The Experts below are selected from a list of 132 Experts worldwide ranked by ideXlab platform
Stephen M. Downs - One of the best experts on this subject based on the ideXlab platform.
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Uptake and salvage of Hypoxanthine mediates developmental arrest in preimplantation mouse embryos.
Biology of reproduction, 1997Co-Authors: Mary Kathryn Dienhart, Marilyn J. O'brien, Stephen M. DownsAbstract:Preimplantation mouse embryos become arrested after first or second cleavage when cultured in Hypoxanthine-supplemented Whitten's medium. We present evidence that the Hypoxanthine-induced arrest is dependent on uptake and salvage of Hypoxanthine and depletion of phosphoribosylpyrophosphate (PRPP) levels. Hypoxanthine uptake increased during the 2-cell stage and was augmented by glucose. HPLC analysis of ['14C]Hypoxanthine metabolism revealed that Hypoxanthine was salvaged and converted to ATP and guanosine triphosphate (GTP), with a shift to more guanyl nucleotide production at the 3- to 4-cell stage. In embryos from mice with a null mutation for the salvage enzyme Hypoxanthine-guanine phosphoribosyltransferase, Hypoxanthine did not block development nor was it taken up by the embryos. Glucose, which is required for the Hypoxanthine-induced arrest, produced a 5.3-fold increase in PRPP levels at the 2-cell stage, which was eliminated by Hypoxanthine. We conclude that metabolism of Hypoxanthine to nucleotides mediates its inhibitory action on preimplantation mouse embryos via negative feedback on PRPP synthetase, ultimately resulting in decreased PRPP availability and arrest of other PRPP-dependent pathways. Finally, reversal of the block by EDTA and cAMP-elevating agents may be mediated by alterations in Hypoxanthine or glucose uptake, or by changes in the relative metabolism of Hypoxanthine.
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Cyclic AMP reversal of Hypoxanthine-arrested preimplantation mouse embryos is EDTA-dependent
Zygote (Cambridge England), 1996Co-Authors: Mary Kathryn Dienhart, Stephen M. DownsAbstract:Hypoxanthine can block preimplantation mouse embryo development in vitro at the 2- to 4-cell stages, and this has recently been shown to be reversed by cAMP-elevating agents. However, the extent of this Hypoxanthine-induced arrest is determined by the culture conditions and strain of mouse. Whitten's and KSOM/AA are two embryo culture media that support preimplantation development to the blastocyst stage. This study was undertaken to examine the influence of several components in these media on Hypoxanthine-arrested preimplantation mouse embryos and to test the hypothesis that reversal of the Hypoxanthine block by cAMP-elevating agents requires cooperative interaction with the chelator, EDTA. Initial experiments demonstrated that embryo development was blocked in the presence of Hypoxanthine in Whitten's medium but not in KSOM/AA; furthermore, removal of EDTA from KSOM/AA rendered this medium incapable of supporting high levels of development to blastocyst (9%), whereas high numbers of blastocysts (80%) formed in Whitten's medium, which does not contain the chelator. Consequently, Whitten's medium was used to test our hypothesis. It has previously been demonstrated that the phosphodiesterase inhibitor, IBMX, can reverse the developmental arrest imposed by Hypoxanthine in EDTA-supplemented Earle's basic salt solution, but in the present study the addition of IBMX to Whitten's medium resulted in a block to development and failed to reverse the Hypoxanthine arrest. These disparate effects can be explained by the presence or absence of EDTA. Supplementing Whitten's medium with EDTA reverses the IBMX effect, but not the Hypoxanthine-induced block. While IBMX alone is unable to reverse the Hypoxanthine block in Whitten's medium, development is greatly enhanced by the simultaneous addition of EDTA and IBMX. Similar results were obtained with the cAMP analogue, 8-AHA-cAMP. The data therefore support our hypothesis that the reversal of the Hypoxanthine-induced arrest by cAMP-elevating agents is critically dependent on the presence of EDTA. We contrast this with the situation in mouse oocytes, where the Hypoxanthine-induced meiotic arrest is not reversed by the addition of EDTA and/or cAMP-elevating agents.
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Hypoxanthine-maintained two-cell block in mouse embryos: dependence on glucose and effect of Hypoxanthine phosphoribosyltransferase inhibitors.
