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Acta Crystallographica. Section D,... Jul 2005Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine-salvage pathway, which allows cells to utilize preformed bases and nucleosides in order to synthesize...
Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine-salvage pathway, which allows cells to utilize preformed bases and nucleosides in order to synthesize nucleotides. PNP is specific for purine nucleosides in the beta-configuration and exhibits a strong preference for purines containing a 6-keto group and ribosyl-containing nucleosides relative to the corresponding analogues. PNP was crystallized in complex with ligands and data collection was performed using synchrotron radiation. This work reports the structure of human PNP in complex with guanosine (at 2.80 A resolution), 3'-deoxyguanosine (at 2.86 A resolution) and 8-azaguanine (at 2.85 A resolution). These structures were compared with the PNP-guanine, PNP-inosine and PNP-immucillin-H complexes solved previously.
Topics: Azaguanine; Binding Sites; Crystallography, X-Ray; Guanine; Guanosine; Humans; Inosine; Ligands; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones; Pyrroles
PubMed: 15983407
DOI: 10.1107/S0907444905005421 -
Guang Pu Xue Yu Guang Pu Fen Xi = Guang... Jul 2004The inclusion complexes of beta-Cyclodextrin (beta-CD) and HP-beta-Cyclodextrin (HP-beta-CD) with 6-Mercaptopurine (6-MP), Azathioprine (BAN) and 8-Azaguanine (Azan)...
The inclusion complexes of beta-Cyclodextrin (beta-CD) and HP-beta-Cyclodextrin (HP-beta-CD) with 6-Mercaptopurine (6-MP), Azathioprine (BAN) and 8-Azaguanine (Azan) were investigated by fluorescence. Various factors affecting the formation of inclusion complexes were discussed in detail including formation time and pH effect. The formation constants of their inclusion complexes were determined. The results indicated that their inclusion was affected significantly by laying time and pH. The formation time of beta-CD inclusion complexes is much longer than that of HP-beta-CD. The optimum pH is about pH = 7.7-12. Their maximum excitation wavelengths are all in the range of 276-285 nm and the maximum emission wavelengths are all in the range of 328-353 nm. The fluorescence signals are intensified with increasing concentration of CD. The stoichiometries of the inclusion complexes of CD with these three anticancer xanthines are all 1:1 and the formation constants are calculated.
Topics: 2-Hydroxypropyl-beta-cyclodextrin; Antineoplastic Agents; Azaguanine; Cell Line, Tumor; Cyclodextrins; Fluorescence; Humans; Hydrogen-Ion Concentration; Inclusion Bodies; Kinetics; Light; Mercaptopurine; Spectrometry, Fluorescence; Spectrum Analysis; Xanthines; beta-Cyclodextrins
PubMed: 15766092
DOI: No ID Found -
Zeitschrift Fur Naturforschung. C,... 2004Interactions of calf spleen purine nucleoside phosphorylase (PNP) with a non-typical substrate, 8-azaguanine (8-azaG), and a bisubstrate analogue inhibitor,...
Interactions of calf spleen purine nucleoside phosphorylase (PNP) with a non-typical substrate, 8-azaguanine (8-azaG), and a bisubstrate analogue inhibitor, 9-(2-phosphonylmethoxyethyl)-8-azaguanine (PME-azaG), were investigated by means of steady-state fluorescence spectroscopy. Both 8-azaG and PME-azaG form fluorescent complexes with the enzyme, and dissociation constants are comparable to the appropriate parameters (Km or Ki) obtained from kinetic measurements. PME-azaG inhibits both the phosphorolytic and synthetic pathway of the reaction in a competitive mode. The complex of 8-azaG with PNP is much weaker than the previously reported Gua-PNP complex, and its dissociation constant increases at pH > 7, where 8-azaG exists predominantly as the monoanion (pKa approximately 6.5). The fluorescence difference spectrum of the PNP/8-azaG complex points to participation of the N(7)H or/and N(8)H tautomers of the neutral substrate, and the 9-(2-phosphonylmethoxyethyl) derivative also exists as a neutral species in the complex with PNP. The latter conclusion is based on spectral characteristics of the PNP/PME-azaG complex, confirmed by fluorimetric determination of dissociation constants, which are virtually pH-independent in the range 6-7. These findings testify to involvement of the neutral purine molecule, and not its monoanion, as the substrate in the reverse, synthetic reaction. It is proposed that, in the reverse reaction pathway, the natural purine substrate is bound to the enzyme as the neutral N(7)H tautomer, which is responsible for the reported strong fluorescence of the guanine-PNP complex.
Topics: Animals; Azaguanine; Cattle; Enzyme Inhibitors; Kinetics; Purine-Nucleoside Phosphorylase; Spectrophotometry; Spleen
PubMed: 15540606
DOI: 10.1515/znc-2004-9-1017 -
Epidemiology and Infection Aug 2004Leptospirosis is endemic in the Andaman Islands, often occurring as outbreaks during the post-monsoon period. Pulmonary involvement is common and associated with high...
