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Journal of Bacteriology Sep 1964Day, Lawrence E., (Michigan State University, East Lansing) and Ralph N. Costilow. Physiology of the sporulation process in Clostridium botulinum. II. Maturation of...
Day, Lawrence E., (Michigan State University, East Lansing) and Ralph N. Costilow. Physiology of the sporulation process in Clostridium botulinum. II. Maturation of forespores. J. Bacteriol. 88:695-701. 1964.-Clostridium botulinum, strain 62-A, did not sporulate endotrophically, but forespores matured to refractile, heat-resistant spores when replaced in solutions containing l-alanine and l-proline, l-isoleucine and l-proline, or l-alanine and l-arginine. Solutions of l-arginine or l-citrulline would not support the maturation process. Acetate, CO(2), and delta-amino valeric acid were produced during sporulation in a replacement solution of l-alanine and l-proline, indicating the operation of the Stickland reaction. There was no large uptake of either exogenous l-alanine or acetate during this process. Similarly, there was no apparent protein or nucleic acid synthesis, since high levels of chloramphenicol, 8-azaguanine, or mitomycin C failed to inhibit, and no significant amount of P(32) was incorporated into the spore nucleic acids. Dipicolinic acid (DPA) was synthesized during forespore maturation. It is believed that these final steps in sporulation of C. botulinum require only an exogenous source of energy which can be obtained via the Stickland reaction system, and that the synthesis of DPA and other unknown materials relies primarily on endogenous substrates.
Topics: Acetates; Alanine; Amino Acids; Arginine; Carbon Dioxide; Carbon Isotopes; Citrulline; Clostridium botulinum; DNA; DNA, Bacterial; Fatty Acids; Fermentation; Hot Temperature; Isoleucine; Metabolism; Ornithine; Phosphorus Isotopes; Picolinic Acids; Proline; RNA; RNA, Bacterial; Research; Spores; Spores, Bacterial
PubMed: 14208509
DOI: 10.1128/jb.88.3.695-701.1964 -
World Journal of Gastroenterology May 2003To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis.
AIM
To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis.
METHODS
The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRT locus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FL-REV3(-) by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3(-) and FL-REV3(-). The blockage effect of REV3 was measured by combination of reverse transcription-polymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3 protein level.
RESULTS
The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27 fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P<0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P<0.05). In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein. MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P<0.05) and 5.8 fold (P<0.01) respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3 was blocked by antisense RNA, and almost recovered to their spontaneous mutation levels. The spontaneous HPRT mutation was disappeared in REV3-disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66 x 10(-6) in FL cells to 0.14 x 10(-6) in transgenic cells as well (P<0.01).
CONCLUSION
The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.
Topics: Base Sequence; Carcinogens; Cell Line; DNA, Antisense; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Humans; Hypoxanthine Phosphoribosyltransferase; Methylnitronitrosoguanidine; Mutagenesis; Plasmids; Stomach Neoplasms; Transfection
PubMed: 12717825
DOI: 10.3748/wjg.v9.i5.888 -
Proceedings of the National Academy of... Jan 1971Double hybridization of Ehrlich ascites tumor cells (ETC) and L cells was performed. In the first step, a hybrid (LE) of ETC and L(AG) (a mutant of L cells, resistant to...
Double hybridization of Ehrlich ascites tumor cells (ETC) and L cells was performed. In the first step, a hybrid (LE) of ETC and L(AG) (a mutant of L cells, resistant to 10 mug/ml of 8-azaguanine) and a hybrid (LL) of L(AG) and L(BrdU) (a mutant of L cells, resistant to 100 mug/ml of 5-bromodeoxyuridine) were prepared by the use of artificial fusion by UV-irradiated HVJ (Sendai virus). In the second step the LE hybrid and ETC, or the LL hybrid and ETC, were fused again by UV-HVJ. The hybrids (LEE and LLE) were segregated during culture. Thus, the series of hybrids L, LLE, LE, LEE, and ETC was obtained. The morphological feature and karyological characters of these hybrids and the distribution of antigens corresponding to each parent on their cell surfaces supported the above identification of the series of hybrids obtained by double hybridization. All three hybrids acquired new characters, such as the ability to form large colonies in soft agar and large tumors on the chorioallantoic membrane of chicken eggs, unlike either parent. The tumor-forming capacity of the series was ETC > LEE > LE > LLE; L cells formed no tumor in mouse abdomen under the test conditions.
