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International Journal of Systematic and... Apr 2009A single Leptospira strain (designated Bejo-Iso9(T)) was isolated from a soil sample taken in Johor, Malaysia. The isolate showed motility and morphology typical of the...
A single Leptospira strain (designated Bejo-Iso9(T)) was isolated from a soil sample taken in Johor, Malaysia. The isolate showed motility and morphology typical of the genus Leptospira under dark-field microscopy. Cells were found to be 10-13 microm in length and 0.2 microm in diameter, with a wavelength of 0.5 microm and an amplitude of approximately 0.2 microm. Phenotypically, strain Bejo-Iso9(T) grew in Ellinghausen-McCullough-Johnson-Harris medium at 13, 30 and 37 degrees C, and also in the presence of 8-azaguanine. Serologically, strain Bejo-Iso9(T) produced titres towards several members of the Tarassovi serogroup, but was found to be serologically unique by cross-agglutinin absorption test and thus represented a novel serovar. The proposed name for this serovar is Malaysia. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the genus Leptospira, with sequence similarities within the range 90.4-99.5% with respect to recognized Leptospira species. DNA-DNA hybridization against the three most closely related Leptospira species was used to confirm the results of the 16S rRNA gene sequence analysis. The G+C content of the genome of strain Bejo-Iso9(T) was 36.2 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Bejo-Iso9(T) represents a novel species of the genus Leptospira, for which the name Leptospira kmetyi sp. nov. is proposed. The type strain is Bejo-Iso9(T) (=WHO LT1101(T)=KIT Bejo-Iso9(T)).
Topics: Base Composition; Cluster Analysis; DNA, Bacterial; DNA, Ribosomal; Leptospira; Locomotion; Malaysia; Molecular Sequence Data; Nucleic Acid Hybridization; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Soil Microbiology
PubMed: 19329592
DOI: 10.1099/ijs.0.002766-0 -
FEBS Letters Jan 2009In plants, nucleobase biochemistry is highly compartmented relying upon a well-regulated and selective membrane transport system. In Arabidopsis two proteins, AtAzg1 and...
In plants, nucleobase biochemistry is highly compartmented relying upon a well-regulated and selective membrane transport system. In Arabidopsis two proteins, AtAzg1 and AtAzg2, show substantial amino acid sequence similarity to the adenine-guanine-hypoxanthine transporter AzgA of Aspergillus nidulans. Analysis of single and double mutant lines harboring T-DNA insertion alleles AtAzg1-1 and AtAzg2-1 reveal a marked resistance to growth in the presence of 8-azaadenine and 8-azaguanine but not to other toxic nucleobase analogues. Conversely, yeast strains expressing AtAzg1 and AtAzg2 gain heightened sensitivity to growth on 8-azaadenine and 8-azaguanine. Radio-labeled purine uptake experiments in yeast and in planta confirm the function of AtAzg1 and AtAzg2 as plant adenine-guanine transporters.
Topics: Adenine; Amino Acid Sequence; Arabidopsis; Azaguanine; Biological Transport; Guanine; Molecular Sequence Data; Nucleobase Transport Proteins; Phylogeny; Saccharomyces cerevisiae
PubMed: 19121308
DOI: 10.1016/j.febslet.2008.12.048 -
International Journal of Systematic and... Oct 2008A single Leptospira strain (designated Khorat-H2(T)) was isolated from the urine of an adult male patient with suspected leptospirosis from the province of...
A single Leptospira strain (designated Khorat-H2(T)) was isolated from the urine of an adult male patient with suspected leptospirosis from the province of Nakornrachasima, Thailand. The isolate showed typical Leptospira motility and morphology under dark-field microscopy. Cells were 10-13 mum long and 0.2 mum in diameter, with a wavelength of 0.5 mum and an amplitude of approximately 0.3 mum. Phenotypically, strain Khorat-H2(T) did not grow at 13 degrees C but grew at 30 and 37 degrees C and in the presence of 8-azaguanine. Serological identification using the microscopic agglutination test revealed that strain Khorat-H2(T) had no cross-reaction with any recognized Leptospira serogroups. Phylogenetic analysis of the 16S rRNA gene sequence placed the novel strain within the radiation of the genus Leptospira, with sequence similarities of 88.1-97.7 % to recognized Leptospira species. DNA-DNA hybridization against the type strains of the three most closely related Leptospira species was used to confirm the results of the 16S rRNA sequence analysis. The G+C content of strain Khorat-H2(T) was 41.8 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Khorat-H2(T) represents a novel species of the genus Leptospira, for which the name Leptospira wolffii sp. nov. is proposed. The type strain is Khorat-H2(T) (=WHO LT1686(T) =KIT Khorat-H2(T)).
Topics: Adult; Bacterial Typing Techniques; Base Composition; DNA, Bacterial; Genes, Bacterial; Genes, rRNA; Humans; Leptospira; Leptospirosis; Male; Molecular Sequence Data; Phenotype; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Thailand
PubMed: 18842846
DOI: 10.1099/ijs.0.64947-0 -
International Journal of Systematic and... Oct 2008This paper reports on a Leptospira isolate of bovine origin and its identification as belonging to a previously unknown serovar, for which the name Topaz is proposed....
