be reproduced by fructose, xylose, or galactose nor by glucose analogs 2-deoxy glucose and 3-O-methyl glucopyranose. These data provide strong evidence for the induction of SREBP-1c mRNA by glucose leading to increased mature protein in the nucleus, thus providing a potential mechanism for the up-regulation of lipogenic genes by glucose in vivo.

Animals↗
PubMedDate not supplied

Quantifying the toxic and mutagenic activity of complex mixtures with Salmonella typhimurium.

The toxicity and mutagenicity of 11 compounds individually and in mixtures were quantified in Salmonella typhimurium strains TA98, TA100, and TA1537 by a modification of the Ames spot test. The distance (millimeters) from the center of the petri dish to the bacterial growth front represented the toxic response. When mutagenicity occurred, the distance from the inner radius to the outer radius of the mutagenic growth represented the mutagenic response. Multiple regression analysis was used to quantify the toxicity and mutagenicity of individual compounds in the mixtures. The results indicate that the effects of compounds in mixtures are generally additive.

Azaserine↗
PubMed1967

Suppression of murine leukemias by L-asparaginase. Incidence of sensitivity among leukemias of various types: comparative inhibitory activities of guinea pig serum L-asparaginase and Escherichia coli L-asparaginase.

A survey of 109 recently derived leukemias of the mouse revealed that sensitivity to suppression by guinea pig serum is a common property of transplanted leukemias of certain classes. The sensitive leukemias included five that arose spontaneously in mice of strains with a low incidence of leukemia and 21 that were induced by X-radiation. Two GPS-sensitive leukemias were not more sensitive than a GPS-resistant leukemia to a range of standard chemotherapeutic agents. The effectiveness of L-asparaginase EC-2 from Escherichia coli in suppression of the GPS-sensitive leukemia EARAD1 depends upon the conditions of assay. Whereas it is not inhibitory when administered as a single dose at the time of inoculation of the leukemia it is considerably more effective than GPS when used in the treatment of established leukemia. Permanent cures of 7-day generalized transplants of EARAD1 can be effected by the administration of 2000 or more units of EC-2. Immunological factors apparently do not contribute to cure as treated survivors are fully susceptible to rechallenge with minimal numbers of cells from the same leukemia. Reinoculated survivors with progressively growing transplants have been successfully retreated with EC-2. The blood clearance of EC-2 L-asparaginase injected into mice is much more rapid than that of GPS L-asparaginase. After intraperitoneal inoculation of the EC-1 L-asparaginase, which does not have leukemia-inhibitory activity, only very low levels of enzyme activity could be detected in the serum. The effectiveness of EC-2 from E. coli and its availability from a virtually limitless source will make it possible to extend the study of inhibition of leukemias and other tumors by L-asparaginase to species other than small rodents.

Amidohydrolases↗
PubMed1998

The primary function of a redundant Sp1 binding site in the mouse aprt gene promoter is to block epigenetic gene inactivation.

The promoter region of the mouse adenine phosphoribosyltransferase (aprt) gene contains one non-consensus Sp1 binding site at its 5' end followed by three consensus Sp1 binding sites. The two 3'-most binding sites are sufficient for maximal expression of aprt , suggesting that the non-consensus and consensus binding sites at the 5' end are redundant. However, the two 3' sites are not sufficient to block epigenetic inactivation, which led to the hypothesis that the redundant consensus and/or non-consensus 5' Sp1 binding sites are required to block inactivation events. To test this hypothesis, promoter region constructs were made in which the two 5' Sp1 binding sites were mutated alone or in tandem, and then each construct was tested for its ability to withstand epigenetic inactivation. A cis -acting methylation center that is normally located 1.2 kb upstream of the promoter was used to induce inactivation. The results demonstrate that the presence of the redundant consensus Sp1 binding site is required to block methylation-associated gene inactivation. Therefore, the Sp1 binding sites comprising the mouse aprt promoter have evolved two distinct functions, one to promote transcription and the other to block epigenetic inactivation.

Adenine↗
PubMed2006

Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco.

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.

Amides↗