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International Journal of Systematic and... Jul 2013Strain Eri-1(T) was isolated from a water sample on the campus of Kyushu University, Fukuoka, Japan. The motility and morphology of the isolate were similar to those of...
Strain Eri-1(T) was isolated from a water sample on the campus of Kyushu University, Fukuoka, Japan. The motility and morphology of the isolate were similar to those of members of the genus Leptospira, but the spiral structure of the isolate was sharper under dark-field microscopy. Cells were 10.6 ± 1.3 µm long and 0.2 µm in diameter, with a wavelength of 0.9 µm and an amplitude of 0.4 µm. Strain Eri-1(T) grew in Korthof's medium at both 13 and 30 °C, and also in the presence of 8-azaguanine. 16S rRNA gene-based phylogenetic analysis placed strain Eri-1(T) within the radiation of the genus Leptospira where it formed a unique lineage within the clade of the known saprophytic species of the genus Leptospira. The strain was not pathogenic to hamsters. Strain Eri-1(T) exhibited low levels (11.2-12.6 %) of similarity by DNA-DNA hybridization to the three most closely related species of the genus Leptospira. The DNA G+C content of the genome of strain Eri-1(T) was 42.5 ± 0.1 mol%. These results suggest that strain Eri-1(T) represents a novel species of the genus Leptospira, for which the name Leptospira idonii sp. nov. is proposed. The type strain is Eri-1(T) ( = DSM 26084(T) = JCM 18486(T)).
Topics: Animals; Azaguanine; Bacterial Typing Techniques; Base Composition; Cricetinae; DNA, Bacterial; Japan; Leptospira; Male; Molecular Sequence Data; Nucleic Acid Hybridization; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Water Microbiology
PubMed: 23203626
DOI: 10.1099/ijs.0.047233-0 -
Journal of Biomolecular Screening Jun 2012Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are...
Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.
Topics: Cell Line; Cysteine Proteinase Inhibitors; Drug Discovery; High-Throughput Screening Assays; Humans; Leupeptins; Mutant Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Stability; Reproducibility of Results; Small Molecule Libraries; Von Hippel-Lindau Tumor Suppressor Protein
PubMed: 22357874
DOI: 10.1177/1087057112436557 -
Cancer Investigation Jun 2012The involvement of apoptosis in the cytotoxicity mediated by nucleoside analogues, namely azaguanine, and its implication in resistance are not well understood. Using...
The involvement of apoptosis in the cytotoxicity mediated by nucleoside analogues, namely azaguanine, and its implication in resistance are not well understood. Using human T-cell acute lymphoblastic leukemia cell lines, sensitive (CEM cells) and resistant to azaguanine (CM3 cells), we observe a decrease in the expression of proapoptotic proteins in CM3 cells, which may be related to the resistance to cell death induced by azaguanine. On the other hand, CM3 cells lack cross resistance with other anticarcinogenic drugs, suggesting that azaguanine may be used alternatively in the presence of chemoresistance. A better knowledge of the apoptotic pathways involved in leukemic cell death resistance may contribute to the development of therapeutic strategies, aimed to prevent chemotherapy resistance.
Topics: Antimetabolites, Antineoplastic; Apoptosis; Azaguanine; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Immunophenotyping; Leukemia
PubMed: 22348536
DOI: 10.3109/07357907.2012.659925 -
Journal of Molecular Modeling Feb 2012The interaction between 8-azaguanine (8-Azan) and bovine serum albumin (BSA) in Tris-HCl buffer solutions at pH 7.4 was investigated by means of fluorescence and...
The interaction between 8-azaguanine (8-Azan) and bovine serum albumin (BSA) in Tris-HCl buffer solutions at pH 7.4 was investigated by means of fluorescence and ultraviolet-visible (UV-Vis) spectroscopy. At 298 K and 310 K, at a wavelength of excitation (λ (ex)) of 282 nm, the fluorescence intensity decreased significantly with increasing concentrations of 8-Azan. Fluorescence static quenching was observed for BSA, which was attributed to the formation of a complex between 8-Azan and BSA during the binding reaction. This was illuminated further by the UV-Vis absorption spectra and the decomposition of the fluorescence spectra. The thermodynamic parameters ∆G, ∆H, ∆S were calculated. The results showed that the forces acting between 8-Azan and BSA were typical hydrophobic forces, and that the interaction process was spontaneous. The interaction distance r between 8-Azan and BSA, evaluated according to fluorescence resonance energy transfer theory, suggested that there is a high possibility of energy transfer from BSA to 8-Azan. Theoretical investigations based on homology modeling and molecular docking suggested that binding between 8-Azan and BSA is dominated by hydrophilic forces and hydrogen bonding. The theoretical investigations provided a good structural basis to explain the phenomenon of fluorescence quenching between 8-Azan and BSA.