This paper reports on a Leptospira isolate of bovine origin and its identification as belonging to a previously unknown serovar, for which the name Topaz is proposed. The isolate (94-79970/3) was cultured from bovine urine from a north Queensland dairy farm in Australia. Strain 94-79970/3 grew at 30 degrees C in Ellinghausen McCullough Johnson Harris (EMJH) medium but failed to grow at 13 degrees C in EMJH medium or in the presence of 8-azaguanine. Serologically, strain 94-79970/3 produced titres against the Leptospira borgpetersenii serovar Tarassovi, the reference strain for the Tarassovi serogroup; however, no significant titres to any other serovars within the serogroup were obtained. Using 16S rRNA and DNA gyrase subunit B gene analysis, strain 94-79970/3 was identified as a member of the species Leptospira weilii. We propose that the serovar be named Topaz, after the location where the original isolate was obtained. The reference strain for this serovar is 94-79970/3 (=KIT 94-79970/3=LT722).
Topics: Animals; Cattle; DNA Gyrase; DNA, Bacterial; Genes, Bacterial; Genes, rRNA; Leptospira; Leptospirosis; Molecular Sequence Data; Phenotype; Queensland; RNA, Ribosomal, 16S; Serotyping
PubMed: 18842835
DOI: 10.1099/ijs.0.64884-0 -
Nucleosides, Nucleotides & Nucleic Acids 2007Phosphorolysis of 7-methylguanosine by calf spleen purine nucleoside phosphorylase (PNP) is weakly inhibited, uncompetitively, by Formycin B (FB) with Ki = 100 micro M...
Phosphorolysis of 7-methylguanosine by calf spleen purine nucleoside phosphorylase (PNP) is weakly inhibited, uncompetitively, by Formycin B (FB) with Ki = 100 micro M and more effectively by its aglycone (7KPP), IC50 35-100 micro M. In striking contrast, 7KPP inhibits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with Ki approximately 2-4 micro M. Formycin B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that both ligands bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-substrate inhibitor of both the phosphorolytic and reverse synthetic pathways.
Topics: Animals; Cattle; Formycins; Guanosine; Kinetics; Purine-Nucleoside Phosphorylase; Pyrimidines; Spectrometry, Fluorescence; Spleen; Substrate Specificity
PubMed: 18066912
DOI: 10.1080/15257770701503993 -
Journal of the American Chemical Society Mar 2007The fluorescent nucleotide analogue 8-azaguanosine-5'-triphosphate (8azaGTP) is prepared easily by in vitro enzymatic synthesis methods. 8azaGTP is an efficient...
The fluorescent nucleotide analogue 8-azaguanosine-5'-triphosphate (8azaGTP) is prepared easily by in vitro enzymatic synthesis methods. 8azaGTP is an efficient substrate for T7 RNA polymerase and is incorporated specifically opposite cytosine in the transcription template, as expected for a nucleobase analogue with the same Watson-Crick hydrogen bonding face as guanine. 8-Azaguanine (8azaG) in oligonucleotides also is recognized as guanine during ribonuclease T1 digestion. Moreover, replacement of guanine by 8azaG does not alter the melting temperature of base-paired RNAs significantly, evidence that 8azaG does not disrupt stacking and hydrogen bonding interactions. 8azaGTP displays a high fluorescent quantum yield when the N1 position is deprotonated at high pH, but fluorescence intensity decreases significantly when N1 is protonated at neutral pH. Fluorescence is quenched 10-fold to 100-fold when 8azaG is incorporated into base-paired RNA and remains pH-dependent, although apparent pKa values determined from the pH dependence of fluorescence intensity shift in the basic direction. Thus, 8azaG is a guanine analogue that does not perturb RNA structure and displays pH-dependent fluorescence that can be used to probe the ionization states of nucleobases in structured RNAs. A key application will be in determining the ionization state of active site nucleobases that have been implicated in the catalytic mechanisms of RNA enzymes.
Topics: Azaguanine; Base Sequence; Guanosine Triphosphate; Hydrogen-Ion Concentration; Ions; Kinetics; Molecular Sequence Data; RNA; Spectrometry, Fluorescence
PubMed: 17326637
DOI: 10.1021/ja067699e -
Journal of Medicinal Chemistry Dec 2006The malarial parasite Plasmodium falciparum depends on the purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) to convert purine bases...
The malarial parasite Plasmodium falciparum depends on the purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) to convert purine bases from the host to nucleotides needed for DNA and RNA synthesis. An approach to developing antimalarial drugs is to use HGXPRT to convert introduced purine base analogs to nucleotides that are toxic to the parasite. This strategy requires that these compounds be good substrates for the parasite enzyme but poor substrates for the human counterpart, HGPRT. Bases with a chlorine atom in the 6-position or a nitrogen in the 8-position exhibited strong discrimination between P. falciparum HGXPRT and human HGPRT. The k(cat)/K(m) values for the Plasmodium enzyme using 6-chloroguanine and 8-azaguanine as substrates were 50 - 80-fold and 336-fold higher than for the human enzyme, respectively. These and other bases were effective in inhibiting the growth of the parasite in vitro, giving IC(50) values as low as 1 microM.
