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Y Natsumeda

Publications and source records attributed to Y Natsumeda.

52 records · Page 3Linked to original sources
PubMed1988

Insulin regulatory effects on purine- and pyrimidine metabolism in alloxan diabetic rat liver.

Aim of this study was to elucidate insulin regulatory effects on purine and pyrimidine metabolism. Livers of alloxan diabetic and insulin treated rats were freeze clamped and nucleotide pools measured using HPLC techniques. Activities of key enzymes of de novo and salvage pathways were analyzed with radioassays. In diabetic liver nucleotide triphosphate pools were reduced between 46 and 75% of controls, nucleotide monophosphate concentrations increased. Activities of de novo biosynthetic enzymes amidophosphoribosyltransferase, FGAM synthase, IMP dehydrogenase, GMP synthase, carbamoylphosphate synthase II were curtailed by 16-61%, those of salvage enzymes hypoxanthine-guanine-phosphoribosyltransferase, adenine-phosphoribosyltransferase, thymidine kinase also decreased to 31-58%. Insulin treatment for 2 and 7 days normalized nucleotide pools, activities of key enzymes of de novo and salvage pathways were increased between 2.4 and 4.1 fold compared to diabetic untreated. Activation of nucleic acid metabolism by insulin can be explained by the requirement for high energy phosphates of certain anabolic key enzymes in carbohydrate and lipid metabolism. Impaired synthesis in insulin deficiency of end products of guanylate and pyrimidine pathway required as substrates for a variety of enzymes synthesising membrane structures throw new light on the pathogenesis of some late complications of diabetic disease.

Animals↗
PubMed1987

The modulation of serine metabolism in hepatoma 3924A during different phases of cellular proliferation in culture.

The activities of 3-phosphoglycerate dehydrogenase and serine hydroxymethyltransferase increased markedly during the transition of hepatoma cells from a resting non-proliferating culture into the proliferating growth phase. Activities declined as cells reached confluency and entered the plateau growth phase. This pattern was paralleled by changes in [14C]serine incorporation into nucleic acids. The experiments support the hypothesis that the biosynthesis of serine is metabolically coupled to its utilization for nucleotide precursor formation in cancer cells.

Animals↗
PubMed1987

Purification of IMP dehydrogenase from rat hepatoma 3924A.

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 +/- 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Animals↗
PubMed1987

Salvage pathways as targets of chemotherapy.

This paper discussed the significance of the activities of purine and pyrimidine salvage enzymes in cancer cells and the targeting against them of chemotherapy. 1. The activities of salvage enzymes in the rat liver were orders of magnitude higher than those of the rate-limiting enzymes of de novo biosynthesis. A similar relationship was observed in rat hepatomas of different growth rates and in primary colon carcinoma in human. 2. The concentrations of nucleosides and nucleobases were measured in plasma, liver and hepatoma 3924A in the rat. The freeze-clamp method was required to determine the concentrations of these precursors in rat liver and hepatoma in a reliable and precise fashion because ischemia markedly altered the concentrations of nucleosides, nucleobases and, as shown earlier, nucleotides in these tissues. The results indicated that the liver markedly concentrated the purine precursors, hypoxanthine, guanine and adenine, but not thymidine, which was one-third that of the plasma. Uridine and deoxycytidine occurred in the same concentration as in plasma, but cytidine was 3-fold higher in liver. In the hepatoma in comparison to the liver the concentrations of the nucleosides and bases were altered and for some of the changes the enzymic differences between liver and hepatoma appeared to be accountable. 3. Kinetic parameters for purine and pyrimidine synthetic enzymes and for the substrates and co-factors were determined in liver and hepatoma 3924A. When enzymic activities were calculated at the tissue steady-state concentrations of the various ligands, the activities of the salvage enzymes were markedly higher than those of the rate-limiting enzymes. 4. Hepatoma cells were highly sensitive to the action of the transport inhibitor, dipyridamole, in lag and log phases. However, plateau phase cells lost their sensitivity to dipyridamole. 5. Amphotericin B rendered plateau phase cells sensitive to the inhibitory action of dipyridamole for the incorporation of thymidine. 6. Amphotericin B enhanced cytotoxicity of dipyridamole in hepatoma and human colon cancer HT-29 cells. 7. In these studies we discovered the decreased responsiveness to dipyridamole of plateau phase cells and the ability of amphotericin B to restore the sensitivity. Moreover, dipyridamole and amphotericin B were synergistic in their cytotoxic action in rat hepatoma cells and human colon cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Amphotericin B↗
PubMed1986

Decreased concentration of xanthine dehydrogenase (EC 1.1.1.204) in rat hepatomas.