Topics: Animals; Antigens; Carcinoma, Ehrlich Tumor; Chromosomes; Clone Cells; Histocompatibility; Hybridization, Genetic; In Vitro Techniques; L Cells; Microscopy, Phase-Contrast; Mutation; Parainfluenza Virus 1, Human; Radiation Effects; Ultraviolet Rays
PubMed: 4322264
DOI: 10.1073/pnas.68.1.38 -
Nucleic Acids Research Nov 2013Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the...
Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.
Topics: Anti-HIV Agents; Azaguanine; CD4-Positive T-Lymphocytes; Cell Line; HIV-1; HeLa Cells; Humans; Quinolines; RNA Splicing; RNA, Viral; Thiophenes; Viral Structural Proteins; Virus Replication; rev Gene Products, Human Immunodeficiency Virus
PubMed: 23945945
DOI: 10.1093/nar/gkt727 -
The Journal of Biological Chemistry Jul 1954
Topics: Azaguanine; Glycosides; Guanine
PubMed: 13192085
DOI: No ID Found -
Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.The Journal of Clinical Investigation Nov 1975The mutation in a young gouty male with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase has been evaluated. The serum uric acid was 11.8 mg/100...
The mutation in a young gouty male with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase has been evaluated. The serum uric acid was 11.8 mg/100 ml, and the urinary uric acid excretion was 1,279 mg/24 h. Erythrocyte hypoxanthine-guanine phosphoribosyltransferase was 34.2 nmol/h/mg, adenine phosphoribosyltransferase was 36.5 nmol/h/mg and phosphoribosylpyrophosphate was 2.6 muM. Hypoxanthine-guanine phosphoribosyltransferase from peripheral leukocytes and cultured diploid skin fibroblasts was within the normal range, but enzyme activity in rectal mucosa was below the normal range. Initial velocity studies of the normal enzyme and the mutant enzyme from erythrocytes with the substrates hypoxanthine, guanine, or phosphoribosylpyrophosphate showed that the Michaelis constants were similar. Product inhibition studies distinguished the mutant enzyme from the normal enzyme. Hyperbolic kinetics with increasing phosphoribosylpyrophosphate were converted to sigmoid kinetics by 0.2 mM GMP with the mutant enzyme but not with the normal enzyme. The mutant erythrocyte hypoxanthine-guanine phosphoribosyltransferase was inactivated normally at 80 degrees C and had a normal half-life in the peripheral circulation. The mol wt of 48,000 was similar to the normal enzyme mol wt of 47,000. With isoelectric focusing, the mutant erythrocyte enzyme had two major peaks with isoelectric pH's of 5.50 and 5.70, in contrast to the isoelectric pH's of 5.76, 5.82, and 6.02 of the normal isozymes. Isoelectric focusing of leukocyte extracts from the patient revealed the presence of the mutant enzyme. Cultured diploid fibroblasts from the propositus appeared to function normally, as shown by the inability to grow in 50-100 muM azaguanine and by the normal incorporation of [14C]hypoxanthine into nucleic acid. In contrast, erythrocytes from the patient displayed abnormal properties, including the increased synthesis of phosphoribosylphyrophosphate and elevated functional activity of orotate phosphoribosyltransferase and orotidylic decarboxylase. These unique kinetic, physical, and functional properties provide support for heterogeneous structural gene mutations in partial deficiencies of hypoxanthine-guanine phosphoribosyltransferase.