Leptospirosis is endemic in the Andaman Islands, often occurring as outbreaks during the post-monsoon period. Pulmonary involvement is common and associated with high morbidity and mortality. During the investigation of an outbreak in North Andaman in 1996 an isolate was recovered from the blood of a patient with fever, headache, body aches and haemoptysis with respiratory distress as presenting symptoms. The isolate was characterized using the cross-agglutination absorption test (CAAT) and monoclonal antibodies (mAbs). The isolate showed typical morphology and characteristic motility of the genus Leptospira. Growth was inhibited at 13 degrees C and in the presence of 8-azaguanine. The isolate could not be identified with grouping sera representing 25 serogroups, CAAT and mAbs. A new serovar of a new serogroup is proposed. Genetic characterization using polymerase chain reaction (PCR) followed by sequencing of the PCR product and randomly amplified polymorphic DNA fingerprinting (RAPD) showed that the isolate was genetically similar to L. interrogans sensu stricto.
Topics: Adult; Agglutination Tests; Amino Acid Sequence; Animals; Disease Outbreaks; Female; Genes, Bacterial; Humans; India; Leptospira; Leptospirosis; Molecular Sequence Data; Polymerase Chain Reaction; Rabbits; Random Amplified Polymorphic DNA Technique; Sequence Alignment; Serotyping
PubMed: 15310168
DOI: 10.1017/s0950268804002328 -
Nucleic Acids Research Feb 2004The use of primary somatic cells in nuclear transfer procedure has opened a new opportunity to manipulate domestic animal genomes via homologous recombination. To date,...
The use of primary somatic cells in nuclear transfer procedure has opened a new opportunity to manipulate domestic animal genomes via homologous recombination. To date, while a few loci have been targeted in somatic cells using similar enrichment strategies as those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells within a colony and difficulties in cloning the cell after targeting. Utilizing the hypoxanthine guanine phosphoribosyl transferase (HPRT) as a test locus, it was determined that while no targeted colonies were identified using a conventional targeting construct, an average of 1 per million targeted cells were identified when a nuclear localization signal (nls) was added to the construct. When the nls was combined with cell synchronization using a thymidine block, targeting efficiency increased 7-fold. Moreover, the number of random integrants decreased by over 54-fold resulting in a 1:3 targeted to random integration ratio. This method should facilitate the application of homologous recombination to primary somatic cells.
Topics: Animals; Azaguanine; Base Sequence; Cattle; Cell Division; Cells, Cultured; Drug Resistance; Fetus; Fibroblasts; Gene Targeting; Genetic Vectors; Hypoxanthine Phosphoribosyltransferase; Male; Molecular Sequence Data; Mutagenesis, Insertional; Nuclear Localization Signals
PubMed: 14960709
DOI: 10.1093/nar/gnh023 -
Journal of Medical Entomology Nov 2003Chemical analysis (high-performance liquid chromatography) and bioassay demonstrated the presence of compounds that seem to be components of the Ixodes scapularis...
Chemical analysis (high-performance liquid chromatography) and bioassay demonstrated the presence of compounds that seem to be components of the Ixodes scapularis arrestment pheromone. Only two purines, guanine and xanthine, were found in acidified saline extracts made from cast skins after molting of fed nymphs, fed larvae, and fecal/excretory exudates deposited by unfed adults on substrates in their environment. The ratio of guanine to xanthine was 10.6:1 in an extract from the nymphal skins versus 0.95:1 in an extract from the larval skins. Guanine, xanthine, and traces of a third purine, tentatively identified as 8-azaguanine, were found in extracts made from filter paper strips or washings from glass vials contaminated with tick feces and excreta left by unfed adults. 8-azaguanine may be a product of microbial degradation of the other purines rather than a natural product from the ticks. Low concentrations of ammonia also were detected in saline extracts of excreta from feeding ticks. Hematin also was found in NH4OH extracts of the black fecal/excretory exudates deposited by the unfed ticks. Hematin was tentatively identified by comparison of spectra with that of the authentic standard. Bioassays demonstrated a strong positive arrestment response to cast skins found to contain a mixture of guanine and xanthine and to black fecal/excretory exudates containing guanine, xanthine, the putative 8-azaguanine, and hematin. A Noldus video tracking system using a CCD video camera and Ethovision Pro tracking software showed statistically significant increases in the frequency of visits to the treated zone versus the control. Ticks were significantly more likely to assemble in response to the tick exudates within as little as 3 h compared with the controls. Previous bioassay studies also showed strong positive responses to guanine, xanthine, other purines, and hematin. Comparisons with the arrestment pheromones of other tick species are described. The inclusion of the pheromone components in a permethrin-impregnated oily matrix, Last Call, increased the lethal activity of the product to 95% compared with only 65% in the formulation with permethrin alone. More detailed knowledge of I. scapularis arrestment pheromone may be useful for improving the efficacy of this tick-killing technology even further.
Topics: Adenine; Animals; Biological Assay; Guanine; Ixodes; Pest Control, Biological; Pheromones; Skin; Xanthine
PubMed: 14765662
DOI: 10.1603/0022-2585-40.6.849 -
Analytical Biochemistry Jan 2004Plasma guanine deaminase (guanase; GD) is well established as an indicator of hepatocellular disease, recently being applied in the detection of hepatitis C in donor...