Xanthine dehydrogenase (EC 1.1.1.204), the rate-limiting enzyme of purine degradation, was purified 642-fold to homogeneity from liver of male Wistar rats. Antibody was generated to the purified enzyme in white rabbits and was partially purified. For the immunotitration a radioassay of high sensitivity was developed to determine low enzyme activities. Titration curves with the antibody showed that the xanthine dehydrogenase enzyme protein amounts in slowly growing hepatoma 20 and rapidly growing hepatoma 3924A were 34 and 4% of those of normal liver, which was in good agreement with the decrease in the activity of the enzyme to 33 and 2%, respectively. The contents of flavin adenine dinucleotide, the essential cofactor of the enzyme, in the immunoprecipitates in hepatomas 20 and 3924A were 27 and 4% of that of the normal liver. This is the first report to provide immunological evidence that a decreased enzyme activity in rat hepatomas, that of xanthine dehydrogenase, was due to a decrease in the enzyme protein amount. The markedly decreased xanthine dehydrogenase activity and amount have far-reaching biochemical and pharmacological implications for the tumors.

Animals↗
PubMed1985

Direct assay method for inosine 5'-monophosphate dehydrogenase activity.

A rapid microassay method for the accurate measurement of the activity of inosine 5'-monophosphate dehydrogenase in crude tissue extracts was described. [8-14C]IMP and the radioactive products were separated by high-voltage electrophoresis in 0.1 M potassium phosphate buffer, pH 7.0, for 45 min. This separation method provides an analysis of the possible interfering reactions such as the metabolic conversion of the substrate IMP to inosine and adenylosuccinate, and the loss of the product XMP to xanthosine or GMP and to other metabolites. Low blank values were consistently obtained with this method because the XMP spot moves faster than the IMP spot. The major advantages of this assay method are direct measurement of IMP dehydrogenase activity in crude extracts, high sensitivity (with a limit of detection of 5 pmol of XMP production), high reproducibility (less than +/- 3.6%), low blank values (60-80 cpm), speed (2 h per 30 assays), and capability to measure activity in small amounts of tissue (10-50 mg wet wt).

Adenosine Diphosphate↗
PubMed1985

Regulation of purine and pyrimidine metabolism by insulin and by resistance to tiazofurin.

The purpose of this investigation was to elucidate the factors that regulate the pattern of gene expression in purine and pyrimidine metabolism in normal liver and hepatoma. For this purpose, the action of a hormone, insulin, and the development of resistance to a chemotherapeutic agent, tiazofurin, were studied. This investigation brought detailed evidence showing that in the rat insulin exerted a profound effect on liver purine and pyrimidine metabolism by regulating the concentrations of nucleotides through controlling the activities of strategic enzymes involved in their biosynthesis. When rats were made diabetic by alloxan treatment, in the average liver cell concentrations of ATP, GTP, UTP and CTP decreased to 66, 62, 54 and 63%, respectively, of those of normal liver. Administration of insulin for 2 days returned the hepatic nucleotide concentrations to normal range; further insulin treatment for an additional 5 days raised the concentrations of ATP, GTP, UTP and CTP to 197, 352, 412 and 792% of values observed in the liver of diabetic rats. In diabetic rats the hepatic activities of OMP decarboxylase, orotate phosphoribosyltransferase, uridine phosphorylase, uridine-cytidine kinase and uracil phosphoribosyltransferase decreased to 44, 48, 70, 36 and 41% of the activities of normal liver. Insulin treatment for 2 days returned activities to normal range. Continued insulin treatment for an additional 5 days increased the enzymic activities to 3.9- to 5.3-fold of those of the liver of the diabetic rats. The regulation by insulin treatment of the activities of enzymes of de novo and salvage synthesis of UMP should explain, in part at least, the decline and increase of the uridylate pool in diabetes and after insulin treatment. In the diabetic rat hepatic CTP synthetase, the rate-limiting enzyme of CTP biosynthesis, decreased to 53% and insulin administration for 2 days restored activity to normal range. Insulin treatment for an additional 5 days increased the synthetase activity to 4-fold of the values of the diabetic liver. Thus, the behavior of liver CTP synthetase activity is tightly linked with that of the CTP pool. In the diabetic rat liver, the activity of IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis, decreased to 24% of that of the normal liver. Insulin administration for 2 days returned the activity to normal range, yielding a 4.5-fold increase in the activity from the diabetic to the insulin-treated state.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Targets and markers of selective action of tiazofurin.

