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Enzima purina nucleosideo fosforilase de Schistosoma Mansoni: estruturas cristalográficas, estudos cinéticos e descoberta de novos ligantes; Purine nucleoside fosforilase from Schistosoma Mansoni: crystal structure, knetics, studies and ligands search

Pereira, Humberto D'Muniz
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 22/12/2003 PT
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O parasita Schistosoma mansoni não possui a via de síntese de bases púricas e depende integralmente da via de salvação de purinas para o seu requerimento de purinas. Uma das enzimas participantes desta via é a Purina Nucleosídeo Fosforilase (PNP) (E.C. 2.4.2.1). A PNP catalisa a fosforólise reversível de nucleosídeos de purina para gerar a base correspondente e ribose-1-fosfato. No Projeto Genoma de Schistosoma mansoni, o gene para esta enzima foi identificado.O cDNA para a PNP de S. mansoni (SmPNP), possui 1055pb e codifica para uma proteína de 287 aminoácidos, que possui 49% de identidade quando comparada a PNP de eritrócitos humana ou de Baco bovino. O gene foi clonado no vetor de expressão pMAL C2G, e expresso na forma de uma proteína de fusão, com MBP (80mg/L). Após a purificação da proteína de fusão, a clivagem proteolítica das duas proteínas foi realizada utilizando-se o Factor Xa. A SmPNP foi então purificada utilizando uma coluna de troca catiônica. Foram determinadas as constantes catalíticas para a fosforólise de inosina pela SmPNP, as quais são 3?M para o KM e 222 s-1para o kcat. O valor para o KM é O menor já descrito para uma PNP de baixa massa molecular. Foram obtidos cristais da SmPNP utilizando 18-24 % de PEG 1500...

Adenosine accumulation in Saccharomyces cerevisiae cultured in medium containing low levels of adenine.

Laten, H M; Valentine, P J; van Kast, C A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1986 EN
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By monitoring the in vivo incorporation of low concentrations of radiolabeled adenine into acid-soluble compounds, we observed the unusual accumulation of two nucleosides in Saccharomyces cerevisiae that were previously considered products of nucleotide degradation. Under the culture conditions used in the present study, radiolabeled adenosine was the major acid-soluble intracellular derivative, and radiolabeled inosine was initially detected as the second most prevalent derivative in a mutant lacking adenine aminohydrolase. The use of yeast mutants defective in the conversion of adenine to hypoxanthine or to AMP renders very unlikely the possibility that the presence of adenosine and inosine is attributable to nucleotide degradation. These data can be explained by postulating the existence of two enzyme activities not previously reported in S. cerevisiae. The first of these activities transfers ribose to the purine ring and may be attributable to purine nucleoside phosphorylase (EC 2.4.2.1) or adenosine phosphorylase (EC 2.4.2.-). The second enzyme converts adenosine to inosine and in all likelihood is adenosine aminohydrolase (EC 3.5.4.4).

Metabolism of Methylated Purines in Escherichia coli: Derepression of Purine Biosynthesis

Love, Samuel H.; Remy, Charles N.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1966 EN
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Love, Samuel H. (Bowman Gray School of Medicine, Wake Forest College, Winston-Salem, N.C.), and Charles N. Remy. Metabolism of methylated purines in Escherichia coli: derepression of purine biosynthesis. J. Bacteriol. 91:1037–1049. 1966.—Various methylated purines were examined for their effects on growth of purine-requiring mutants of Escherichia coli, strains W-11 and B-96, and for their effects on purine biosynthesis. 6-Methylaminopurine and 6-methoxypurine stimulated the accumulation of purine precursor derivatives (ribosyl-5-aminoimidazole and ribosyl-5-amino-4-imidazole carboxamide) beyond their ability to support growth. Information obtained from in vivo and in vitro systems demonstrated that the metabolism of 6-methylaminopurine and 6-methoxypurine utilized identical pathways. The riboside derivatives are formed either by direct ribosidation via nucleoside phosphorylase or, indirectly, by dephosphorylation of the 5′-phosphoribosyl derivatives which are synthesized via adenylate pyrophosphorylase. Information obtained with the aid of strain W-11/DAP (lacking adenylate pyrophosphorylase) demonstrated that both pathways were important to the growing cells. Regardless of the metabolic pathway by which they are synthesized...

Genetic analysis of nucleoside transport in Leishmania donovani.

Iovannisci, D M; Kaur, K; Young, L; Ullman, B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1984 EN
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Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.

Effects of aspirin and dipyridamole on the degradation of adenosine diphosphate by cultured cells derived from bovine pulmonary artery.

