Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Azaserine”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources
PubMed1987

Human cell lines secreting lymphokines. I. Establishment of T-T hybridomas.

Lymphokines (Lks) are conventionally produced by mitogen stimulation of T lymphocytes. However, this procedure results in a mixture of factors, some of which may have antagonistic effects. An alternative method of producing Lks is the construction of monoclonal T-T hybridomas which secrete distinct Lks. By adapting the hybridoma technology for T cells, one can select distinct T hybridomas which may serve as constant sources for the production of uniform and well defined Lks. Since the compatibility of a certain cell line to serve as a fusion partner is unpredictable, several lines were tried in the hybridization procedure. An obligatory requirement of such a mutant line is its sensitivity to a selective medium, in which only the hybrid cell would survive. For this purpose three 8-azaguanine (8AG) resistant mutant lines (Jurkat/12 CEM/14 and Molt-4/10) were established from the respective T leukemic cell lines. This was achieved by culturing the cells in the presence of 200 microM 8AG. The surviving resistant cells were sensitive to aminopterin and azaserine inhibitors. Depending on the cell line and the inhibitor, death of these mutants was complete in 7 to 14 days. Non-adherent peripheral blood lymphocytes (PBL) stimulated for 48 hours with 1 microgram/ml phytohemagglutinin (PHA), were fused with the CEM/14 line. Fifteen hybridomas secreted a substance with B cell growth factor (BCGF) activity, nineteen hybridomas secreted T cell growth factor (IL-2), and eight hybridomas secreted gamma-interferon (gamma-IFN). The six lines which exhibited BCGF activity only, were expanded and cloned. The BCGF activity in the supernatant of a positive clone, designated TH-5, was found to be 3-fold more potent than a preparation of BCGF obtained by stimulation of PBL with PHA.

Cells, Cultured↗
PubMed1977

The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica.

The principal initial product of metabolism of 13N-labeled ammonium by Anabaena cylindrica grown with either NH4+ or N2 as nitrogen source is amide-labeled glutamine. The specific activity of glutamine synthetase is approximately half as great in NH4+-grown as in N2-grown filaments. After 1.5 min of exposure to 13NH4+, the ratio of 13N in glutamate to 13N in glutamine reaches a value of approximately 0.1 for N2- and 0.15 for NH4+-grown filaments, whereas after the same period of exposure to [13N]N2, that ratio has reached a value close to unity and is rising rapidly. During pulse-chase experiments, 13N is transferred from the amide group to glutamine into glutamate, and then apparently into the alpha-amino group of glutamine. Methionine sulfoximine, an inhibitor of glutamine synthetase, inhibits the formation of glutamine. In the presence of the inhibitor, direct formation of glutamate takes place, but accounts for only a few per cent of the normal rate of formation of that amino acid; and alanine is formed about as rapidly as glutamate. Azaserine reduces formation of [13N]glutamate approximately 100-fold, with relatively little effect on the formation of [13N]glutamine. Aminooxyacetate, an inhibitor of transaminase reactions blocks transfer of 13N to aspartate, citrulline, and arginine. We conclude, on the basis of these results and others in the literature, that the glutamine synthetase/glutamate synthase pathway mediates most of the initial metabolism of ammonium in A. cylindrica, and that glutamic acid dehydrogenase and alanine dehydrogenase have only a very minor role.

Ammonia↗
PubMed1980

Gamma-glutamyl transpeptidase-mediated transport of amino acid in lecithin vesicles.

Gamma-Glutamyl transpeptidase, a membrane-bound enzyme purified from hog kidney cortex following solubilization with detergent, was incorporated by sonication procedure into large phospholipid vesicles using a 1:2 molar ratio of deoxycholate to phospholipid. tthese vesicles, when containing entrapped glutathione, exhibited the uptake of L-[14C]glutamate, but not of L-proline. The glutamate uptake did not occur to a significant extent in these vesicles when glutathione was added externally. The uptake of glutamate in the reconstituted system was sensitive to the temperature of incubation and to the inhibitors (serine-borate, azaserine, and 6-diazo-5-oxo-L-norleucine) of gamma-glutamyl transpeptidase activity. The accumulated product during the transport of glutamate in the gamma-glutamyl transpeptidase reconstituted system containing intravesicular glutathione was identified as gamma-glutamyl-glutamate. These results indicate that gamma-glutamyl transpeptidase mediates the translocation of glutamate in vitro and provides evidence in support of its role in the transport of amino acids in natural membranes.