The molecular correlation concept proposed that IMP dehydrogenase activity should be a sensitive target of chemotherapy. This hypothesis received support from an array of evidence. IMP dehydrogenase has the lowest activity in purine biosynthesis; it is the rate-limiting enzyme in GTP production; the enzymic activity is transformation-and progression-linked; it is elevated in all examined animal and human neoplastic cells. The activity of GMP synthetase and the concentrations of GMP and dGTP were increased in cancer cells. Whereas guanine salvage has a high potential activity, the low guanine content may well curtail actual salvage capacity. Ribonucleotide reductase activity was two orders of magnitude lower than that of IMP dehydrogenase. Tiazofurin, a C-nucleoside, had marked cytotoxicity on hepatoma cells in vitro and was the first drug that as a single agent profoundly inhibited the proliferation of the subcutaneously inoculated solid hepatoma 3924A in the rat. The impact of tiazofurin administration in hepatoma cells was revealed in a cascade of biochemical alterations involving primary, secondary and tertiary targets and markers of this drug action. The primary target was IMP dehydrogenase where the active metabolite of tiazofurin, TAD, was thought to be absorbed to the NADH site of the enzyme. As a consequence, the enzymic activity declined rapidly to about 30-40% and returned to normal range by 36 to 48 hr after injection. The secondary targets and markers are the profoundly decreased pools of guanylates (GMP, GDP, GTP). Concurrently, the concentrations of IMP and PRPP were increased 8- to 15-fold. The elevated IMP pools were attributed to the de-inhibition of the AMP deaminase activity subsequent to the decline in GTP concentration. The rise in PRPP pools was attributed to the selective inhibition of GPRT and HPRT activities by the high IMP pool which did not affect APRT activity. This interpretation is supported by the 6- to 8-fold increase in the concentrations of guanine and hypoxanthine and the lack of change in the adenine pools inthe hepatomas after tiazofurin administration. The marked drop in NAD concentration which was drug dose- and time-dependent is attributed to the competition for NAD pyrophosphorylase activity by the precursors of NAD and tiazofurin monophosphate. The tertiary targets were dominated by the profound alterations in the concentrations of the dNTPs. This was characterized by a rapid and persistent drop (for 3 days) of the dGTP pool. The concentrations of dATP and dCTP also declined, but these alterations were less pronounced and the pools returned to normal after 2 days.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1985

Purine enzymology of human colon carcinomas.

The purpose of this study was to elucidate the purine enzymic programs of human primary colorectal carcinomas. Marked alteration in the enzymology of the human colon neoplasm clearly distinguished it from that of the normal colon mucosa. In the human colon mucosa, the activities of ribonucleotide reductase, inosine phosphate dehydrogenase, formylglycinamidine ribonucleotide synthetase, guanosine phosphate synthetase, and amidophosphoribosyltransferase were 0.042, 5.2, 5.6, 8.2 and 36.0 nmol/h/mg protein, respectively, and in the colon carcinomas the activities increased to 755, 575, 295, 280, and 294% of the normal values. The activities of the salvage enzymes, adenine and hypoxanthine-guanine phosphoribosyltransferases, were 310, 249, and 602 nmol/h/mg protein, respectively, whereas in the tumors, only the activity of adenine phosphoribosyltransferase was increased (2-fold). The markedly higher absolute enzymic capacity for salvage in the tumors accounts, in part at least, for the lack of chemotherapeutic success of inhibitors of enzymes of de novo synthesis that have been used in the clinical treatment of colorectal carcinomas. Combinations of inhibitors of de novo biosynthesis and blockers of the salvage enzymes or of salvage transport (e.g., dipyridamole) should improve the chemotherapy of colon neoplasms. Since in the colon carcinoma the activities of glutamine-utilizing enzymes (guanosine phosphate and formylglycinamidine ribonucleotide synthetase and amidophosphoribosyltransferase) were markedly increased, and the glutamine concentration was decreased (50%), treatment with an antiglutamine agent (e.g., acivicin) should be of relevance. Since the activity of ribonucleotide reductase, the rate-limiting enzyme of nucleic acid biosynthesis, was markedly increased in the colon neoplasms, combination chemotherapy might include drugs against this enzyme.

