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PloS One 2014The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the...
The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.
Topics: Bacterial Proteins; Isocitrate Dehydrogenase; Ketoglutaric Acids; NADP; Oxidation-Reduction; Prochlorococcus
PubMed: 25061751
DOI: 10.1371/journal.pone.0103380 -
Cardiovascular Research Feb 2013Post-translational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is cardioprotective but its role in cardioprotection by remote ischaemic...
AIMS
Post-translational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is cardioprotective but its role in cardioprotection by remote ischaemic preconditioning (rIPC) and the reduced efficacy of rIPC in type 2 diabetes mellitus is unknown. In this study we achieved mechanistic insight into the remote stimulus mediating and the target organ response eliciting the cardioprotective effect by rIPC in non-diabetic and diabetic myocardium and the influence of O-GlcNAcylation.
METHODS AND RESULTS
The cardioprotective capacity and the influence on myocardial O-GlcNAc levels of plasma dialysate from eight healthy volunteers and eight type 2 diabetic patients drawn before and after subjection to an rIPC stimulus were tested on human isolated atrial trabeculae subjected to ischaemia/reperfusion injury. Dialysate from healthy volunteers exposed to rIPC improved post-ischaemic haemodynamic recovery (40 ± 6 vs. 16 ± 2%; P < 0.01) and increased myocardial O-GlcNAc levels. Similar observations were made with dialysate from diabetic patients before exposure to rIPC (43 ± 3 vs. 16 ± 2%; P < 0.001) but no additional cardioprotection or further increase in O-GlcNAc levels was achieved by perfusion with dialysate after exposure to rIPC (44 ± 4 and 42 ± 5 vs. 43 ± 3%; P = 0.7). The glutamine:fructose-6-phosphate amidotransferase (GFAT) inhibitor azaserine abolished the cardioprotective effects and the increment in myocardial O-GlcNAc levels afforded by plasma from diabetic patients and healthy volunteers treated with rIPC.
CONCLUSIONS
rIPC and diabetes mellitus per se influence myocardial O-GlcNAc levels through circulating humoral factors. O-GlcNAc signalling participates in mediating rIPC-induced cardioprotection and maintaining a state of inherent chronic activation of cardioprotection in diabetic myocardium, restricting it from further protection by rIPC.
Topics: Acetylglucosamine; Aged; Diabetes Mellitus, Type 2; Female; Hemodynamics; Humans; Ischemic Preconditioning, Myocardial; Male; Middle Aged; N-Acetylglucosaminyltransferases; beta-N-Acetylhexosaminidases
PubMed: 23201773
DOI: 10.1093/cvr/cvs337 -
Frontiers in Microbiology 2017Previous studies showed differences in the regulatory response to C/N balance in with respect to other cyanobacteria, but no information was available about its causes,...
Previous studies showed differences in the regulatory response to C/N balance in with respect to other cyanobacteria, but no information was available about its causes, or the ecological advantages conferred to thrive in oligotrophic environments. We addressed the changes in key enzymes (glutamine synthetase, isocitrate dehydrogenase) and the gene (the global nitrogen regulator) involved in C/N metabolism and its regulation, in three model strains: MED4, SS120, and MIT9313. We observed a remarkable level of diversity in their response to azaserine, a glutamate synthase inhibitor which increases the concentration of the key metabolite 2-oxoglutarate, used to sense the C/N balance by cyanobacteria. Besides, we studied the binding between the global nitrogen regulator (NtcA) and the promoter of the gene in the same strains, and its dependence on the 2-oxoglutarate concentration, by using isothermal titration calorimetry, surface plasmon resonance, and electrophoretic mobility shift. Our results show a reduction in the responsiveness of NtcA to 2-oxoglutarate in , especially in the MED4 and SS120 strains. This suggests a trend to streamline the regulation of C/N metabolism in late-branching strains (MED4 and SS120), in adaptation to the rather stable conditions found in the oligotrophic ocean gyres where this microorganism is most abundant.