Crutchley, D J; Ryan, U S; Ryan, J W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1980 EN
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To improve understanding of the mechanisms by which ADP is degraded during passage through the pulmonary vascular bed, we examined cultured endothelial and smooth muscle cells of bovine pulmonmary artery for their abilities to metabolize [8-14C]ADP. ADP is rapidly converted to AMP and then to adenosine, hypoxanthine, and inosine. Inosine is the major metabolite produced by endothelial cells. Radioactivity (5-10%) is accumulated intracellularly primarily as ATP. Medium containing 50 micro M ADP incubated with endothelial cells rapidly loses its ability to aggregate platelets and becomes antiaggregatory under conditions in which prostacyclin is absent. The antiaggregatory activity is probably the result of accumulated adenosine. 10 micro M dipyridamole inhibits cellular uptake of radioactivity by greater than 90%, and inosine in the medium is largely replaced by adenosine. This is accompanied by increased anti-aggregatory activity of conditioned medium, which can be matched by authentic adenosine at the same concentration. 1 mM aspirin had no effect on the metabolism of ADP by endothelial cells. Our results suggest: (a) Metabolism of ADP during passage through the lung is mainly the result of endothelial ADPase. (b) ADP released from aggregating platelets can be converted to the antiaggregatory substance...

Overproduction of Uric Acid in Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: CONTRIBUTION BY IMPAIRED PURINE SALVAGE

Edwards, N. Lawrence; Recker, David; Fox, Irving H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1979 EN
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The contribution of reduced purine salvage to the hyperuricemia associated with hypoxanthine-guanine phosphoribosyltransferase deficiency was measured by the intravenous administration of tracer doses of [8-14C]adenine to nine patients with normal enzyme activity, three patients with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, and six patients with the Lesch-Nyhan syndrome. The mean cumulative excretion of radioactivity 7 d after the adenine administration is 5.6±2.4, 12.9±0.9, and 22.3±4.7% of infused radioactivity for control subjects, partial hypoxanthine-guanine phosphoribosyltransferase-deficient subjects, and Lesch-Nyhan patients, respectively. To assess relative rates of nucleotide degradation in control and hypoxanthine-guanine phosphoribosyltransferase-deficient patients two separate studies were employed. With [8-14C]inosine administration, three control subjects excreted 3.7-8.5% and two enzyme-deficient patients excreted 26.5-48.0% of the injected radioactivity in 18 h. The capacity of the nucleotide catabolic pathway to accelerate in response to d-fructose was evaluated in control and enzyme-deficient patients. The normal metabolic response to intravenous fructose is a 7.5±4.2-mmol/g creatinine increase in total urinary purines during the 3-h after the infusion. The partial hypoxanthine-guanine phosphoribosyltransferase-deficient subjects and Lesch-Nyhan patients show increases of 18.6±10.8 and 17.3±11.8 mmol/g creatinine...

Pronouced instability of tandem IU base pairs in RNA

Serra, Martin J.; Smolter, Patricia E.; Westhof, Eric
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
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Optical melting was used to determine the stabilities of three series of RNA oligomers containing tandem XU base pairs, GGCXUGCC (5′XU3′), GGCUXGCC (5′UX3′) and GGCXXGGC/CCGUUCCG (5′XX3′), where X is either A, G or I (inosine). The helices containing tandem AU base pairs were the most stable in the first two series (5′XU3′ and 5′UX3′), with an average melting temperature ∼11°C higher than the helices with tandem 5′GU3′ base pairs and 25°C higher than the helices with tandem 5′IU3′ base pairs. For the third series (5′XX3′), the helix containing tandem GG is the most stable, with an average melting temperature ∼2°C higher than the helix with tandem AA base pairs and ∼24°C higher than the helix with tandem II base pairs. The thermodynamic stability of the oligomers with tandem IU base pairs was also investigated as a function of magnesium ion concentration. As with normal A–U or G–U tandem duplexes, the data could best be interpreted as non-specific binding of magnesium ions to the inosine-containing RNA oligonucleotides.

Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.

Maynes, J T; Yam, W; Jenuth, J P; Gang Yuan, R; Litster, S A; Phipps, B M; Snyder, F F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/12/1999 EN
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Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM...

Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes

Watkins, Norman E.; SantaLucia, John
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
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Nearest-neighbor thermodynamic parameters of the ‘universal pairing base’ deoxyinosine were determined for the pairs I·C, I·A, I·T, I·G and I·I adjacent to G·C and A·T pairs. Ultraviolet absorbance melting curves were measured and non-linear regression performed on 84 oligonucleotide duplexes with 9 or 12 bp lengths. These data were combined with data for 13 inosine containing duplexes from the literature. Multiple linear regression was used to solve for the 32 nearest-neighbor unknowns. The parameters predict the Tm for all sequences within 1.2°C on average. The general trend in decreasing stability is I·C > I·A > I·T ≈ I· G > I·I. The stability trend for the base pair 5′ of the I·X pair is G·C > C·G > A·T > T·A. The stability trend for the base pair 3′ of I·X is the same. These trends indicate a complex interplay between H-bonding, nearest-neighbor stacking, and mismatch geometry. A survey of 14 tandem inosine pairs and 8 tandem self-complementary inosine pairs is also provided. These results may be used in the design of degenerate PCR primers and for degenerate microarray probes.

Nucleoside transport in sheep erythrocytes: genetically controlled transport variation and its influence on erythrocyte ATP concentrations.

Young, J D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1978 EN
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1. The permeability of sheep erythrocytes to purine and pyrimidine nucleosides was investigated. Erythrocytes from most sheep (nucleoside-impermeable) were almost completely impermeable to 5 mM inosine whereas cells from approximately 5% of the animals studied (nucleoside-permeable) showed a rapid inosine uptake. Cells from both types of animal were permeable to 5 mM adenosine, although transport was slower in nucleoside-impermeable erythrocytes. 2. Two distinct nucleoside transport routes were present in nucleoside-permeable erythrocytes; a high affinity (apparent Km congruent to 0.2 mM) facilitated diffusion system which transported both purine and pyrimidine nucleosides, and a non-saturable uptake route selective for adenosine. The high affinity system was the major route of adenosine transport at physiological concentrations. 3. Transport by the high affinity system was completely inhibited by micromolar concentrations of dipyridamole and nitrobenzylthioinosine. Dipyridamole had no effect on the non-saturable component of adenosine uptake. 4. The transport differences between nucleoside-permeable and impermeable erythrocytes were due to the absence of the high affinity system from nucleoside-impermeable cells. 5. Nucleoside-permeable cells had a higher intracellular ATP concentration than nucleoside-impermeable erythrocytes...

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase†

Murkin, Andrew S.; Birck, Matthew R.; Rinaldo-Matthis, Agnes; Shi, Wuxian; Taylor, Erika A.; Schramm, Vern L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition state analogues show His257 within hydrogen-bonding distance to the 5′-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (Km/Ki) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His257 serves an important role in the early stages of transition state formation. Whereas mutation of His257 resulted in little variation in the PNP·DADMe-ImmH·SO4 structures, His257Phe·ImmH·PO4 showed distortion at the 5′-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies on the remote 5′-3H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe...

Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)*

Arendt, Cassandra S.; Ullman, Buddy
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
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Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability...

HIF-1 alpha is an essential effector for purine nucleoside-mediated neuroprotection against hypoxia in PC12 cells and primary cerebellar granule neurons

zur Nedden, Stephanie; Tomaselli, Bettina; Baier-Bitterlich, Gabriele
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Hypoxia-inducible factor-1 alpha (HIF-1α) and purine nucleosides adenosine and inosine are critical mediators of physiological responses to acute and chronic hypoxia. The specific aim of this paper was to evaluate the potential role of HIF-1α in purine-mediated neuroprotection. We show that adenosine and inosine efficiently rescued PC12 cells (up to 43.6%) as well as primary cerebellar granule neurons (up to 25.1%) from hypoxic insult, and furthermore, that HIF-1α is critical for purine-mediated neuroprotection. Next, we studied hypoxia- or purine nucleoside-increased nuclear accumulation of HIF-1α in PC12 cells. As a possible result of increased protein stabilization or synthesis an up to 2.5 fold induction of HIF-1α accumulation was detected. In cerebellar granule neurons, purine nucleosides induced an up to 3.1 fold HIF-1α accumulation in cell lysates. Concomitant with these results, siRNA-mediated reduction of HIF-1α completely abolished adenosine- and inosine-mediated protection in PC12 cells and severely hampered purine nucleoside-mediated protection in primary neurons (up to 94.2%). Data presented in this paper thus clearly demonstrate that HIF-1α is a key regulator of purine nucleoside-mediated rescue of hypoxic neuronal cells.

Identification and Characterization of Two Adenosine Phosphorylase Activities in Mycobacterium smegmatis▿

Buckoreelall, Kajal; Wilson, Landon; Parker, William B.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2011 EN
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Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine + phosphate ↔ adenine + alpha-d-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent Km (Km app) of 98 ± 6 μM (with Ado) and a native molecular mass of 125 ± 7 kDa. The second Ado cleavage activity was identified as 5′-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade...