Colon↗
PubMed1984

Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide).

Tiazofurin, a C-nucleoside, was cytotoxic in hepatoma 3924A cells grown in culture with an LC50 = 7.5 microM. In the culture, a closely linked dose-related response of tumor cell-kill and depletion of GTP pools was observed after tiazofurin treatment. In rats carrying subcutaneously transplanted hepatoma 3924A solid tumors, a single intraperitoneal injection of tiazofurin (200 mg/kg) caused a rapid inhibition of IMP dehydrogenase (EC 1.2.1.14) activity and depleted GDP, GTP, and dGTP pools in the tumor; concurrently, the 5-phosphoribosyl 1-pyrophosphate (PRPP) and IMP pools expanded 8- and 15-fold, respectively. Tiazofurin decreased tumoral IMP dehydrogenase activity and dGTP pools in a dose-dependent manner over a range of 50-200 mg/kg; by contrast, the depletion of GTP and the accumulation of IMP and PRPP pools were near maximum at 50 mg/kg. The increase in PRPP pools may be attributed to an inhibition by IMP of the activity of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). The IMP dehydrogenase activity and the pools of ribonucleotides returned to the normal range by 24-48 h after the single injection of tiazofurin. However, the markedly depleted dGTP pools remained low for 72 h. Tiazofurin treatment resulted in significant anti-tumor activity in rats inoculated with hepatoma 3924A. The decrease in GTP levels and particularly the sustained depletion in the dGTP pools may explain, in part at least, the chemo-therapeutic action of tiazofurin on hepatoma 3924A. This is the first report showing that a marked therapeutic response was achieved against rapidly growing hepatoma 3924A by treatment with a single anti-metabolite.

Animals↗
PubMed1984

Control of enzymic programs and nucleotide pattern in cancer cells by acivicin and tiazofurin.

The mechanism of action of acivicin and tiazofurin was compared in hepatoma 3924A. The results were evaluated by assessing the impact of these drugs on primary targets, the activities of key enzymes, and on secondary and tertiary targets, the concentrations of pools of ribonucleotides and deoxyribonucleotides. The action of acivicin entails inhibition and inactivation of the key enzymes of glutamine utilization in the biosynthesis of purines and pyrimidines. As a result, the GTP and CTP pools were markedly depleted, whereas those of ATP and UTP were unaffected. Acivicin also markedly decreased the concentrations of all 4 deoxynucleoside triphosphates. The nucleotide pools returned to normal or near normal range within 2 to 3 days after a single acivicin injection. The pharmacologic targets of acivicin in anticancer chemotherapy include prominently the activities of glutamine-utilizing enzymes and the pools of GTP and CTP and all 4 dNTP's. These biochemical targets also serve as indicators of acivicin action in cancer cells. The action of tiazofurin in hepatoma cells entails the primary target, IMP dehydrogenase. The subsequent effects include marked enlargement of IMP and PRPP pools and depletion of the pools of GDP and GTP. The increased IMP concentration selectively inhibited the activities of hypoxanthine-guanine phosphoribosyltransferase, but did not affect that of adenine phosphoribosyltransferase. The markedly decreased GTP pool de-inhibited the activity of AMP deaminase which permitted the channeling of AMP to IMP. An important indicator of tiazofurin action is the prolonged depletion of dGTP pools and similar but less pronounced declines in the pools of dCTP and dATP. In contrast, dTTP pools were increased. The crucial biochemical targets and indicators of tiazofurin action in sensitive cancer cells include inhibition of IMP dehydrogenase, a decrease in the concentrations of GDP, GTP, dGTP, dCTP, dATP and marked rise in the pools of IMP, PRPP and dTTP. Measurements of the molecular targets and indicators of drug action should be helpful in identifying cancer cells and tissues sensitive or resistant to the action of acivicin or tiazofurin. Identification of the targets and indicators should also be helpful in the design of frequency of administration of the drugs in combatting animal and human neoplasia.