PubMed: 29375510
DOI: 10.3389/fmicb.2017.02641 -
Molecular and Cellular Biology Jan 1986HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase,...
HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).
Topics: Alleles; Azacitidine; Azaserine; Ethyl Methanesulfonate; Genes; HeLa Cells; Humans; Hypoxanthine Phosphoribosyltransferase; Methylation; Methylnitronitrosoguanidine; Mutation; Thioguanine
PubMed: 2431268
DOI: 10.1128/mcb.6.1.97-104.1986 -
Chemical Science Aug 2023Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative...
characterization of nonribosomal peptide synthetase-dependent -(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine.
Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative azaserine biosynthetic gene () cluster, which contains 21 genes, including those responsible for hydrazinoacetic acid (HAA) synthesis, was discovered using bioinformatics analysis of the genome. Azaserine was produced by the heterologous expression of the cluster in . enzyme assays using recombinant Azs proteins revealed the azaserine biosynthetic pathway as follows. AzsSPTF and carrier protein (CP) AzsQ are used to synthesize the 2-hydrazineylideneacetyl (HDA) moiety attached to AzsQ from HAA. AzsD transfers the HDA moiety to the C-terminal CP domain of AzsN. The heterocyclization (Cy) domain of the nonribosomal peptide synthetase AzsO synthesizes -(2-hydrazineylideneacetyl)serine (HDA-Ser) attached to its CP domain from l-serine and HDA moiety-attached AzsN. The thioesterase AzsB hydrolyzes it to yield HDA-Ser, which appears to be converted to azaserine by oxidation. Bioinformatics analysis of the Cy domain of AzsO showed that it has a conserved DxxxxD motif; however, two conserved amino acid residues (Thr and Asp) important for heterocyclization are substituted for Asn. Site-directed mutagenesis of two Asp residues in the DxxxxD motif (D193 and D198) and two substituted Asn residues (N414 and N447) indicated that these four residues are important for ester bond synthesis. These results showed that the diazo ester of azasrine is synthesized by the stepwise oxidation of the HAA moiety and provided another strategy to biosynthesize the diazo group.
PubMed: 37621439
DOI: 10.1039/d3sc01906c -
Lipids in Health and Disease Sep 2015We previously demonstrated that hyperglycemia could suppress apolipoprotein M (apoM) synthesis both in vivo and in vitro; however, the mechanism of hyperglycemia-induced...
BACKGROUND
We previously demonstrated that hyperglycemia could suppress apolipoprotein M (apoM) synthesis both in vivo and in vitro; however, the mechanism of hyperglycemia-induced downregulation of apoM expression is unknown yet.
METHODS
In the present study we further examined if hexosamine pathway, one of the most important pathways of glucose turnover, being involved in modulating apoM expression in the hyperglycemia condition. We examined the effect of glucosamine, a prominent component of hexosamine pathway and intracellular mediator of insulin resistance, on apoM expression in HepG2 cells and in rat's models. In the present study we also determined apolipoprotein A1 (apoA1) as a control gene.
RESULTS
Our results demonstrated that glucosamine could even up-regulate both apoM and apoA1 expressions in HepG2 cell cultures. The glucosamine induced upregulation of apoM expression could be blocked by addition of azaserine, an inhibitor of hexosamine pathway. Moreover, intravenous infusion of glucosamine could enhance hepatic apoM expression in rats, although serum apoM levels were not significantly influences.
CONCLUSIONS
It is concluded that both exogenous and endogenous glucosamine were essential for the over-expression of apoM, which may suggest that the increased intracellular content of glucosamine does not be responsible for the depressed apoM expression at hyperglycemia condition.