Animals↗
PubMed1981

Transport of amino acids in gamma-glutamyl transpeptidase-implanted human erythrocytes.

gamma-Glutamyl transpeptidase purified from hog kidney cortex was implanted in the human erythrocyte membrane by incubation of erythrocytes at 37 degrees c with gamma-glutamyl transpeptidase-incorporated dipalmitoyl phosphatidylcholine vesicles. Membranes prepared from these implanted cells exhibited 4- to 5-fold increase in gamma-glutamyl transpeptidase activity. The association/insertion of gamma-glutamyl transpeptidase into erythrocyte membrane was further demonstrated by antibody to gamma-glutamyl transpeptidase. Implantation of gamma-glutamyl transpeptidase into erythrocyte membrane led to stimulation of uptake of glutamate and alanine, which are normally transported at a slow rate in human erythrocytes. The uptake of these amino acids in the implanted system was inhibited by inhibitors (serine-borate and azaserine) of transpeptidase activity as well as by antibody to gamma-glutamyl transpeptidase. These results in the implanted human erythrocytes demonstrate that gamma-glutamyl transpeptidase enzyme can mediate the translocation of amino acids and provide further evidence in support of its postulated role in the transport of amino acids in natural membranes.

Alanine↗
PubMed1981

In vitro chemical mutagenesis and viral transformation of a human endothelial cell strain.

We have established and characterized a diploid cell strain of normal human endothelial cells, RuBa 7E. RuBa 7E cells have an average cloning efficiency of 20% during early passages and undergo approximately 50 doublings in vitro before senescing spontaneously. At confluence, RuBa 7E cells form a homogeneous monolayer of flat polygonal cells. RuBa 7E cells react positively with antibody to human endothelial-specific Factor VIII. The toxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine on RuBa 7E cells were studied and are similar to those reported for diploid human fibroblasts. Mutant cells lacking hypoxanthine-guanine phosphoribosyltransferase were selected by their resistance to 6-thioguanine. The spontaneous incidence of mutants was less than or equal to 6 X 10(-6), and the induced incidence was 4.4 X 10(-4) at a survival frequency of 0.05. All 17 mutants that were tested lacked detectable hypoxanthine-guanine phosphoribosyltransferase activity, and none grew in medium containing azaserine and hypoxanthine. Autoradiography showed that mutant cells incorporated radioactive adenine but did not incorporate radioactive hypoxanthine. Unlike human fibroblasts, in which the recovery of 6-thioguanine-resistant mutants is reduced by contact feeding when the inoculum size during selection is increased above 10(4) cells per P60 dish, 5 to 10 X 10(4) RuBa 7E cells can be plated per P60 dish without reducing mutant recovery. This apparent lack of plating density suppression of mutant recovery makes RuBa 7E cells a comparatively compact and economical system for quantifying mutagenesis in diploid human cells. In order to determine whether RuBa 7E cells would undergo a distinct morphological transformation toward cancer in vitro, we infected them with SV40. As early as 14 days postinfection, discrete foci of morphologically transformed, mitotically active cells were seen against a monolayer background of normal cells when cultures were maintained in medium with low serum. Seven of the 33 foci which were obtained were studied for SV40-specific viral T-antigen, and all were positive. The facility with which RuBa 7E cells can be mutagenized and the ease with which morphological transformants can be identified make these cells potentially useful for studies comparing the mutagenic and transforming effects of chemicals and other agents on diploid human cells.

Cell Division↗
PubMed1984

A new method for the detection of Lesch-Nyhan heterozygotes by peripheral blood T cell culture using T cell growth factor.

