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Canadian Journal of Veterinary Research... Jan 1990In studies to develop an oral rabies vaccine for wildlife, the immune response to and pathogenicity of two types of mutants of rabies viruses were examined. Forty-five...
In studies to develop an oral rabies vaccine for wildlife, the immune response to and pathogenicity of two types of mutants of rabies viruses were examined. Forty-five small plaque mutants were selected from cultures of ERA rabies virus treated with 8-azaguanine or 5-fluorouracil and tested for pathogenicity in mice. Two of these mutants AZA 1 and AZA 2 (low pathogenicity in mice) were given to skunks by oral (bait), intestinal (endoscope) and intramuscular routes. Additionally, challenge virus standard (CVS) rabies virus and mutants of this and ERA rabies virus (CVS 3766 and 3713, and ERA 3629) that were resistant to neutralization by specific antiglycoprotein monoclonal antibodies (and apathogenic in mice) were tested by various routes in skunks. Skunks given AZA 1 and AZA 2 were challenged at three months postinoculation with street rabies virus. After oral administration, there were very low rates of seroconversion with AZA 1 and AZA 2 and on challenge only 2/7 given AZA 1 and 1/8 given AZA 2 survived. None of the skunks given the other mutants orally seroconverted. AZA 2 produced a high rate of seroconversion (8/8) by the intestinal route and all challenged skunks in this group survived (7/7). All skunks vaccinated intramuscularly with AZA 1 (4/4) or AZA 2 (4/4) developed high levels of rabies neutralizing antibodies and survived challenge. The mutant CVS 3766, while apathogenic when given intracerebrally to adult mice, was consistently pathogenic by this route (and intranasally) in skunks. These results demonstrate that skunks are highly resistant to oral immunization by live rabies virus vaccines and that pathogenicity (by intracerebral route) of the mutant CVS 3766 is markedly different in mice and skunks.
Topics: Animals; Carnivora; Female; Male; Mephitidae; Mice; Mutation; Rabies Vaccines; Rabies virus
PubMed: 2306670
DOI: No ID Found -
Scientific Reports Feb 2019Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have...
Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have uncovered recurrent somatic mutations within RNA splicing factors, including SF3B1, SRSF2, U2AF1 and ZRSR2. The most frequently mutated gene is SF3B1, mutated in 17% of MDS patients. While SF3B1 mutations and their effects on splicing have been well characterised, much remains to be explored about their more far-reaching effects on cellular homeostasis. Given that mRNA splicing and nuclear export are coordinated processes, we hypothesised that SF3B1 mutation might also affect export of certain mRNAs and that this may represent a targetable pathway for the treatment of SF3B1-mutant MDS. We used CRISPR/Cas9-genome editing to create isogenic cellular models. Comprehensive transcriptome and proteome profiling of these cells identified alterations in the splicing and export of components of the translational machinery, primarily tRNA synthetases, in response to the SF3B1 K700E mutation. While steady-state protein synthesis was unaffected, SF3B1 mutant cells were more sensitive to the clinically-relevant purine analogue, 8-azaguanine. In this study, we also demonstrated that 8-azaguanine affects splicing. Our results suggest that the simultaneous targeting of RNA metabolism and splicing by 8-azaguanine represents a therapeutic opportunity for SF3B1-mutant myelodysplastic syndromes.
Topics: Amino Acyl-tRNA Synthetases; Cell Line, Tumor; Cytoplasm; Gene Editing; Gene Expression Profiling; HEK293 Cells; Humans; K562 Cells; Mutation; Myelodysplastic Syndromes; Phosphoproteins; Protein Biosynthesis; Proteome; Proteomics; RNA Splicing; RNA Splicing Factors
PubMed: 30804405
DOI: 10.1038/s41598-019-39591-7 -
Molecules (Basel, Switzerland) Dec 2015Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst,...
Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-β-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-β-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.
Topics: Azaguanine; Catalysis; Catalytic Domain; Fluorescent Dyes; Humans; Molecular Structure; Point Mutation; Purine-Nucleoside Phosphorylase
PubMed: 26729076
DOI: 10.3390/molecules21010044 -
Asian Pacific Journal of Cancer... 2013Prostate cancer caused by the abnormal disorderly growth of prostatic acinar cells is the most prevalent cancer of men in western countries. We aimed to screen out...
PURPOSE
Prostate cancer caused by the abnormal disorderly growth of prostatic acinar cells is the most prevalent cancer of men in western countries. We aimed to screen out differentially expressed genes (DEGs) and explore small molecule drugs for prostate cancer.
MATERIALS AND METHODS
The GSE3824 gene expression profile of prostate cancer was downloaded from Gene Expression Omnibus database which including 21 normal samples and 18 prostate cancer cells. The DEGs were identified by Limma package in R language and gene ontology and pathway enrichment analyses were performed. In addition, potential regulatory microRNAs and the target sites of the transcription factors were screened out based on the molecular signature database. In addition, the DEGs were mapped to the connectivity map database to identify potential small molecule drugs.