Topics: Animals; Antimetabolites, Antineoplastic; Apolipoprotein A-I; Apolipoproteins; Apolipoproteins M; Azaserine; Gene Expression Regulation; Glucosamine; Hep G2 Cells; Humans; Hyperglycemia; Infusions, Intravenous; Lipocalins; Liver; Male; Rats; Rats, Sprague-Dawley; Signal Transduction
PubMed: 26377577
DOI: 10.1186/s12944-015-0103-5 -
The Biochemical Journal Jun 19731. The effects of azaserine and nicotinamide, agents which inhibit and stimulate hepatic NAD synthesis respectively, on the content of NAD(+) and NADH in isolated rat...
1. The effects of azaserine and nicotinamide, agents which inhibit and stimulate hepatic NAD synthesis respectively, on the content of NAD(+) and NADH in isolated rat islets of Langerhans incubated in vitro were studied. The effects of these compounds on the rates of insulin release, from isolated islets incubated in vitro, in response to various secretagogues were also measured. 2. Preincubation of islets in the presence of azaserine (0.3mm) caused a marked depletion of the normal islet-cell content of both NAD(+) and NADH and prevented the secretion of insulin in response to stimulatory concentrations of d-glucose, xylitol, d-xylulose, l-arginine hydrochloride and l-leucine. 3. Preincubation of islets in the presence of nicotinamide (2mm) increased the islet content of NAD(+) and enhanced the rate of release of insulin in response to d-glucose. Also when nicotinamide was present the inhibitory effect of azaserine on insulin release and the azaserine-induced depletion of the islet content of NAD(+) and NADH was prevented. 4. Preincubation with azaserine was without effect on the stimulation of insulin release caused by theophylline or dibutyryl cyclic AMP. 5. It is suggested that insulin release caused by sugars and amino acids is dependent on the maintenance of NAD concentrations, though this may not be the case for release due to theophylline and dibutyryl cyclic AMP.
PubMed: 16742817
DOI: 10.1042/bj1340557 -
Oncotarget Dec 2016The hexosamine biosynthetic pathway (HBP) requires two key nutrients glucose and glutamine for O-linked N-acetylglucosamine (O-GlcNAc) cycling, a post-translational...
The hexosamine biosynthetic pathway (HBP) requires two key nutrients glucose and glutamine for O-linked N-acetylglucosamine (O-GlcNAc) cycling, a post-translational protein modification that adds GlcNAc to nuclear and cytoplasmic proteins. Increased GlcNAc has been linked to regulatory factors involved in cancer cell growth and survival. However, the biological significance of GlcNAc in diffuse large B-cell lymphoma (DLBCL) is not well defined. This study is the first to show that both the substrate and the endpoint O-GlcNAc transferase (OGT) enzyme of the HBP were highly expressed in DLBCL cell lines and in patient tumors compared with normal B-lymphocytes. Notably, high OGT mRNA levels were associated with poor survival of DLBCL patients. Targeting OGT via small interference RNA in DLBCL cells inhibited activation of GlcNAc, nuclear factor kappa B (NF-κB), and nuclear factor of activated T-cells 1 (NFATc1), as well as cell growth. Depleting both glucose and glutamine in DLBCL cells or treating them with an HBP inhibitor (azaserine) diminished O-GlcNAc protein substrate, inhibited constitutive NF-κB and NFATc1 activation, and induced G0/G1 cell-cycle arrest and apoptosis. Replenishing glucose-and glutamine-deprived DLBCL cells with a synthetic glucose analog (ethylenedicysteine-N-acetylglucosamine [ECG]) reversed these phenotypes. Finally, we showed in both in vitro and in vivo murine models that DLBCL cells easily take up radiolabeled technetium-99m-ECG conjugate. These findings suggest that targeting the HBP has therapeutic relevance for DLBCL and underscores the imaging potential of the glucosamine analog ECG in DLBCL.