Topics: Animals; Azaguanine; Binding Sites; Cattle; Fluorescence Resonance Energy Transfer; Humans; Models, Molecular; Protein Binding; Protein Structure, Secondary; Serum Albumin, Bovine; Spectrometry, Fluorescence; Spectrum Analysis; Thermodynamics
PubMed: 21541747
DOI: 10.1007/s00894-011-1069-5 -
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 -
Metabolic Engineering Jan 2011Currently, the mutant of the flavinogenic yeast Candida famata dep8 isolated by classic mutagenesis and selection is used for industrial riboflavin production. Here we...
Currently, the mutant of the flavinogenic yeast Candida famata dep8 isolated by classic mutagenesis and selection is used for industrial riboflavin production. Here we report on construction of a riboflavin overproducing strain of C. famata using a combination of random mutagenesis based on the selection of mutants resistant to different antimetabolites as well as rational approaches of metabolic engineering. The conventional mutagenesis involved consecutive selection for resistance to riboflavin structural analog 7-methyl-8-trifluoromethyl-10-(1'-d-ribityl)isoalloxazine), 8-azaguanine, 6-azauracil, 2-diazo-5-oxo-L-norleucine and guanosine as well as screening for yellow colonies at high pH. The metabolic engineering approaches involved introduction of additional copies of transcription factor SEF1 and IMH3 (coding for IMP dehydrogenase) orthologs from Debaryomyces hansenii, and the homologous genes RIB1 and RIB7, encoding GTP cyclohydrolase II and riboflavin synthetase, the first and the last enzymes of riboflavin biosynthesis pathway, respectively. Overexpression of the aforementioned genes in riboflavin overproducer AF-4 obtained by classical selection resulted in a 4.1-fold increase in riboflavin production in shake-flask experiments. D. hansenii IMH3 and modified ARO4 genes conferring resistance to mycophenolic acid and fluorophenylalanine, respectively, were successfully used as new dominant selection markers for C. famata.
Topics: Candida; Cloning, Molecular; Fungal Proteins; Genetic Enhancement; Recombinant Proteins; Riboflavin; Signal Transduction; Species Specificity
PubMed: 21040798
DOI: 10.1016/j.ymben.2010.10.005 -
Journal of Microbiology, Immunology,... Feb 2010The genus Leptospira comprises pathogenic and saprophytic strains. Conventional methods for the identification of pathogenic leptospiral isolates are cumbersome and... (Comparative Study)
Comparative Study
BACKGROUND/PURPOSE
The genus Leptospira comprises pathogenic and saprophytic strains. Conventional methods for the identification of pathogenic leptospiral isolates are cumbersome and laborious. In view of these limitations, the search for alternative methods have been focused on DNA based techniques. In this study, we have developed an effective method for the rapid identification of pathogenic and saprophytic leptospiral isolates based on DNA-based techniques.
METHODS
A polymerase chain reaction(PCR)-based approach was developed using specific primer sets (flaB, G1-G2, B64I-II, and A-B) to differentiate pathogenic and saprophytic leptospiral strains. Fifty-five leptospiral isolates were used for this study. The pathogenic status of the isolates was compared with the results obtained using conventional techniques, which included growth in the presence of 8-azaguanine and growth at 13 degrees C.
RESULTS
In this analysis, 46 leptospiral isolates were confirmed as pathogenic and nine were confirmed as saprophytic. PCR with the A-B primer set yielded an amplified product of 331 bp in all of the pathogenic and saprophytic isolates. The other primer sets, G1-G2, B64I-II and flaB, yielded products of 258 bp, 568 bp, and 793 bp, respectively, exclusively for the pathogenic leptospiral strains. None of the saprophytic strains yielded products with these primer sets.
CONCLUSION
The flaB-specific primers consistently yielded an amplification product for all of the pathogenic leptospiral isolates, indicating the presence of the flaB gene only among pathogenic leptospires, and making this a useful tool for distinguishing between pathogenic and saprophytic leptospires. The efficiency of PCR-based identification corroborates the implementation of these techniques for the identification of pathogenic and saprophytic leptospiral strains.
Topics: Animals; Bacterial Proteins; Bacteriological Techniques; DNA Primers; Flagellin; Humans; Leptospira; Leptospirosis; Polymerase Chain Reaction; Rats; Sensitivity and Specificity; Water Microbiology
PubMed: 20434125
DOI: 10.1016/S1684-1182(10)60009-6 -
Anticancer Research Nov 2009Despite improvements in the treatment of patients with Ewing family tumours (EFT) during the past decades, the prognosis for patients with advanced disease is still...