Topics: Animals; Antimalarials; Guanine; Humans; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Kinetics; Pentosyltransferases; Plasmodium falciparum; Purines; Structure-Activity Relationship
PubMed: 17149876
DOI: 10.1021/jm061012j -
Pathophysiology : the Official Journal... May 2007Dipeptidyl peptidase IV, a cell membrane surface protease also known as CD26 (CD26/DPPIV), is known to play multiple functions in human organism, where it is largely...
Dipeptidyl peptidase IV, a cell membrane surface protease also known as CD26 (CD26/DPPIV), is known to play multiple functions in human organism, where it is largely expressed, for instance, in the development of human cancer and metastasis as well as in chemotherapy response. The objective of this work was to study the CD26 membrane expression and DPPIV activity in T-acute leukaemia cell lines (CEM and MOLT3) in culture, in order to observe the modification of its expression under the 8-azaguanine treatment. Cell line samples were incubated, some without different azaguanine concentration and others with, ranging from 10 to 100muM. Cell surface CD26 expression has been identified by flow cytometry and DPPIV activity, in cultured medium, was fluorimetrically measured. Results we have observed showed that 8-azaguanine induced a decrease in cell viability in a dose, time and cell type dependent manner with MOLT3 cells being the most sensitive to 8-azaguanine citotoxic effects (24h IC50: +/-10muM) when compared with CEM cells (24h IC50: +/-100muM). In the same experimental conditions, MOLT3 cell treated with 8-azaguanine shows an increase in CD26 expression (MIF) compared with that of CEM cell submitted to the same conditions (65.4+/-1.3 versus 18.7+/-1.7). DPPIV activity in culture medium supernatant of CEM versus MOLT3 controls cells (1.91+/-0.43 versus 2.06+/-0.50) and of CEM versus MOLT3 treated cells (2.10+/-0.16 versus 1.89+/-0.04) did not show a significant difference. These preliminary results suggest that 8-azaguanine stimulates CD26 expression which may be related to cellular sensitivity to 8-azaguanine.
PubMed: 17055708
DOI: 10.1016/j.pathophys.2006.09.003 -
Environmental and Molecular Mutagenesis Jun 2006Endogenous DNA damage clusters--two or more oxidized bases, abasic sites, or strand breaks within about 20 base pairs on opposing strands--can accumulate in unirradiated...
Endogenous DNA damage clusters--two or more oxidized bases, abasic sites, or strand breaks within about 20 base pairs on opposing strands--can accumulate in unirradiated mammalian cells, and may be significant origins of spontaneous detrimental biological effects. Factors determining the levels of such endogenous clusters are largely unknown. To determine if cellular repair genotype can affect endogenous cluster levels in mammalian cells, the authors examined cluster levels, growth rates, and mutant frequencies in Chinese hamster ovary cells expressing the Escherichia coli glycosylase fpg protein, whose principal substrates are oxidized purines. In cells expressing high levels of fpg protein, the levels of oxypurine clustered damages were decreased while those of oxypyrimidine clusters and abasic clusters were unchanged. Furthermore, in these cells, the growth rates were increased and the level of spontaneous background mutants in the hypoxanthine guanine phosphoribosyl transferase gene was decreased. These results suggest that endogenous clusters are potentially detrimental DNA damages, and that their levels-as well as the detrimental consequences of their presence-can be effectively reduced by increased cellular activity of specific DNA repair proteins.
Topics: Animals; Azaguanine; CHO Cells; Cricetinae; Cricetulus; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Escherichia coli Proteins; Hypoxanthine Phosphoribosyltransferase; Mutation; Transfection
PubMed: 16518838
DOI: 10.1002/em.20208 -
Nucleosides, Nucleotides & Nucleic Acids 2005Spectroscopic and kinetic studies of interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, an excellent fluorescent/fluorogenic substrate for...
Spectroscopic and kinetic studies of interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, an excellent fluorescent/fluorogenic substrate for the synthetic pathway of the reaction, and its 9-(2-phosphonylmethoxyethyl) derivative, a bisubstrate analogue inhibitor, were carried out. The goal was to clarify the catalytic mechanism of the enzymatic reaction by identification of ionic/tautomeric forms of these ligands in the complex with PNP.
Topics: Animals; Antimetabolites, Antineoplastic; Azaguanine; Cattle; Drug Interactions; Hydrogen-Ion Concentration; Kinetics; Ligands; Macromolecular Substances; Models, Chemical; Purine-Nucleoside Phosphorylase; Spectrometry, Fluorescence; Spleen; Substrate Specificity
PubMed: 16247971
DOI: 10.1081/ncn-200060004