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At least 343 records · Page 19Linked to original sources
PubMed1992

Molecular and biochemical elucidation of a cellular phenotype characterized by adenine analogue resistance in the presence of high levels of adenine phosphoribosyltransferase activity.

A mouse embryonal carcinoma cell line isolated for resistance to the adenine analogue 2,6-diaminopurine (DAP) was found to have near-wild-type levels of adenine phosphoribosyltransferase (APRT) activity in a cell-free assay. This DAP-resistant (DAPr) cell line, termed H29D1, also exhibited near-wild-type levels of adenine accumulation and the ability to grow in medium containing azaserine and adenine. Growth in this medium requires high levels of intracellular APRT activity. Using the polymerase chain reaction (PCR) and the dideoxy chain termination sequencing technique, an A-->G transition was discovered in exon 3 of the aprt gene in H29D1. This mutation resulted in an Arg-to-Gln change at amino acid 87 of the APRT protein that, in turn, resulted in a decreased affinity for adenine. An increased sensitivity of APRT to inhibition by AMP was observed when comparing H29D1 to P19, the parental cell line. Using a transgene containing the A-->G mutation, we demonstrated that this mutation is responsible for the biochemical and cellular phenotypes observed for the H29D1 cell line. The approach used in this study provides a definitive method for linking a mutation to a specific cellular phenotype.

2-Aminopurine↗
PubMed2002

Glutamate synthesis in barley roots: the role of the plastidic glucose-6-phosphate dehydrogenase.

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.

Adenosine Triphosphate↗
PubMed1977

Evidence for cycloheximide acting as a glutamine analogue in plant tissue.

In growing maize root tissue [14C]asparagine formation in inhibited and [14C]glutamine accumulation stimulated by treatment with cycloheximide or glutamine analogs such as azaserine. In contrast, puromycin enhances the accumulation of [14C]asparagine but not [14C]glutamine. Cycloheximide and puromycin alone inhibit protein synthesis. This is interpreted to mean that the alteration in amide metabolism following cycloheximide treatment is a direct result of the antibiotic acting as a glutamine analog. While cycloheximide is often the cytoplasmic protein synthesis inhibitor of choice due to its potency and rapid action, its assumed specificity of action of eukaryotes is doubtful.

Asparagine↗
PubMed1981

Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. I. Inhibitors of de novo purine synthesis and a comparison with the effects of caffeine.

The effect of pre- and posttreatment incubation of UV-irradiated and ethyl methanesulphonate (EMS) treated cells with non-toxic concentrations of inhibitors of de novo purine synthesis (dnPS) on expression of potentially lethal and premutational damage at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 cells has been examined. The concentrations of inhibitors used were shown to profoundly perturb de novo DNA synthesis, by measurements of [14C]formate uptake, and cell cycle progression by flow cytofluorimetry. Postincubation in 6-methyl mercapto-purine ribonucleoside (MMPR) usually but not invariably potentiated the cytotoxic effect of UV and EMS but azaserine (AZS) and methotrexate (MTX) were without effect. No effects on mutant frequencies were observed on posttreatment with any of these agents. Caffeine produced the least effect on dnPS, but invariably potentiated lethal damage. This potentiation of lethal damage is not mediated by dnPS inhibition as has been suggested for Chinese hamster ovary (CHO) cells.

Animals↗
PubMed1985

An improved assay for the detection of interleukin 1.

A 24 h, highly sensitive assay for detection of interleukin 1 (IL-1) is described. A thioguanine-resistant mutant of the murine lymphoma cell line LBRM 33 was selected (LBRM TG6). When this cell line was incubated with low concentrations of PHA and IL-1 it produced interleukin 2 (IL-2). IL-2-dependent HT2 cells were co-cultured with the LBRM cells to measure the released IL-2. Prior to addition of tritiated thymidine to the co-culture, hypoxanthine and azaserine were added to metabolically block DNA synthesis by LBRM TG6 cells. This resulted in a sensitive, short term assay requiring minimal technical manipulations and characterized by a high signal to noise ratio.

Animals↗
PubMed1979

Relationship between cytotoxicity and induction of sister-chromatid exchanges in mouse foetal cells exposed to several doses of carcinogenic and non-carcinogenic chemicals.