Topics: Acetylglucosamine; Animals; Antineoplastic Agents; Apoptosis; Azaserine; Cell Line, Tumor; Cell Proliferation; Contrast Media; Cysteine; Enzyme Inhibitors; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glycosylation; Hexosamines; Humans; Lymphoma, Large B-Cell, Diffuse; Mice, Inbred NOD; Mice, SCID; N-Acetylglucosaminyltransferases; NF-kappa B; NFATC Transcription Factors; Organotechnetium Compounds; RNA Interference; RNA, Messenger; RNAi Therapeutics; Signal Transduction; Transfection
PubMed: 27716624
DOI: 10.18632/oncotarget.12413 -
The Biochemical Journal Jan 2001L-Glutamine is a physiological inhibitor of endothelial NO synthesis. The present study was conducted to test the hypothesis that metabolism of glutamine to glucosamine...
L-Glutamine is a physiological inhibitor of endothelial NO synthesis. The present study was conducted to test the hypothesis that metabolism of glutamine to glucosamine is necessary for glutamine inhibition of endothelial NO generation. Bovine venular endothelial cells were cultured for 24 h in the presence of 0, 0.1, 0.5 or 2 mM D-glucosamine, or of 0.2 or 2 mM L-glutamine with or without 20 microM 6-diazo-5-oxo-L-norleucine (DON) or with 100 microM azaserine. Both DON and azaserine are inhibitors of L-glutamine:D-fructose-6-phosphate transaminase (isomerizing) (EC 2.6.1.16), the first and rate controlling enzyme in glucosamine synthesis. Glucosamine at 0.1, 0.5 and 2 mM decreased NO production by 34, 45 and 56% respectively compared with controls where glucosamine was lacking. DON (20 microM) and azaserine (100 microM) blocked glucosamine synthesis and prevented the inhibition of NO generation by glutamine. Neither glutamine nor glucosamine had an effect on NO synthase (NOS) activity, arginine transport or cellular tetrahydrobiopterin and Ca(2+) levels. However, both glutamine and glucosamine inhibited pentose cycle activity and decreased cellular NADPH concentrations; these effects of glutamine were abolished by DON or azaserine. Restoration of cellular NADPH levels by the addition of 1 mM citrate also prevented the inhibiting effect of glutamine or glucosamine on NO synthesis. A further increase in cellular NADPH levels by the addition of 5 mM citrate resulted in greater production of NO. Collectively, our results demonstrate that the metabolism of glutamine to glucosamine is necessary for the inhibition of endothelial NO generation by glutamine. Glucosamine reduces the cellular availability of NADPH (an essential cofactor for NOS) by inhibiting pentose cycle activity, and this may be a metabolic basis for the inhibition of endothelial NO synthesis by glucosamine.
Topics: Animals; Calcium; Cattle; Cells, Cultured; Endothelium, Vascular; Glucosamine; Glutamine; Models, Chemical; NADP; Nitric Oxide; Nitric Oxide Synthase; Pentose Phosphate Pathway
PubMed: 11139387
DOI: 10.1042/0264-6021:3530245 -
American Journal of Physiology. Cell... Jan 2007Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to...
Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in protein O-GlcNAcylation resulted in improved survival and viability following ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of ischemia and 16 h of reperfusion, and cell viability, necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with glucosamine, hyperglycemia, or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate(PUGNAc), an inhibitor of O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both necrosis and apoptosis compared with untreated cells following I/R. Alloxan, an inhibitor of O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with hyperglycemia was attenuated by azaserine, which inhibits glucose metabolism via the hexosamine biosynthesis pathway. Reperfusion in the absence of glucose reduced O-GlcNAc levels on reperfusion compared with normal glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of glucosamine and PUGNAc on cellular viability were associated with reduced calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca(2+). These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection.
Topics: Acetylglucosamine; Animals; Animals, Newborn; Apoptosis; Biological Transport; Cell Nucleus; Cell Survival; Cells, Cultured; Glucosamine; Glycoproteins; Glycosylation; Heart; Heart Ventricles; Hexosamines; Hyperglycemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; NFATC Transcription Factors; Necrosis; Rats; Rats, Sprague-Dawley; Time Factors
PubMed: 16899550
DOI: 10.1152/ajpcell.00162.2006