Topics: Adult; Erythrocytes; Female; Fibroblasts; Genes; Gout; Humans; Hypoxanthine Phosphoribosyltransferase; In Vitro Techniques; Mutation; Pedigree; Uric Acid
PubMed: 1184748
DOI: 10.1172/JCI108200 -
Genetics Nov 2000We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes). We show that bra3 mutations are alleles of the GUK1...
We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes). We show that bra3 mutations are alleles of the GUK1 gene, which encodes GMP kinase. The bra3 mutants have a low GMP kinase activity, excrete purines in the medium, and show vegetative growth defects and resistance to purine base analogs. The bra3 locus also corresponds to the previously described pur5 locus. Several lines of evidence indicate that the decrease in GMP kinase activity in the bra3 mutants results in GMP accumulation and feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), encoded by the HPT1 gene. First, guk1 and hpt1 mutants share several phenotypes, such as adenine derepression, purine excretion, and 8-azaguanine resistance. Second, overexpression of HPT1 allows suppression of the deregulated phenotype of the guk1 mutants. Third, we show that purified yeast HGPRT is inhibited by GMP in vitro. Finally, incorporation of hypoxanthine into nucleotides is similarly diminished in hpt1 and guk1 mutants in vivo. We conclude that the decrease in GMP kinase activity in the guk1 mutants results in deregulation of the ADE gene expression by phenocopying a defect in HGPRT. The possible occurrence of a similar phenomenon in humans is discussed.
Topics: Adenosine Monophosphate; Adenylate Kinase; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Genes, Fungal; Genotype; Guanylate Kinases; Hypoxanthine Phosphoribosyltransferase; Kinetics; Mutation; Nucleoside-Phosphate Kinase; Phenotype; Recombinant Fusion Proteins; Saccharomyces cerevisiae
PubMed: 11063676
DOI: 10.1093/genetics/156.3.953 -
The Journal of Experimental Medicine Jun 1980Cells of two teratocarcinoma stem cell lines (PCC4 azaguanine [aza] 1 and F9 5-bromodeoxyuridine [BrdU]) were fused with normal mouse spleen cells and mouse...
Cells of two teratocarcinoma stem cell lines (PCC4 azaguanine [aza] 1 and F9 5-bromodeoxyuridine [BrdU]) were fused with normal mouse spleen cells and mouse thymoma-derived cells (BW 5147), respectively. Hybrid clones were tested for the expression of molecules coded by the H-2K and -2D genes both by absorption analysis of conventional H-2 sera and by indirect antibody-binding radioimmunoassay with monoclonal antibodies. Somatic cell hybrids between PCC4 aza 1 and spleen cells morphologically resemble teratocarcinoma stem cells and do not express H-2 antigens. However, after differentiation in vitro, one of these hybrid clones expresses the H-2K and -2D gene products of both parental cell lines, one close expresses H-2-D- but not H-2K-coded antigenic determinants, and one clone remains H-2 negative. Somatic cell hybrids between F9 BrdU and BW 5147 resemble fibroblasts. Analysis of a series of hybrid clones revealed some clones that express both the H-2K- and H-2D-coded antigenic specificities of both parental alleles, some that express H-2D gene products strongly and the H-2K gene products very weakly, and some that express H-2D- but not H-2K-coded molecules. These results imply independent regulation of expression of the H-2K and -2D genes. The H-2D gene products appear to be preferentially expressed if the hybrid cells are capable of expressing H-2. The results suggest complex regulatory mechanisms that are H-2K and H-2D specific.
Topics: Animals; Antibody Specificity; Cell Differentiation; Clone Cells; Genes; H-2 Antigens; Hybrid Cells; Isoantibodies; Major Histocompatibility Complex; Mice; Neoplasms, Experimental; Spleen; Teratoma
PubMed: 7381363
DOI: 10.1084/jem.151.6.1349 -
Virology Sep 2002Monocyte-derived dendritic cells (DCs) from adult T cell leukemia are impaired in taking up exogenous antigens. To overcome this impairment, we fused unpulsed DCs to...