The increase in sister-chromatid exchanges induced by 5 chemicals, with different DNA damaging and carcinogenic activities, was studied in short-term foetal-mouse cultures. A significant increase in SCE was induced by N-methyl-N'-nitro-N-nitrosoguanidine, N-diazoacetylglycine-amide, azaserine and methotrexate. k-Strophantin, on the contrary, was totally inactive. On a molar basis, MNNG was the most active chemical followed by MTX, AZS and DGA, in that order. At equitoxic concentrations (D37), the order of SCE-inducing abilities was MNNG, DGA, AZS and MTX. Compared with previous data, at equitoxic concentrations, the most DNA-damaging agents were also the most effective in inducing SCE. The SCE increase seems to correlate not with unspecific cytotoxicity but more with DNA damage or other damage at the genome level. MTX, a non-mutagen, which induced SCE only at toxic levels, could be considered a false positive because this positivity may reflect an enhancement of incorporation of 5-BrdUrd into DNA. The positive results obtained with AZS suggest a sufficient sensitivity of the method for detecting relatively weak carcinogens.

Animals↗
PubMed1985

Enzyme targets of antiglutamine agents in cancer chemotherapy.

The modes of action of azaserine and acivicin were compared. The results were evaluated by assessing the impact of these drugs on primary targets, the activities of key enzymes, and on secondary and tertiary targets, the concentrations of pools of ribonucleotides and deoxyribonucleotides. It was observed that both drugs act as competitive inhibitors for glutamine-utilizing enzymes involved in the biosynthesis of purines and pyrimidines, but in addition acivicin exerts a direct inactivating effect (probably by alkylation) on the enzymes. The different tissues examined displayed varying sensitivity to the drugs which may be attributed in part at least to the tissue glutamine content. Acivicin markedly depleted the CTP pools, but ATP and UTP were unaffected. It also decreased the concentration of all 4 deoxynucleoside triphosphates. These biochemical targets serve as indicators of acivicin action in cancer cells and should also be helpful in the design of combination chemotherapy. On the basis of the biochemical action of acivicin, actinomycin and dipyridamole were selected for testing in combination chemotherapy. Both drugs acted synergistically with acivicin.

Animals↗
PubMed1984

Salvage of 5'-deoxy-methylthioadenosine into purines and methionine by lymphoid cells and inhibition of cell proliferation.

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine metabolism, is a potent inhibitor of cell proliferation. MTA phosphorylase cleaves MTA into adenine and 5'-methylthioribose-1-P. We studied MTA inhibition and salvage into purine compounds and methionine in concanavalin A-stimulated rat T lymphocytes and in Raji cells. When de novo purine synthesis was inhibited by azaserine (20 microM), low concentrations of MTA, (less than or equal to 20 microM), were able to completely restore cell proliferation in both types of cells. When cells were cultured in a methionine-free medium, MTA (15 microM) completely fulfilled the methionine requirement of Raji cells but only 50% of that of rat T lymphocytes. MTA displayed a dose-dependent inhibition of the proliferation of both types of cells, but in the case of MTA salvage into purines or methionine, the curves were shifted to higher MTA concentrations. In vitro studies by Backlund et al. (Backlund, P.S., Chang, C.P. and Smith, R.A. (1982) J. Biol. Chem. 257, 4196-4202) on rat liver homogenates, suggested that the last step of MTA salvage into methionine may be the transamination of 2-keto-4-methylthiobutyrate to methionine. We present evidence that this is a step physiologically efficient in intact cells.

Adenosine↗
PubMed1988

Estradiol regulation of the synthesis of uterine proteins with clusters of proline- and glycine-rich peptide sequences.

Estradiol (E2) regulates the synthesis of uterine proteins at both the transcriptional and translational levels. E2 induces an increase in the specific amino acid acceptor activity of uterine tRNA, with the largest increases seen for proline, glycine and methionine. The synthesis of three uterine proteins that are rich in proline and glycine, estrogen receptor, progesterone receptor and glucose-6-phosphate dehydrogenase, is induced by E2. E2-induced increases in these proteins were preceded by an correlated with stimulation of tRNA acceptor activity for proline and glycine and these responses were specifically and simultaneously inhibited by prior azaserine treatment, which inhibits the E2-induced repair and synthesis of the 3'-CCA acceptor terminus of tRNAs. The high frequency and clustering of proline and glycine residues in estrogen receptor, progesterone receptor and glucose-6-phosphate dehydrogenase suggests that the translating ribosomes may slow down during synthesis of these proteins due to limiting levels of these tRNAs in E2-deprived uteri.

Amino Acid Sequence↗
PubMed1996

15N-NMR study of ammonium assimilation in Agaricus bisporus.