Optimal guideRNAs for re-directing deaminase activity of hADAR1 and hADAR2 in trans

Schneider, Marius F.; Wettengel, Jacqueline; Hoffmann, Patrick C.; Stafforst, Thorsten
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
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Adenosine deaminases that act on RNA (ADAR) are a class of enzymes that catalyze the conversion of adenosine to inosine in RNA. Since inosine is read as guanosine ADAR activity formally introduces A-to-G point mutations. Re-addressing ADAR activity toward new targets in an RNA-dependent manner is a highly rational, programmable approach for the manipulation of RNA and protein function. However, the strategy encounters limitations with respect to sequence and codon contexts. Selectivity is difficult to achieve in adenosine-rich sequences and some codons, like 5′-GAG, seem virtually inert. To overcome such restrictions, we systematically studied the possibilities of activating difficult codons by optimizing the guideRNA that is applied in trans. We find that all 5′-XAG codons with X = U, A, C, G are editable in vitro to a substantial amount of at least 50% once the guideRNA/mRNA duplex is optimized. Notably, some codons, including CAG and GAG, accept or even require the presence of 5′-mismatched neighboring base pairs. This was unexpected from the reported analysis of global editing preferences on large double-stranded RNA substrates. Furthermore, we report the usage of guanosine mismatching as a means to suppress unwanted off-site editing in proximity to targeted adenosine bases. Together...

ESI-MS[n] of anticancer pt[iv] organoamido complexes and their interactions with DNA-model compounds /

Gurgi, Mohamedkamal Ahmed.
Fonte: Brock University Publicador: Brock University
Tipo: Electronic Thesis or Dissertation
ENG
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The general solution behaviour and" the major fragmentation pathways of the anticanceractive PtIV coordination complexes, trans, trans, cis, cis-[PtCIOH{N(pFC6F4) CH2h(pY)2] (1), trans, cis, cis-[Pt(OH)2{N(p-FC6F4)CH2h(Py)2] (2), trans, cis, cis-[Pt(OH)2{N(p-HC6F4)CH2h(Py)2] (3), trans, trans, cis, cis-[PtCIOH{N(pHC6F4) CH2h(Py)2] (4), and trans, trans, cis, cis-[PtOH(OCH3){N(p-HC6F4)CH2h(PY)2] (5) (Py = pyridine) have been deduced by positive-ion tandem-in-time ESI-MS. Overall, the acquired full-scan, positive-ion ESI-MS spectra of 2, 3, and 5 were characterized by the presence of relatively low-intensity [M+Nar and [M+Kt mass spectral peaks, whereas those of 1 and 4 were dominated by extremely intense [M+Hr peaks. Complexes 2 and 3 were also noted to form [2M+Ht and [2M+Nat dilneric cations. The source of Na + and K+ ions is believed to be the sample, the solvent systems used or the transport line carrying the sample solutions into the ES ion source. Further, the fragmentation pathway of all complexes studied was found to be almost identical with concurrent loss of py and H20 molecules, loss of a {N(p-YC6F4)CH2} (Y = F, H) group and/or concomitant release of the latter group and a py ligand being the most conunon. The photochemical degradation behaviour of 1 and 2 was also investigated using either fluorescent or ultraviolet light and some products of that degradation were positively identified. Altogether...

Effects of some purine derivatives on the guinea-pig trachea and their interaction with drugs that block adenosine uptake.

Coleman, R A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1976 EN
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1 Adenosine, adenosine 5'-triphosphate (ATP), adenine, inosine and guanosine all caused concentration-dependent relaxations of guinea-pig tracheal smooth muscle in vitro. The relative potencies in descending order were: adenine greater than or equal to guanosine greater than inosine greater than or equal to adenosine greater than or equal to ATP. 2 Responses to the purine compounds were unaffected by propranolol (1 mug/ml). 3 The spasmolytic potencies of adenosine and ATP were greatly enhanced in the presence of the adenosine uptake blocking drugs dipyridamole, hexobendine or Dilazep, whereas responses to adenine were unaffected and those to inosine and guanosine were reduced. 4 The spasmolytic potencies of noradrenaline, aminophylline, prostaglandin E2 and glyceryl trinitrate were unaffected by dipyridamole, hexobendine and Dilazep. 5 It is suggested that an adenosine uptake process may exist in the trachea of the guinea-pig and that this process is inhibited by dipyridamole, hexobendine and Dilazep.