RESULTS
A total of 6,588 genes were filtered as DEGs between normal and prostate cancer samples. Examples such as ITGB6, ITGB3, ITGAV and ITGA2 may induce prostate cancer through actions on the focal adhesion pathway. Furthermore, the transcription factor, SP1, and its target genes ARHGAP26 and USF1 were identified. The most significant microRNA, MIR-506, was screened and found to regulate genes including ITGB1 and ITGB3. Additionally, small molecules MS-275, 8-azaguanine and pyrvinium were discovered to have the potential to repair the disordered metabolic pathways, abd furthermore to remedy prostate cancer.
CONCLUSIONS
The results of our analysis bear on the mechanism of prostate cancer and allow screening for small molecular drugs for this cancer. The findings have the potential for future use in the clinic for treatment of prostate cancer.
Topics: Computational Biology; Databases, Genetic; Drug Discovery; Humans; Male; MicroRNAs; Pharmaceutical Preparations; Prostatic Neoplasms; Small Molecule Libraries; Transcriptome
PubMed: 24175814
DOI: 10.7314/apjcp.2013.14.9.5281 -
Journal of Bacteriology Dec 1964Johnson, Russell C. (University of Minnesota, Minneapolis), and Palmer Rogers. Differentiation of pathogenic and saprophytic leptospires with 8-azaguanine. J. Bacteriol....
Johnson, Russell C. (University of Minnesota, Minneapolis), and Palmer Rogers. Differentiation of pathogenic and saprophytic leptospires with 8-azaguanine. J. Bacteriol. 88:1618-1623. 1964.-The use of the purine analogue, 8-azaguanine, as a differential agent for the separation of pathogenic and saprophytic leptospires was investigated. Growth of strains of the saprophyte Leptospira biflexa was almost insensitive to the bacteriostatic action of 8-azaguanine at concentrations varying from 25 to 600 mug/ml; these saprophytic leptospires were serially transferred five times in media containing 225 mug without any change in growth rate or cell yield. In contrast, decreased growth rate and cell yield of the pathogenic serotypes were observed with 25 to 50 mug/ml of 8-azaguanine. Complete inhibition of growth occurred at concentrations of 100 mug/ml and above. A medium containing 225 mug/ml of 8-azaguanine was successfully used to differentiate 20 serotypes of pathogenic leptospires and 10 saprophytic strains. L. andaman CH11, L. semarang Veldrat S1 73, and L. andaman Correa, were classified with the L. biflexa strains on the basis of their growth response to 8-azaguanine.
Topics: Antimetabolites; Azaguanine; Fluorouracil; Leptospira; Leptospira interrogans serovar icterohaemorrhagiae; Leptospirosis; Pharmacology; Research
PubMed: 14244050
DOI: 10.1128/jb.88.6.1618-1623.1964 -
The Journal of Biological Chemistry Apr 1985Purine nucleoside phosphorylase (EC 2.4.2.1, purine nucleoside:orthophosphate ribosyltransferase) was purified and characterized from the malarial parasite, Plasmodium...
Purine nucleoside phosphorylase (EC 2.4.2.1, purine nucleoside:orthophosphate ribosyltransferase) was purified and characterized from the malarial parasite, Plasmodium lophurae, using a chromatofocusing (Pharmacia) column and a formycin B affinity column. The apparent isoelectric point of the native protein, as determined by chromatofocusing, was 6.80. By gel filtration and both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the native enzyme appeared to be a pentamer with a native molecular weight of 125,300 and a subunit molecular weight of 23,900. The enzyme had a broad pH optimum, pH 5.5-7.5, with maximum activity at pH 6.0-6.5. The enzyme reaction was readily reversible with a Km for inosine of 33 microM and a Km for hypoxanthine of 82 microM. Thioinosine, guanosine, and guanine were also substrates for the plasmodial enzyme, but allopurinol and adenine were not. The parasite enzyme was competitively inhibited by formycin B (Ki = 0.39 microM). Formycin A, azaguanine, and 8-aminoguanosine were not inhibitors of the enzyme.
Topics: Animals; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Formycins; Isoelectric Focusing; Kinetics; Molecular Weight; Pentosyltransferases; Plasmodium; Purine-Nucleoside Phosphorylase
PubMed: 3920217
DOI: No ID Found -
Journal of Biochemistry Mar 1979A pterin deaminase catalyzing the hydrolytic deamination of various pteridines was found in the bacterium, Bacillus megaterium, and partially purified from bacterial...
A pterin deaminase catalyzing the hydrolytic deamination of various pteridines was found in the bacterium, Bacillus megaterium, and partially purified from bacterial extract. The specific activity was raised 90-fold over that of the crude extract. The pH optimum is around 7.3, and the Km value for 6-carboxypterin is 1.3 mM. The molecular weight of the enzyme was estimated by gel filtration to be about 110,000. The enzyme deaminated pterin, 6-carboxypterin, biopterin, 6-methylpterin, 7-methylpterin, xanthopterin, 6-hydroxymethylpterin, sepiapterin, isosepiapterin, folic acid, and 6,7-dimethylpterin to their corresponding lumazines, whereas guanine, 7-carboxypterin, leucopterin, isoxanthopterin, and 6-methylisoxanthopterin did not serve as substrates. The enzyme was inhibited by PCMB and 8-azaguanine.