Ammonium assimilation was studied by feeding [15N]ammonium to actively growing mycelium of Agaricus bisporus. Products of ammonium assimilation were analysed using 15N-NMR. Participation of glutamine synthetase, glutamate synthase and NADP-dependent glutamate dehydrogenase was determined by inhibiting glutamine synthetase with phosphinothricin and glutamate synthase with azaserine. Our results clearly indicate that, under the conditions used, ammonium assimilation is mainly catalysed by the enzymes of the glutamine synthetase/glutamate synthase pathway. No indications were found for participation of NADP-dependent glutamate dehydrogenase. Furthermore, 15N-labelling shows that transamination of glutamate with pyruvate to yield alanine is a major route in nitrogen metabolism. Another major route is the formation of N-acetylglucosamine. Compared to the formation of N-acetylglucosamine there was only a limited formation of arginine.

Agaricus↗
PubMed1990

Evaluation of promotion of pancreatic carcinogenesis in rats by benzyl acetate.

Benzyl acetate was found to induce liver tumours and gastric squamous neoplasms in mice in a chronic bioassay conducted through the National Toxicology Program. An increased incidence of acinar cell adenomas of the pancreas of F344 rats was noted in the bioassay, but the significance of these lesions was confounded because the benzyl acetate was given by gavage in corn oil. The use of corn oil as a vehicle has been shown to enhance the growth of such lesions in the rat pancreas. The current studies were undertaken to evaluate benzyl acetate alone as an initiator and promoter of carcinogenesis in the pancreas. Alkaline elution analysis of acinar cell DNA showed no evidence of damage 1 hr after administration of benzyl acetate. Significant stimulation of growth of azaserine-induced foci was observed in a 6-month study, and a low but significant incidence of carcinoma in situ was observed in rats fed benzyl acetate in the diet for 2 yr. These experiments suggest that benzyl acetate is a weak promoter of the growth of carcinogen-induced and spontaneous pre-neoplastic foci in the pancreas.

Adenoma↗
PubMed1985

Effects of melphalan on the development of experimental pancreatic cancer.

The effects of melphalan were studied in rats fed raw soya flour and injected with azaserine in order to determine the suitability of this experimental model for testing drugs potentially useful for the treatment of pancreatic cancer. While melphalan did not prevent or delay the development of pancreatic cancer in these rats, the drug significantly lessened the number and size of the premalignant proliferative lesions in the pancreas. It seems that the model is useful both for testing potentially useful therapeutic agents and for analysing some of the processes involved in the development of pancreatic cancer.

Animals↗
PubMed1986

Effects of feeding partial and intermittent raw soya flour diets on the rat pancreas.

Continuous administration of a diet consisting of raw soya flour produces pancreatic cancer in rats and sensitises the rat pancreas to the action of genotoxic carcinogens. We have therefore studied the effects of continuous feeding of diets containing lesser amounts of raw soya flour (5%, 25% and 50%) and feeding raw soya flour intermittently (2 days each week). The study has shown that a diet containing as little as 5% raw soya flour stimulates focal proliferation of the pancreatic acinar cells and sensitises to the action of azaserine. Similarly, intermittent feeding of raw soya flour induced focal proliferation of the acinar pancreas and, when 100% raw soya flour diet was fed for 2 days each week, resulted in the development of pancreatic cancer in some of the rats. We conclude that raw soya flour must be excluded from the diets of rats used in toxicological and carcinogenicity studies.

Adenoma↗
PubMedDate not supplied

Inhibition of dietary fat promoted development of (pre)neoplastic lesions in exocrine pancreas of rats and hamsters by supplemental selenium and beta-carotene.

The modulating effects of selenium and beta-carotene on the development of putative preneoplastic foci in exocrine pancreas were investigated in rats and hamsters, treated with azaserine and N-nitrosobis(2-oxopropyl)amine (BOP), respectively. The animals were fed a semipurified diet high in saturated fat (20% lard) either supplemented or not with beta-carotene or selenium. A separate group maintained on a diet low in saturated fat (5% lard) was incorporated as an extra control group. The animals were given their diets 12 days after the last treatment with carcinogen. Four months postinitiation, the pancreata were quantitatively examined for the number and size of putative (pre)neoplastic lesions. Rats as well as hamsters maintained on a low-fat diet had significantly less putative preneoplastic pancreatic lesions as compared to animals fed a diet containing 20% lard. Selenium and beta-carotene both inhibited the growth of basophilic foci in rat pancreas. In hamster pancreas, beta-carotene and selenium caused a significant decrease in the number of early ductal complexes. The number of carcinomas was decreased in the beta-carotene group and increased in the group fed a diet high in selenium.

Animals↗
PubMed1984

Consequences of the salvage of purine compounds on the proliferation of rat T-lymphocytes with normal or inhibited purine de novo synthesis.