Role of CD73 in renal sympathetic neurotransmission in the mouse kidney

Jackson, Edwin K; Cheng, Dongmei; Mi, Zaichuan; Verrier, Jonathan D; Janesko-Feldman, Keri; Kochanek, Patrick M
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
EN
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Adenosine formed during renal sympathetic nerve stimulation (RSNS) enhances, by activating A1 receptors, the postjunctional effects of released norepinephrine and participates in renal sympathetic neurotransmission. Because in many cell types CD73 (ecto-5′-nucleotidase) is important for the conversion of 5′-AMP to adenosine, we investigated whether CD73 is necessary for normal renal sympathetic neurotransmission. In isolated kidneys from CD73 wild-type mice (CD73+/+; n = 17) perfused at a constant rate with Tyrode's solution, RSNS increased perfusion pressure by 17 ± 4, 36 ± 8, and 44 ± 10 mm Hg at 3, 5, and 7 Hz, respectively. Similar responses were elicited from kidneys isolated from CD73 knockout mice (CD73−/−; n = 13; 28 ± 11, 43 ± 10, and 44 ± 10 mm Hg at 3, 5, and 7 Hz, respectively); and a high concentration (100 μmol/L) of α,β-methyleneadenosine 5′-diphosphate (CD73 inhibitor) did not alter responses to RSNS in C57BL/6 mouse kidneys (n = 5; 21 ± 5, 36 ± 8, and 43 ± 9 at 3, 5, and 7 Hz, respectively). Measurements of renal venous adenosine and inosine (adenosine metabolite) by liquid chromatography-tandem mass spectrometry demonstrated that the metabolism of exogenous 5′-AMP to adenosine and inosine was similar in CD73−/− versus CD73+/+ kidneys. A1 receptor mRNA expression was increased in CD73−/− kidneys...

The control of adenosine concentration in polymorphonuclear leucocytes, cultured heart cells and isolated perfused heart from the rat.

Newby, A C; Holmquist, C A; Illingworth, J; Pearson, J D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/08/1983 EN
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Rat polymorphonuclear leucocytes or neonatal-rat heart cells in culture were treated with 2'-deoxycoformycin and 5-iodotubercidin at concentrations that inhibited adenosine deaminase (EC 3.5.4.4) and adenosine kinase (EC 2.7.1.20) inside the intact cells, and the rate of adenosine accumulation was determined. The basal rate of adenosine formation was 2% (polymorphonuclear leucocytes) or 9% (heart cells) of the maximal activity of adenosine kinase also measured in intact cells. Greatly increased rates of adenosine formation were observed during adenine nucleotide catabolism. This condition also led to a decrease in adenosine kinase activity. When isolated rat hearts were perfused with 5-iodotubercidin alone at a concentration which inhibited adenosine kinase, no increase in tissue or perfusate adenosine or inosine concentration was observed. However, perfusion with hypoxic buffer or infusion of adenosine into the coronary circulation at a rate (20 nmol/min) equivalent to 40% of the activity of adenosine kinase caused large increases in effluent perfusate adenosine and inosine concentrations. These data argue unanimously against the existence of a substrate cycle controlling adenosine concentration. They suggest instead that an increase in the rate of adenosine formation is the principal cause of elevations in adenosine concentration during ATP catabolism.

beta-deuterium kinetic isotope effects in the purine nucleoside phosphorylase reaction.

Guo, X M; Ashwell, M; Sinnott, M L; Krenitsky, T A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/09/1991 EN
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1. [2'-2H]Inosine was made from inosine by tetraisopropyldisiloxanyl protection of the 3'- and 5'-positions, oxidation with dimethyl sulphoxide and acetic anhydride, immediate NaB2H4 reduction of the oxo sugar product and inversion at C-2' of the resultant protected [2'-2H]arabino-inosine by trifluoromethanesulphonylation and reaction with caesium propionate, followed by deprotection. 2. The equilibrium-perturbation technique was used to measure beta 2H(V/K) for phosphorolysis of this compound by the purine nucleoside phosphorylase of Escherichia coli as a function of pH. 3. The pH variation indicates an intrinsic effect of 1.068 masked by isotopically silent steps near the pH optimum. 4. The similar pH variation of these beta-deuterium effects and the alpha-deuterium effects measured previously [Stein & Cordes (1981) J. Biol. Chem. 256, 767-772; Lehikoinen, Sinnott & Krenitsky (1989) Biochem. J. 257, 355-359] for this reaction provides the first experimental reassurance for the common assumption that pH changes merely mask and unmask the chemical steps in an enzyme-catalysed reaction, and do not detectably alter transition-state structure. 5. The dihedral angle between the C-H-2' bond and the electron-deficient p-orbital at the transition state is in the range 32-48 degrees...