Biology of reproduction, 1991Co-Authors: Stephen M. Downs, Mark P. D. DowAbstract:The culture conditions under which Hypoxanthine maintains a two-cell block in preimplantation mouse embryos were assessed. Hypoxanthine prevented embryo development past the two-cell stage at concentrations as low as 30 nM, and this inhibitory activity required the presence of n-glucose. The action of Hypoxanthine plus n-glucose was reversed by glutamine and higher lactate. n-mannose substituted for n-glucose in supporting the inhibitory action of Hypoxanthine, but L-glucose, D-fructose, and 2-deoxyglucose were much less effective. Other purine derivatives such as inosine and adenosine, but not xanthosine or uric acid, also blocked development at the two-cell stage at a concentration of 30 1aM, and guanosine was inhibitory at higher doses. Assays of Hypoxanthine phosphoribosyltransferase (HPRT) activity in lysates of four-cell embryos determined that the drugs 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP), but not 6-azauridine (6-AzaU), prevented salvage of Hypoxanthine. In addition, MPTF and 6-MP produced a significant two-cell block, which did not depend upon the presence of Hypoxanthine or n-glucose; whereas 6-AzaU was without effect. When embryos were cultured 2 days in the presence or absence of n-glucose, Hypoxanthine salvage was significantly reduced in lysates of four-cell embryos exposed to n-glucose. n-glucose had no effect when added directly to the assay mixture. These data demonstrate that the ability of Hypoxanthine to block embryo development at the two-cell stage depends on the presence of n-glucose or other glycolyzable sugars and suggest that inhibition of the purine salvage pathway promotes the two-cell block.
Simon M. Jarvis - One of the best experts on this subject based on the ideXlab platform.
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Adenosine and Hypoxanthine transport in horse erythrocytes: evidence for a polymorphism in the transport of Hypoxanthine via a sodium‐dependent cotransporter
Experimental physiology, 1998Co-Authors: Simon M. Jarvis, Roger HarrisAbstract:The inward transport of two purines, adenosine and Hypoxanthine, at 37 degrees C by horse erythrocytes was compared. No mediated transport of adenosine was detected in horse erythrocytes, nor was saturable, high-affinity binding of the potent facilitated-diffusion inhibitor nitrobenzylthioinosine demonstrable in horse erythrocyte membranes. In contrast, erythrocytes from most horses possessed a saturable sodium-dependent Hypoxanthine transporter (apparent K(m), 100 +/− 28 microM; Vmax, 0.20 +/− 0.08 mmol (l cells)-1 h-1; means +/− S.E.M., n = 5). Guanine inhibited Hypoxanthine influx (apparent Ki, 24 +/− 6 microM), but adenine and xanthine had no effect. Unlike human erythrocytes, no sodium-independent Hypoxanthine transporter was detected in horse erythrocytes. There are, however, a small number of animals (approximately 15%) whose erythrocytes fail to transport Hypoxanthine. This variation appears to be under genetic control, but the precise nature of the control is unknown.
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Hypoxanthine uptake through a purine selective nucleobase transporter in trypanosoma brucei brucei procyclic cells is driven by protonmotive force
FEBS Journal, 1997Co-Authors: Harry P De Koning, Simon M. JarvisAbstract:The mechanism of purine nucleobase transport in procyclic cells of the protozoan parasite Trypanosoma brucei brucei was investigated. Hypoxanthine uptake at 22°C was rapid and saturable, exhibiting an apparent Km, of 9.3±2.0 μM and a Vmax of 4.5±0.8 pmol · (107cells)−1· s−1. All the natural purine nucleobases tested (Km 1.8–7.2 μM), as well as the purine analogues oxypurinol and allopurinol, inhibited Hypoxanthine influx in a manner consistent with the presence of a single high-affinity carrier. Nucleosides and pyrimidine nucleobases had little or no effect on Hypoxanthine influx. The uptake process was independent of extracellular sodium, but inhibited by ionophores inducing cytosolic acidification (carbonyl cyanide chlorophenylhydrazone, nigericin, valinomycin) or membrane depolarisation (gramicidin) as well as by the adenosine triphosphatase inhibitors N-ethylmaleimide and N, N′-dicyclohexylcarbodiimide. Using the fluorescent dyes bisoxonol and 2′, 7′-bis-(carboxyethyl)-5, 6-carboxy-fluorescein to determine membrane potential and intracellular pH (pHi, the rate of Hypoxanthine uptake was shown to be directly proportional to the protonmotive force. Similarly, under alkaline extracellular conditions Hypoxanthine uptake was reversibly inhibited alongside a reduction in protonmotive force. In addition, Hypoxanthine accelerated the rate of pH, recovery to pH7 after base-loading with NH4Cl, indicative of a proton influx concurrent with Hypoxanthine transport. Finally, after pretreatment of cells with N-ethylmaleimide, Hypoxanthine induced a slow membrane depolarisation, demonstrating that Hypoxanthine transport is electro-genic. These data show that Hypoxanthine uptake in T b. brucei procyclic cells is dependent on the protonmotive force, and are consistent with a nucleobase/H+-symporter model for this transporter.