Plasma guanine deaminase (guanase; GD) is well established as an indicator of hepatocellular disease, recently being applied in the detection of hepatitis C in donor blood and in the diagnosis of hepatoma. No totally efficient, simple method for the estimation of plasma GD activity is routine since both guanine and 8-azaguanine, the substrates of the enzyme, are scarcely soluble in water. This difficulty in preparing stable substrates of sufficient concentration has resulted in methods that are both troublesome and inaccurate. Here we describe the development of new colorimetric and high-performance liquid chromatography (HPLC) methods utilizing guanosine as a "prosubstrate." After an initial breakdown of the guanosine to guanine using purine nucleoside phosphorylase, the ammonia formed as a result of the breakdown of the guanine by GD was estimated colorimetrically by the Berthelot reaction. As an alternative or a complementary assay, the xanthine also formed was measured using an isocratic HPLC method. These methods are suitable for routine assays for measuring plasma GD over a wide range of activities.
Topics: Ammonia; Chromatography, High Pressure Liquid; Clinical Enzyme Tests; Colorimetry; Guanine Deaminase; Guanosine; Humans; Purine-Nucleoside Phosphorylase; Solubility; Xanthine
PubMed: 14690689
DOI: 10.1016/j.ab.2003.09.041 -
Metabolic Engineering Oct 2003Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals. In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I...
Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals. In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I (GCHI), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SPR). BH4 is a medicine used to treat atypical hyperphenylalaninemia. It is currently synthesized by chemical means, which consists of many steps, and requires costly materials and complicated procedures. To explore an alternative microbial method for BH4 production, we utilized recombinant DNA technology to construct recombinant Escherichia coli (E. coli) strains carrying genes expressing GCHI, PTPS and SPR enzymes. These strains successfully produced BH4, which was detected as dihydrobiopterin and biopterin, oxidation products of BH4. In order to increase BH4 productivity we made further improvements. First, to increase the de novo GTP supply, an 8-azaguanine resistant mutant was isolated and an additional guaBA operon was introduced. Second, to augment the activity of GCHI, the folE gene from E. coli was replaced by the mtrA gene from Bacillus subtilis. These modifications provided us with a strain showing significantly higher productivity, up to 4.0 g of biopterin/L of culture broth. The results suggest the possibility of commercial BH4 production by our method.
Topics: Alcohol Oxidoreductases; Biopterins; Escherichia coli; GTP Cyclohydrolase; Gene Expression Regulation, Bacterial; Genetic Engineering; Multienzyme Complexes; Phosphorus-Oxygen Lyases; Recombinant Proteins
PubMed: 14642352
DOI: 10.1016/s1096-7176(03)00046-6 -
Spectrochimica Acta. Part A, Molecular... Nov 2003A comparative study, luminescence behavior of 6-Mercaptopurine (6-MP), Azathiopurine (BAN), and 8-Azaguanine (8-Azan) have been investigated including the low...
A comparative study, luminescence behavior of 6-Mercaptopurine (6-MP), Azathiopurine (BAN), and 8-Azaguanine (8-Azan) have been investigated including the low temperature phosphorescence, the low temperature fluorescence, the room temperature phosphorescence (RTP) and the room temperature fluorescence (RTF). The effect of pH on the luminescence intensity is discussed. Analytical characteristics of RTF and RTP of 6-MP, BAN, and 8-Azan have been studied. The lifetime of phosphorescence and the polarity of RTF and RTP have been examined.
Topics: Azaguanine; Hydrogen-Ion Concentration; Luminescence; Mercaptopurine; Spectrometry, Fluorescence; Spectrophotometry
PubMed: 14583288
DOI: 10.1016/s1386-1425(03)00121-5 -
Bioorganic Chemistry Oct 2003Watson-Crick optimized geometries and the energies of base pairing for the natural pairs of nucleic bases: adenine-thymine (AT) and guanine-cytosine (GC) have been...
Watson-Crick optimized geometries and the energies of base pairing for the natural pairs of nucleic bases: adenine-thymine (AT) and guanine-cytosine (GC) have been recalculated by ab initio methods in order to compare results to those found for the non-natural azaadenine-thymine (AAT) and azaguanine-cytosine (AGC) pairs. Geometry optimizations carried out at the HF/6-31G** level and energies obtained at MP2/6-31G**, show that AAT and AGC have hydrogen bonding patterns similar to the natural AT and GC and that the interaction energies (DeltaH0int) for the former are ca. 7 kcal/mol more stable than the latter. Accordingly, the pairs based on azapurines would be favored with respect to the natural pairs. Some possible explanations why nature does not use extensively the azabases in base pairing are given.
Topics: Azaguanine; Base Pairing; Cytosine; Hydrogen Bonding; Molecular Structure; Nucleic Acids; Thermodynamics; Thymine
PubMed: 12941289
DOI: 10.1016/s0045-2068(03)00083-x