Monocyte-derived dendritic cells (DCs) from adult T cell leukemia are impaired in taking up exogenous antigens. To overcome this impairment, we fused unpulsed DCs to human T-lymphotropic virus type I (HTLV-I)-infected CD4(+) T cells (fusion DC-T cells). The efficiency of fusion was 50% and the fusion cells expressed higher HLA-ABC and CD86 Ags than HTLV-I-infected DCs. The fusion DC-T cells stimulated autologous CD4(+) and CD8(+) T cells, but DCs fused to itself or PHA-blasts did not stimulate any subsets. The functionally highest fusion DC-T cells was obtained when a DC and HTLV-I-infected T cells were fused at a ratio of 3:1. Expression of HTLV-Igag Ag on CD4(+) T cells was up-regulated when infected in the presence of 8-azaguanine, and these fusion DC-T cells were quite efficient in induction of higher CD8(+) T cell response. The results suggest that fusion DC-T cells produce functionally competent Ag-presenting cells and may be a likely candidate for immunotherapeutic use.
Topics: Antigen Presentation; Antigen-Presenting Cells; Cell Fusion; Dendritic Cells; Human T-lymphotropic virus 1; Humans; Immunophenotyping; Immunotherapy; Leukemia-Lymphoma, Adult T-Cell; T-Lymphocytes
PubMed: 12359442
DOI: 10.1006/viro.2002.1546 -
Proceedings of the National Academy of... Feb 1976The mutagenicity of benzo[a]pyrene and 15 of its derivatives, which included phenols, the benzo[a]yrene-4,5-epoxide (the K-region epoxide), dihydrodiols, two isomeric...
The mutagenicity of benzo[a]pyrene and 15 of its derivatives, which included phenols, the benzo[a]yrene-4,5-epoxide (the K-region epoxide), dihydrodiols, two isomeric 7,8-diol-9,10-epoxides, a 6-methyl derivative, and a 6-hydroxymethyl derivative, were tested with Chinese hamster V79 cells in order to identify the mutagenic metabolites of benzo[a]pyrene. Mutations were characterized by resistance to ouabain or 8-azaguanine. Since V79 cells do not metabolize polycyclic hydrocarbons, mutagenesis was tested both in the presence and absence of benzo[a]pyrene-metabolizing normal golden hamster cells. All the tested phenols, 4,5-diols, trans-9,10-diol, 6-methyl, and 6-hydroxymethyl derivatives of benzo[a]pyrene showed little or no mutagenicity for both genetic markers. The (+/-)7alpha,8beta-dihydroxy-9alpha,10alpha-epoxy-7,8;9,10-tetrahydrobenzo[a]pyrene and K-region 4,5-epoxide exhibited similar and moderate mutagenicity in the absence of benzo[a]pyrene-metabolizing cells, but the (+/-)7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene showed a 2000- and 270-fold higher mutation frequency for ouabain and 8-azaguanine resistance, respectively, than did the K-region 4,5-epoxide. The trans-7,8-diol which was not mutagenic in the absence of benzo[a]pyrene-metabolizing cells was more mutagenic than benzo[a]pyrene after metabolism and mutagenesis by trans-7,8-diol in these cells was inhibited by 7,8-benzoflavone, an inhibitor of mixed-function oxidases. Metabolically formed trans-7,8-diol was isolated and incubated with rat liver microsomes in the presence of co-factors. High-pressure liquid chromatography analysis indicated that the major metabolite of trans-7,8-diol is 7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. The results indicate that the latter compound is metabolically formed and the major mutagenic intermediate of benzo[a]yrene metabolism.
Topics: Benzopyrenes; Cell Line; Microsomes; Mutation; Structure-Activity Relationship
PubMed: 1061161
DOI: 10.1073/pnas.73.2.607