Topics: Aminohydrolases; Ammonia; Azaguanine; Bacillus megaterium; Chloromercuribenzoates; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Pterins; Substrate Specificity
PubMed: 34599
DOI: No ID Found -
Nitric Oxide : Biology and Chemistry Nov 2013Nitric oxide (NO) is a very effective radiosensitizer of hypoxic mammalian cells, at least as efficient as oxygen in enhancing cell death in vitro. NO may induce cell...
Nitric oxide (NO) is a very effective radiosensitizer of hypoxic mammalian cells, at least as efficient as oxygen in enhancing cell death in vitro. NO may induce cell death through the formation of base lesions which are difficult to repair, and if they occur within complex clustered damage common to ionizing radiation, they may lead to replication-induced DNA strand breaks. It has previously been shown that 8-azaguanine and xanthine result from the reaction of guanine radicals with nitric oxide. We have now shown that adenine radicals also react with NO to form hypoxanthine and 8-azaadenine. Cells irradiated in exponential growth in the presence of NO are twice as radiosensitive compared to those irradiated in anoxia alone, whereas confluent cells are less radiosensitive to (•)NO. In addition, the numbers of DNA double strand breaks observed as γH2AX staining following radiosensitization by NO, are higher in exponential cells than in confluent cells. DNA damage, detected as 53BP1 foci, is also higher in HF-19 cells expressing Cyclin A, a marker for cells in S and G2 phases of the cell cycle, following radiosensitization by NO. RAD51 foci are highest in V79-4 cells irradiated in the presence of NO compared to in anoxia, 24h after radiolysis. This work presents evidence that radiosensitization of cells by NO is in part through the formation of specific DNA damage, difficult to repair, which in dividing cells may induce the formation of stalled replication forks and as a consequence replication-induced DNA strand breaks which may lead to cell death.
Topics: Adenine; Animals; Cell Cycle; Cell Line; Cell Survival; Cricetinae; DNA; DNA Damage; DNA Replication; Humans; Intracellular Signaling Peptides and Proteins; Nitric Oxide; Rad51 Recombinase; Radiation, Ionizing; Radiation-Sensitizing Agents
PubMed: 23623927
DOI: 10.1016/j.niox.2013.04.005 -
Proceedings of the National Academy of... Mar 1979Salmonella typhimurium strain TM677 was mutagenized with aflatoxin B1 (AFB1) in liquid suspension culture in the presence of a rat liver postmitochondrial supernatant....
Salmonella typhimurium strain TM677 was mutagenized with aflatoxin B1 (AFB1) in liquid suspension culture in the presence of a rat liver postmitochondrial supernatant. Forward mutation to 8-azaguanine resistance was measured in the treated cultures and was found to increase linearly with AFB1 concentration. DNA purified from mutagenized cells was analyzed for AFB1 adduct formation by high-pressure liquid chromatography after adduct liberation. AFB1 exposures at 0.16 and 0.32 micrometer for 35 min produced 15 and 22 AFB1--DNA adducts per genome, respectively, and induced 8-azaguanine-resistant fractions of 4.9 X 10(-4) and 9.6 X 10(-4). Approximately 70% of the AFB1 bound to DNA was chromatographically identical to 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 at the two AFB1 levels used.
Topics: Aflatoxins; Animals; DNA, Bacterial; Microsomes, Liver; Mutation; Protein Binding; Rats; Salmonella typhimurium
PubMed: 375236
DOI: 10.1073/pnas.76.3.1343 -
Tropical Biomedicine Dec 2010Leptospirosis is recognized as one of the important zoonotic diseases in the world including Malaysia. A total of 145 soil and water samples were collected from selected...
Leptospirosis is recognized as one of the important zoonotic diseases in the world including Malaysia. A total of 145 soil and water samples were collected from selected National Service Training Centres (NSTC) in Kelantan and Terengganu. The samples were inoculated into modified semisolid Ellinghausen McCullough Johnson Harris (EMJH) medium, incubated at room temperature for 1 month and examined under the dark-field microscope. Positive growth of the leptospiral isolates were then confirmed with 8-Azaguanine Test, Polymerase Chain Reaction (PCR) assay and Microscopic Agglutination Test (MAT). Fifteen cultures (10.34%) exhibited positive growths which were seen under dark field microscope whilst only 20% (3/15) were confirmed as pathogenic species. based on 8-Azaguanine Test and PCR. Serological identification of the isolates with MAT showed that hebdomadis was the dominant serovar in Terengganu. Pathogenic leptospires can be detected in Malaysian environment and this has the potential to cause an outbreak. Therefore, precautionary steps against leptospirosis should be taken by camp authorities to ensure the safety of trainees.
Topics: Agglutination Tests; Animals; Antimetabolites; Azaguanine; Bacteriological Techniques; Culture Media; Humans; Leptospira; Malaysia; Microscopy; Polymerase Chain Reaction; Serotyping; Soil Microbiology; Water Microbiology
PubMed: 21399605
DOI: No ID Found