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CHARACTERIZATION OF A SODIUM-DEPENDENT CONCENTRATIVE NUCLEOBASE-TRANSPORT SYSTEM IN GUINEA-PIG KIDNEY CORTEX BRUSH-BORDER MEMBRANE VESICLES
Biochemical Journal, 1994Co-Authors: Douglas A. Griffith, Simon M. JarvisAbstract:The characteristics of Hypoxanthine transport were examined in purified brush-border membrane vesicles isolated from guinea-pig kidney. Hypoxanthine uptake in the vesicles was specifically stimulated by both Na+ and an inside-negative potential, resulting in a transient accumulation of intravesicular Hypoxanthine. Na(+)-dependent Hypoxanthine influx was saturable (apparent Km 4.4 +/- 2.1 microM, Vmax. 128 +/- 29 pmol/min per mg of protein at 100 mM NaCl and 22 degrees C). Guanine, thymine, 5-fluorouracil and uracil inhibited Hypoxanthine uptake (Ki values 1-30 microM), but adenine and the nucleosides inosine and thymidine were without effect. Guanine competitively inhibited Na(+)-dependent Hypoxanthine influx, suggesting that it was a substrate for the active nucleobase transporter in guinea-pig renal membrane vesicles. A sigmoidal dependence between Hypoxanthine influx and Na+ concentration was obtained (KNa 13 +/- 2 mM; Hill coefficient, h, 2.13 +/- 0.14), suggesting that at least two Na+ ions are transported per Hypoxanthine molecule. This system differs from the Na(+)-nucleobase carrier in cultured LLC-PK1 renal cells, which has a stoichiometric coupling ratio of 1:1. These results represent the first demonstration of an active electrogenic nucleobase carrier in renal apical membrane vesicles.
Motoharu Kondo - One of the best experts on this subject based on the ideXlab platform.
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A Novel Cancer Therapy Based on Oxygen Radicals
Cancer research, 1995Co-Authors: Toshikazu Yoshikawa, Satoshi Kokura, Tainaka K, Yuji Naito, Motoharu KondoAbstract:The antitumor effect of oxygen radicals produced by Hypoxanthine and xanthine oxidase reaction was studied in an experimental rabbit model. VX2 carcinomas were transplanted into rabbit hind legs. Hypoxanthine was administered continuously through the ear vein, while xanthine oxidase was administered simultaneously through the femoral artery. As a result, Hypoxanthine and xanthine oxidase reacted only in the hind leg, and superoxide was produced in that area. The volume of the VX2 carcinoma was measured immediately prior to treatment and 7 days later. As an index of lipid peroxidation, thiobarbituric acid-reactive substances in the tumor tissue were measured 60 min following infusion of Hypoxanthine and xanthine oxidase. Tumor growth was suppressed significantly by the Hypoxanthine-xanthine oxidase reaction, and thiobarbituric acid-reactive substances in the tumor tissue infused with Hypoxanthine and xanthine oxidase were significantly increased. In addition, the antitumor effect of the Hypoxanthine and xanthine oxidase reaction was significantly inhibited by the administration of superoxide dismutase and catalase. Pathological examination showed that oxygen radicals produced by Hypoxanthine and xanthine oxidase reaction were selectively more destructive for VX2 carcinoma tissue than muscle tissue surrounding the tumor region. These results suggest that oxygen radicals produced by Hypoxanthine and xanthine oxidase reaction produce an anticancer effect and that the VX2 carcinoma used in this study was more sensitive to oxygen radicals than normal muscle tissue.
Mary Kathryn Dienhart - One of the best experts on this subject based on the ideXlab platform.
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Uptake and salvage of Hypoxanthine mediates developmental arrest in preimplantation mouse embryos.
Biology of reproduction, 1997Co-Authors: Mary Kathryn Dienhart, Marilyn J. O'brien, Stephen M. DownsAbstract:Preimplantation mouse embryos become arrested after first or second cleavage when cultured in Hypoxanthine-supplemented Whitten's medium. We present evidence that the Hypoxanthine-induced arrest is dependent on uptake and salvage of Hypoxanthine and depletion of phosphoribosylpyrophosphate (PRPP) levels. Hypoxanthine uptake increased during the 2-cell stage and was augmented by glucose. HPLC analysis of ['14C]Hypoxanthine metabolism revealed that Hypoxanthine was salvaged and converted to ATP and guanosine triphosphate (GTP), with a shift to more guanyl nucleotide production at the 3- to 4-cell stage. In embryos from mice with a null mutation for the salvage enzyme Hypoxanthine-guanine phosphoribosyltransferase, Hypoxanthine did not block development nor was it taken up by the embryos. Glucose, which is required for the Hypoxanthine-induced arrest, produced a 5.3-fold increase in PRPP levels at the 2-cell stage, which was eliminated by Hypoxanthine. We conclude that metabolism of Hypoxanthine to nucleotides mediates its inhibitory action on preimplantation mouse embryos via negative feedback on PRPP synthetase, ultimately resulting in decreased PRPP availability and arrest of other PRPP-dependent pathways. Finally, reversal of the block by EDTA and cAMP-elevating agents may be mediated by alterations in Hypoxanthine or glucose uptake, or by changes in the relative metabolism of Hypoxanthine.