Thioguanine-resistant T lymphoblast populations were selectively amplified using T cell growth factor in the cultures of peripheral blood T cells from four Lesch-Nyhan heterozygotes. Although Lesch-Nyhan T lymphoblasts were all thioguanine-resistant, none of the cultures from 13 control subjects yielded the growth of such defective cell populations. These data provide direct evidence for the existence of a small percentage (5%-40%) of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient T cells in the heterozygotes, but not in normal individuals. Conversely, culture of the T lymphoblasts with azaserine plus hypoxanthine permitted the growth of the other part of the cell population that was enzyme positive. The low percentages of HGPRT-negative cells among T cells in heterozygotes suggest that the presence of this enzyme is beneficial for differentiation of lymphocytes of T cell linkage. Considering the ease and the reliability, culture of the peripheral T cells with thioguanine and T cell growth factor is very likely of practical use for detecting Lesch-Nyhan syndrome carriers among predisposed females.

Adolescent↗
PubMed1984

Genotoxicity of a variety of mycotoxins in the hepatocyte primary culture/DNA repair test using rat and mouse hepatocytes.

Twenty-eight mycotoxins were studied in the hepatocyte primary culture/DNA repair test using rat and mouse hepatocytes. DNA repair synthesis was elicited by several compounds of unknown carcinogenicity, 5,6- dimethoxysterigmatocystin , versicolorins A and B, averufin , xanthomegnin , luteosporin , and chrysazin , as well as by the carcinogenic myocotoxins , aflatoxin B1, sterigmatocystin, luteoskyrin , ochratoxin A, azaserine, mitomycin C, and actinomycin D. The positive results with compounds of unknown carcinogenicity suggest that they are possibly genotoxic carcinogens. The carcinogenic mycotoxins, penicillic acid, patulin, griseofulvin, and rugulosin , which did not elicit repair synthesis may be nongenotoxic carcinogens.

Animals↗
PubMed1982

Antitumor activity of N-diazoacetyl derivatives of glycine and phenylalanine against P388 leukemia and B16 melanoma in mice.

N-Diazoacetyl derivatives of glycine and phenylalanine show antitumor activity in mice bearing P388 leukemia or B16 melanoma. The presented data indicate that antitumor activity is shown by diazomethylamide derivatives of glycine and phenylalanine, in addition to that already established for the amino acid derivatives having an O-diazoacetyl group (azaserine) or a diazoketone structure (DON and azotomycin) and for 1,2-bis-diazoacetyl ethane.

Animals↗
PubMed1982

Adenine phosphoribosyltransferase deficiency in cultured mouse mammary tumor FM3A cells resistant to 4-carbamoylimidazolium 5-olate.

4-Carbamoylimidazolium 5-olate (CIO), the aglycone of the nucleoside antibiotic, bredinin (4-carbamoyl-1-beta-D-ribofuranosylimidazolium 5-olate), exhibited potent cytotoxic effects of subclonal line F28-7 of C3H mouse mammary carcinoma FM3A cells in culture. We isolated 11 cell lines resistant to CIO from wild-type F28-7 cells mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine. These resistant (cio') lines were 160- to 400-fold less sensitive to CIO than were the wild-type cells and inherited the resistant phenotypes during subculture for more than 3 months in the drug-free medium. They were cross-resistant to an adenine analog, 2,6-diaminopurine, while 2,6-diaminopurine-resistant (dap') lines, isolated independently, were cross-resistant to CIO. Neither of the cio' lines tested were able to form colonies in agar medium containing azaserine and adenine, nor were they able to incorporate tritiated adenine into the macromolecular fraction, indicating that they could not utilize exogenous adenine for growth. Enzyme assays using cell-free extracts revealed that all the cio' lines had undetectable levels of adenine phosphoribosyltransferase (EC 2.4.2.7) activity, but they, except one, had normal levels of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20) activities. These results demonstrate that the CIO resistance in these lines is attributed to deficient adenine phosphoribosyltransferase activity and therefore that CIO is activated by adenine phosphoribosyltransferase to form a cytotoxic nucleotide within the drug-sensitive cells.