We studied the ability of purine compounds to restore the proliferation of concanavalin-A-stimulated rat T-lymphocytes under conditions of purine de novo synthesis inhibition and, on the other hand, the inhibition by purine nucleosides of the response of these cells to a mitogenic stimulation under conditions of normal purine de novo synthesis. The use of 50 microM azaserine, a potent inhibitor of purine de novo synthesis, allowed us to define the physiologically active salvage pathways of purine bases, ribo- and deoxyribonucleosides in concanavalin-A-stimulated rat T-lymphocytes. Except for guanylic compounds, all purines completely restored cell proliferation at a concentration of 50 microM. Guanine, guanosine and 2'-deoxyguanosine at concentrations up to 500 microM did not allow us to restore more than 50% of the cell proliferation. In conditions of normal purine de novo synthesis, the addition of 1000 microM adenine, adenosine, 2'-deoxyadenosine or 100 microM 2'-deoxyguanosine inhibited rat T-lymphocyte proliferation. The differences between the degree of inhibition of cell proliferation could be explained only in part by the differences between the capacities of salvage of these compounds. Furthermore, the fact that 2'-deoxyguanosine toxicity was dependent and 2'-deoxyadenosine toxicity independent on the activation state of the cells provided more evidence that the biochemical mechanisms of inhibition of cell proliferation should be different for these two nucleosides.

AMP Deaminase↗
PubMed2006

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum: effects of metabolic inhibitors.

Poly-beta-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied in presence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation in phosphate-limited N. muscorum under photoautotrophic growth condition. PHB accumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dry weight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4 -dinitrophenol (DNP) supplementation depicted insignificant effect on PHB pool of the test cyanobacterium. Supplementation of l-methionine-dl-sulfoximine (MSX) and azaserine was also found to increase PHB accumulation in N(2) -fixing and NH(4)(+) -grown N. muscorum, but not in NO(3)(-) -grown cells. The stimulatory action of monofluoroacetate on PHB accumulation was suppressed in presence of alpha-ketoglutarate and DCMU. Interestingly, 2,3 -butanedione supplementation was not only found inhibitory for accumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grown N. muscorum but suppression of PHB synthesis was also evident in control cultures in presence of 2,3 -butanedione. The possible mechanisms are discussed.

Azaserine↗
PubMed2004

Nitrogen-15 NMR studies of nitrogen metabolism in Picea glauca buds.

In vivo (15)N nuclear magnetic resonance (NMR) as well as (15)N solid-state magic angle spinning (MAS) NMR spectroscopy were used to investigate nitrogen metabolism in cultured white spruce (Picea glauca) buds. Long-term as well as short-term experiments were carried out involving the use of inhibitors of the nitrogen pathways such as methionine sulfoximine (MSO), azaserine (AZA) and aminooxyacetate (AOA). Both in vivo and solid-state NMR showed that when MSO blocked glutamine synthetase (GS) no NH(4)(+) is incorporated. When glutamate synthase (GOGAT) is blocked by AZA there is some incorporation into glutamine (Gln), but very little into alpha-amino groups (glutamate, Glu). The transamination inhibitor AOA does not affect the metabolism of (15)NH(4)(+) into Gln and Glu, but blocks the production of arginine (Arg), as would be expected. Proline (Pro) and gamma-aminobutyric acid (GABA), which are produced directly from Glu without a transamination step, were not affected. The solid-state NMR experiments showed that protein synthesis occurred. Collectively, our results show that NH(4)(+) can only be assimilated through the GS/GOGAT pathway in P. glauca buds.

Aminooxyacetic Acid↗
PubMed2006

Glutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels.

It has been shown that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have not been identified. Glutamine:fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP), and activation of this pathway has been shown to be cardioprotective. Glutamine is required for metabolism of glucose via GFAT; therefore, the goal of this study was to determine whether glutamine cardioprotection could be attributed to increased flux through the HBP and elevated levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5 mM glucose, and global, no-flow ischemia was induced for 20 min followed by 60 min of reperfusion. Thirty-minute pre-treatment with 2.5 mM glutamine significantly improved functional recovery (RPP: 15.6+/-5.7% vs. 59.4+/-6.1%; p<0.05) and decreased cardiac troponin I release (25.4+/-3.0 vs. 4.7+/-1.9 ng/ml; p<0.05) during reperfusion. This protection was associated with a significant increase in the levels of protein O-GlcNAc and ATP. Pre-treatment with 80 muM azaserine, an inhibitor of GFAT, completely reversed the protection seen with glutamine and prevented the increase in protein O-GlcNAc. O-GlcNAc transferase (OGT) catalyzes the formation of O-GlcNAc, and inhibition of OGT with 5 mM alloxan also reversed the protection associated with glutamine. These data support the hypothesis that in the ex vivo perfused heart glutamine cardioprotection is due, at least in part, to enhanced flux through the HBP and increased protein O-GlcNAc levels.

Acetylglucosamine↗