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Cyclic AMP reversal of Hypoxanthine-arrested preimplantation mouse embryos is EDTA-dependent
Zygote (Cambridge England), 1996Co-Authors: Mary Kathryn Dienhart, Stephen M. DownsAbstract:Hypoxanthine can block preimplantation mouse embryo development in vitro at the 2- to 4-cell stages, and this has recently been shown to be reversed by cAMP-elevating agents. However, the extent of this Hypoxanthine-induced arrest is determined by the culture conditions and strain of mouse. Whitten's and KSOM/AA are two embryo culture media that support preimplantation development to the blastocyst stage. This study was undertaken to examine the influence of several components in these media on Hypoxanthine-arrested preimplantation mouse embryos and to test the hypothesis that reversal of the Hypoxanthine block by cAMP-elevating agents requires cooperative interaction with the chelator, EDTA. Initial experiments demonstrated that embryo development was blocked in the presence of Hypoxanthine in Whitten's medium but not in KSOM/AA; furthermore, removal of EDTA from KSOM/AA rendered this medium incapable of supporting high levels of development to blastocyst (9%), whereas high numbers of blastocysts (80%) formed in Whitten's medium, which does not contain the chelator. Consequently, Whitten's medium was used to test our hypothesis. It has previously been demonstrated that the phosphodiesterase inhibitor, IBMX, can reverse the developmental arrest imposed by Hypoxanthine in EDTA-supplemented Earle's basic salt solution, but in the present study the addition of IBMX to Whitten's medium resulted in a block to development and failed to reverse the Hypoxanthine arrest. These disparate effects can be explained by the presence or absence of EDTA. Supplementing Whitten's medium with EDTA reverses the IBMX effect, but not the Hypoxanthine-induced block. While IBMX alone is unable to reverse the Hypoxanthine block in Whitten's medium, development is greatly enhanced by the simultaneous addition of EDTA and IBMX. Similar results were obtained with the cAMP analogue, 8-AHA-cAMP. The data therefore support our hypothesis that the reversal of the Hypoxanthine-induced arrest by cAMP-elevating agents is critically dependent on the presence of EDTA. We contrast this with the situation in mouse oocytes, where the Hypoxanthine-induced meiotic arrest is not reversed by the addition of EDTA and/or cAMP-elevating agents.
Toshikazu Yoshikawa - One of the best experts on this subject based on the ideXlab platform.
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A Novel Cancer Therapy Based on Oxygen Radicals
Cancer research, 1995Co-Authors: Toshikazu Yoshikawa, Satoshi Kokura, Tainaka K, Yuji Naito, Motoharu KondoAbstract:The antitumor effect of oxygen radicals produced by Hypoxanthine and xanthine oxidase reaction was studied in an experimental rabbit model. VX2 carcinomas were transplanted into rabbit hind legs. Hypoxanthine was administered continuously through the ear vein, while xanthine oxidase was administered simultaneously through the femoral artery. As a result, Hypoxanthine and xanthine oxidase reacted only in the hind leg, and superoxide was produced in that area. The volume of the VX2 carcinoma was measured immediately prior to treatment and 7 days later. As an index of lipid peroxidation, thiobarbituric acid-reactive substances in the tumor tissue were measured 60 min following infusion of Hypoxanthine and xanthine oxidase. Tumor growth was suppressed significantly by the Hypoxanthine-xanthine oxidase reaction, and thiobarbituric acid-reactive substances in the tumor tissue infused with Hypoxanthine and xanthine oxidase were significantly increased. In addition, the antitumor effect of the Hypoxanthine and xanthine oxidase reaction was significantly inhibited by the administration of superoxide dismutase and catalase. Pathological examination showed that oxygen radicals produced by Hypoxanthine and xanthine oxidase reaction were selectively more destructive for VX2 carcinoma tissue than muscle tissue surrounding the tumor region. These results suggest that oxygen radicals produced by Hypoxanthine and xanthine oxidase reaction produce an anticancer effect and that the VX2 carcinoma used in this study was more sensitive to oxygen radicals than normal muscle tissue.