Adenine↗
PubMed1980

Acinar cell carcinoma of the rat pancreas grown in cell culture and in nude mice.

An acinar cell carcinoma of the pancreas, which was induced in a male Wistar rat by repeated injections of azaserine, was propagated in cell culture. Sheets of epithelial-like cells grew within 2 weeks and were subcultured serially. Initially acinar cell carcinoma in the culture medium produced a high level of amylase, but the secretion ceased rapidly as cells began to proliferate. Only negligible amounts of trypsinogen and chymotrypsinogen were detected in cell homogenates at passages 7 and 9. The chromosome distribution ranged from hypodiploid to hypertetraploid. When cultured cells were transplanted s.c. into nude mice, palpable tumors appeared within 4 weeks and could be transplanted serially. Histological examination of the tumor showed poorly differentiated carcinoma without acinar structures. Tumor homogenate contained amylase, trypsinogen, and chymotrypsinogen, and the electron microscopic examination revealed that many tumor cells contained zymogen-like granules. These results indicate that pancreatic acinar cell carcinoma in cell cultures, in which there was no differentiated function, can be activated to synthesize tissue-specific enzymes when transplanted into nude mice by yet undefined factors present in the host animals. The cell line and transplantable tumors of pancreatic acinar cell carcinoma may be useful in the analysis of the biological behavior of this type of tumor and in the study of the control mechanisms of the synthesis of tissue-specific products in cells.

Animals↗
PubMed1995

Partial purification and some properties of gamma-glutamyl peptide-hydrolysing enzyme from Actinobacillus actinomycetemcomitans.

A gamma-glutamyl peptide-hydrolysing enzyme was partially purified from Actinobacillus actinomycetemcomitans. The enzyme required metal ions, and 1 to 2 mM Mn2+ ions, especially, were essential for hydrolytic reaction. Its distribution by treatment of cells with lysozyme-EDTA suggested that the enzyme was a membrane-bound protein. The pI of the enzyme was in range of pH 5.1 to 5.6, and the apparent Km value for gamma-glutamyl-p-nitroanilide was 3.0 x 10(-4) M. The enzyme hydrolysed specifically gamma-glutamyl residues from the N-terminal of gamma-glutamyl compounds such as gamma-Glu-Met, gamma-Glu-Ala, gamma-Glu-Leu and gamma-Glu-Tyr, but did not catalyse the transpeptidation reaction. Neither free amino acids such as Ala-, Pro-, Gly- and Leu-p-nitroanilide nor alpha-glutamyl derivatives were hydrolysed. Its activity was strongly inactivated by metal chelators (EDTA or o-phenanthroline) and amino acids (Glu, Gln). In addition, the activity was specifically inactivated by gamma-glutamyl affinity-labelling reagents such as AT-125, 6-diazo-5-oxo-L-norleucine and azaserine, which are inhibitors of the gamma-glutamyl donor sites of mammalian gamma-glutamyl transpeptidase. Antibodies against bovine kidney gamma-glutamyl transpeptidase decreased the activity of the bacterial enzyme by 65%. These results suggested that the active sites in the bacterial enzyme were similar to those in mammalian gamma-glutamyl transpeptidase.

Aggregatibacter actinomycetemcomitans↗
PubMed1976

Pathway of nitrogen metabolism after fixation of 13N-labeled nitrogen gas by the cyanobacterium, Anabaena cylindrica.

Methods have been developed for identifying the pathway of assimilation of N2-derived nitrogen. The products of fixation of 13N-labeled nitrogen gas ([13N]N2), and the distribution of 13N within glutamine, were determined after short periods of labeling (approximately 1 to 120 s) and also in pulse-chase experiments. Ammonia, the amide nitrogen of glutamine, and the alpha-amino nitrogen of glutamate, in that order, were the first observed products of fixation of N2 by the cyanobacterium (blue-green alga), Anabaena cylindrica. This sequence of the formation of nitrogenous products was confirmed by the use of inhibitors. The presence of 1 mM methionine sulfoximine permitted continued formation of 13NH3, while virtually preventing 13N-labeling of amino acids. In the presence of 1 mM azaserine, glutamine was labeled, but not other amino acids. Our observations demonstrate unequivocally that N2-derived nitrogen fixed by this organism is metabolized initially by the glutamine synthetase/glutamate synthase pathway.