BACKGROUND
Despite improvements in the treatment of patients with Ewing family tumours (EFT) during the past decades, the prognosis for patients with advanced disease is still unsatisfying. New treatment strategies have to be developed.
MATERIALS AND METHODS
A hypoxanthine/aminopterin/thymidine (HAT)-sensitive EFT cell line was developed by repetitive treatment of the EFT cell line SK-N-MC with 8'-azaguanine (8AG). By using DNA microarrays, the gene expression profile of this cell line was characterized. Immunostimulatory activity was assessed by mixed lymphocyte/tumour cell culture (MLTC). Artificial fusion of tumour cells and dendritic cells was visualized by flow cytometry.
RESULTS
After selection of 8AG-resistant cells, a cell line with high sensitivity for treatment with HAT was obtained. Expression of the X chromosome inactivation specific transcript XIST was higher in HAT-sensitive cells. Nevertheless, HAT-sensitive cells retained the EFT-associated gene expression profile. Moreover, in the presence of HAT, it was possible to use these cells without irradiation as stimulatory cells in MLTC or as fusion partner for dendritic cells.
CONCLUSION
HAT-sensitive EFT cells might be an interesting tool for the development of new immunotherapeutic approaches for the treatment of EFT.
Topics: Aminopterin; Antigens, Neoplasm; Azaguanine; Calmodulin-Binding Proteins; Cancer Vaccines; Cell Line, Tumor; Gene Expression Profiling; HLA-A2 Antigen; Humans; Hypoxanthine; Immunotherapy, Active; Oligonucleotide Array Sequence Analysis; Peptides; Proto-Oncogene Protein c-fli-1; RNA-Binding Protein EWS; RNA-Binding Proteins; Sarcoma, Ewing; Thymidine
PubMed: 20032396
DOI: No ID Found -
The Journal of Physical Chemistry. A Nov 2009Excited state characteristics of aza analogues of nucleic acid bases, 8-azaadenine (8AA), 5-azacytosine (5AC), 8-azaguanine (8AG), and 6-azauracil (6AU), in acetonitrile...
Excited state characteristics of aza analogues of nucleic acid bases, 8-azaadenine (8AA), 5-azacytosine (5AC), 8-azaguanine (8AG), and 6-azauracil (6AU), in acetonitrile solution were comprehensively investigated with steady state absorption and emission spectra, transient absorption measurements, emission measurements for the singlet oxygen molecule, and time-dependent density functional theory (TD-DFT) calculations. The triplet-triplet absorption spectrum of 8AA whose peak was 455 nm was observed for the first time. Sensitized singlet oxygen formation of 8AA was also observed in O(2)-saturated acetonitrile with quantum yields of 0.15 +/- 0.02. It was concluded that there were two kinds of aza analogues of nucleic acid bases: type A had substantial quantum yield for the intersystem crossing and potential of O2 (1Delta(g)) formation (8AA and 6AU), and type B did not (5AC and 8AG). TD-DFT calculations indicated that type A molecules had a dark 1npi* state below the first allowed 1pipi* state, while both S1 and S2 states for type B molecules had a pipi* character. It strongly suggested that the dark 1npi* state below the 1pipi* state would play an important role in the ISC process of aza analogues of nucleic acid bases.
Topics: Absorption; Acetonitriles; Adenine; Aza Compounds; Azaguanine; Cytosine; Nucleic Acids; Quantum Theory; Singlet Oxygen; Solvents; Spectrum Analysis; Uracil
PubMed: 19795828
DOI: 10.1021/jp905433s -
Nature Chemical Biology May 2009Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise functions in catalysis are unclear. To learn whether protonated or...
Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise functions in catalysis are unclear. To learn whether protonated or deprotonated forms of guanine predominate in the active site, microscopic pKa values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme in order to determine microscopic pKa values for 8-azaguanine deprotonation from the pH dependence of fluorescence. Microscopic pKa values above 9 for deprotonation of 8-azaguanine in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self-cleavage kinetics. Thus, the increase in activity with increasing pH does not correlate with deprotonation of G8, and most of G8 is protonated at neutral pH. These results do not exclude a role in proton transfer, but a simple interpretation is that G8 functions in the protonated form, perhaps by donating hydrogen bonds.
Topics: Base Sequence; Catalytic Domain; Guanine; Hydrogen-Ion Concentration; Kinetics; Microscopy, Fluorescence; Models, Molecular; Nucleic Acid Conformation; RNA, Catalytic
PubMed: 19330013
DOI: 10.1038/nchembio.156