Cyanobacteria↗
PubMed1993

The pyruvate kinase gene as a model for studies of glucose-dependent regulation of gene expression in the endocrine pancreatic beta-cell type.

The insulinoma beta-cell line INS-1 expresses the L-type pyruvate kinase gene at high level and responds to a rise in extracellular glucose by strong induction of gene expression. Following the addition of glucose to the culture medium in the 3.5-33 mM concentration range, the cellular level of L-type pyruvate kinase mRNA increases within 2 h and reaches a maximum 15-fold above basal in 8-12 h. By run-on nuclear assay, the relative transcription rate of the pyruvate kinase gene is shown to increase 4-fold at maximal stimulation, suggesting that both transcriptional and post-transcriptional effects contribute to mRNA accumulation. The glucose effect is totally suppressed by the hexokinase inhibitor mannoheptulose, indicating a requirement for glucose phosphorylation. The mRNA induction is not inhibited in glutamine-free culture medium or by azaserine, suggesting that the hexosamine biosynthetic pathway is not involved. Moreover, metabolism along the glycolytic pathway does not appear to be an absolute requisite, since 2-deoxyglucose partly mimics the inductive effect of glucose. The glucose effect on the pyruvate kinase gene is reversibly antagonized by agents increasing intracellular cAMP. In addition, the effect is highly specific to the pyruvate kinase gene. Neither proinsulin I mRNA nor glucokinase mRNA are increased in glucose-stimulated INS-1 cells. Short term transfection with CAT plasmids driven by the pyruvate kinase L promoter reveals specific glucose-inducible reporter activity with the 183-base pair promoter region upstream of the cap site. Within this region, the previously described L4 cis-acting element is crucial for glucose responsiveness, as demonstrated by the fact that a plasmid with a mutation in this element does not elicit glucose-inducible CAT activity. Induction of L-type pyruvate kinase mRNA occurs in the islets of rats subjected to fasting and carbohydrate refeeding. In conclusion, the L-type pyruvate kinase gene provides an interesting model of glucose-regulated gene in the endocrine beta-cell type.

Animals↗
PubMed1993

[Effect of cholecystokinin and secretin on insulin binding to rat pancreatic acini and pancreatic cancer cell line AR42J cells].

In order to clarify the interaction of hormones which exert various effects on the exocrine pancreas, we investigated the effect of cholecystokinin (CCK) and secretin on subsequent insulin binding to pancreatic acini and cultured AR42J cells derived from azaserine-induced acinar cell carcinoma of the pancreas. CCK at concentrations of 100pM-10nM inhibited subsequent 125I-insulin binding to pancreatic acini. 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited 125I-insulin binding whereas A23187 had little effect, suggesting that the inhibitory effect of CCK is mediated by protein kinase C. On the other hand, 100pM-10nM secretin had no effect on subsequent 125I-insulin binding to pancreatic acini, although higher concentrations of forskolin and 8 bromoadenosine 3', 5'-cyclic monophosphate inhibited 125I-insulin binding. In addition, secretin exerted no potentiating effect on the inhibitory effect of CCK on 125I-insulin binding to pancreatic acini. Based on these results, we further investigated the effect of CCK and TPA on subsequent 125I-insulin binding to AR42J cells. In this carcinoma cell line, inhibitory effect of CCK and TPA on insulin binding was completely abolished. The present results suggest, therefore, that hormonal interaction may play an important role in the regulation of exocrine pancreatic function including acinar cell growth.

Animals↗
PubMed1996

Relief of ornithine decarboxylase messenger RNA translational repression induced by alternative splicing of its 5' untranslated region.

The ornithine decarboxylase enzyme (ODC) is the key regulator of polyamine synthesis and is a member of the cellular proto-oncogene family. Its expression becomes constitutively activated by carcinogens, viruses, and oncogenes. ODC mRNA has a long 5' untranslated region that could be important in the regulation of enzyme levels by affecting translation. To test this hypothesis, we have determined the role of this region on the constitutive ODC hyperexpression measured in AR4-2J cells, an azaserine-induced, tumor-derived pancreatic acinar cell line. Construction of expression vectors in which ODC 5' leader sequence was placed flanking the chloramphenicol acetyltransferase reporter gene allowed us to identify three AR4-2J specific, different alternatively spliced ODC 5' leaders. The 5' ends of exons 2 and 3 were lengthened by 17 and 13 bases, respectively. Translation performed in a cell-free system as well as in COS7 transient transfection experiments demonstrated that AR4-2J isoforms induce a strong increase in the rate of translation. These results provide evidence that alternative splicing observed in tumoral cells, coupled with translation regulation, relieves the translation repression mediated by the long and structured 5' untranslated region of the ODC proto-oncogene.

Alternative Splicing↗
PubMed1997

Some properties of gamma-glutamyl transpeptidase from Fusobacterium nucleatum.

A series of chromatographic techniques was used to purify gamma-glutamyl transpeptidase from Fusobacterium nucleatum ATCC 23726. The purified enzyme was homogenous with a molecular weight (MW) of approximately 101 kD consisting of two different subunits with MW of 67 kD and 31 kD. Its distribution by treatment of lysozyme-EDTA suggested that the enzyme was a periplasmic protein. The pl of the enzyme was 5.9 to 6.2, and the optimum pH of the transpeptidation and the hydrolytic reaction was 8.0. Glycylglycine, glycine and methionine were good acceptors, and the enzyme reaction, in the presence of glycylglycine, was especially efficient. Glutamic acid, glutamine and serine were poor acceptors, and the enzyme activity was inhibited by these amino acids. The apparent Km value for the gamma-glutamyl donor (gamma-glutamyl-p-nitroanilide) was 4.9 x 10(-4)M and that for the acceptor (glycylglycine) was 0.19 M. Affinity-labelling reagents, such as DON, azaserine and AT-125 strongly inhibited the enzyme activity, and its activity was inhibited by L-serine plus borate, as is mammalian gamma-glutamyl transpeptidase. The antibodies against gamma-glutamyl transpeptidase from bovine kidney reduced the activity of the bacterial enzyme by 65%. These results indicate that the catalytic sites in F. nucleatum gamma-glutamyl transpeptidase were similar to those in mammalian gamma-glutamyl transpeptidase.

Animals↗
PubMed1997

Glutamine synthetase from N2-fixing cyanobacterium Nostoc muscorum--purification, biochemical and immunological characteristics.

A modified procedure for purification of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming)] from N2-fixing cyanobacterium N. muscorum, to homogeneity is described using DEAE-Sephadex and Blue-Sepharose affinity chromatography. Specific activities of the purified enzyme in biosynthetic and transferase assays were 8.5 and 28 mumole product formed min-1 mg-1 protein. Apparent molecular mass of native GS enzyme was about 610 kDa as estimated by gel filtration. On SDS-PAGE the enzyme protein migrated as single band with molecular weight of 51 kDa. Apparent Michaelis Menten constant (Km) for glutamate, glutamine, ATP and ammonium were 2.8, 4.0, 0.35 and 0.82 mM respectively. Ammonium and structural analogues of glutamine and ammonium viz, methionine sulfone (MSO), methionine-DL-sulfoximine (MSX), ethylenediamine (EDA) and glyphosine significantly inactivated the enzyme activity while azaserine showed partial inhibition. Polyclonal antibodies raised against purified GS protein of N. muscorum showed high specificity to both crude and pure GS preparations in different immunological assays like double diffusion, rocket immunoelectrophoresis, ELISA and western blotting. With serological procedure a microquantity of GS-antigen upto 2 ng in vivo and immunological relationship of the protein with different strains have been documented.

Cyanobacteria↗