Animals↗
PubMed1984

Enzymic capacities of purine de Novo and salvage pathways for nucleotide synthesis in normal and neoplastic tissues.

The enzymic capacities of the de novo and the salvage pathways for purine nucleotide synthesis were compared in rat in normal, differentiating, and regenerating liver, and in three hepatomas of widely different growth rates. The activities of the key de novo and salvage enzymes were also determined in mouse lung and Lewis lung carcinoma, in human kidney and liver, and in renal cell carcinoma and hepatocellular carcinomas. A precise and reproducible assay was worked out for measuring the activities of adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) in crude liver and hepatoma systems. Kinetic studies on the salvage enzymes were carried out in the crude 100,000 X g supernatant fluid from normal liver and rapidly growing hepatoma 3924A. In both tissue extracts, Michaelis-Menten kinetics was observed for adenine phosphoribosyltransferase and HGPRT. The reciprocal plots for 5-phosphoribosyl-1-pyrophosphate (PRPP) of liver and hepatoma enzymes gave apparent KmS of 2 microM for adenine phosphoribosyltransferase and 4 microM for HGPRT, showing two orders of magnitude higher affinities for PRPP than that of the rate-limiting enzyme of de novo purine synthesis, amidophosphoribosyltransferase (EC 2.4.2.14) (Km = 400 to 900 microM). The apparent Km values for adenine of liver and hepatoma adenine phosphoribosyltransferase were 0.6 to 0.9 microM, respectively. For both liver and hepatoma HGPRT, the reciprocal plots for hypoxanthine and guanine yielded the same Km of 3 microM. The specific activities of purine phosphoribosyltransferases were markedly higher than that of amidophosphoribosyltransferase in rat thymus, spleen, testis, bone marrow, colon, liver, kidney cortex, lung, heart, brain, and skeletal muscle, but were lower in the small intestine. In hepatomas and regenerating and differentiating liver, the activities of the salvage enzymes were 2.1- to 32-fold higher than that of amidophosphoribosyltransferase. The purine phosphoribosyltransferase activities were also higher than that of amidophosphoribosyltransferase in Lewis lung carcinoma (8.2- to 32-fold), human renal cell carcinoma (3.5- to 22-fold), and hepatocellular carcinoma (3.4- to 30-fold). The high activities and the high affinity to PRPP of the purine phosphoribosyltransferases might explain the lack of linkage of the behavior of these enzymic activities with proliferation in normal, regenerating, differentiating, or neoplastic tissues. In contrast, the specific activity of the amidophosphoribosyltransferase, which is lower than that of the salvage enzymes, is linked with transformation as it is increased in all examined tumors.4

Adenine Phosphoribosyltransferase↗
PubMed1983

Salvage capacity of hepatoma 3924A and action of dipyridamole.

The role and behavior of the salvage enzymes in the biosynthesis of purines (adenine and hypoxanthine-guanine phosphoribosyltransferases) and pyrimidines (uridine-cytidine, deoxycytidine and thymidine kinases) were elucidated. In liver purine metabolism the transferase activities were orders of magnitude higher than the activities of the enzymes of de novo biosynthesis. In both purine and pyrimidine biosynthesis the activities of the enzymes of the de novo pathways were low (23 pmol to 70 nmol/hr/mg protein), whereas those of salvage synthetic pathways ranged from 0.8 to 1,470 nmol/hr/mg protein. In purine metabolism the salvage enzymes had markedly higher affinity to the shared substrate PRPP (4 to 40 microM) than the rate-limiting enzyme of de novo synthesis, amidophosphoribosyltransferase (900 microM). In rapidly growing hepatoma 3924A the activities of the enzymes of de novo purine biosynthesis increased, whereas those of the salvage pathway changed little. However, the activities of the enzymes of the salvage pathways remained much higher than those of the enzymes of de novo purine production. In pyrimidine production in the hepatomas the activities of both de novo and salvage enzymes markedly increased. However, the activities of the salvage enzymes far outstripped those of the enzymes of the de novo pathways. To inhibit the operation of the salvage pathways, the action of the transport inhibitor, dipyridamole, was examined. In tissue culture, dipyridamole inhibited the transport of purine and pyrimidine nucleosides with an IC50 of 10(-6) or 10(-7) M. As measured by colony-forming assay, dipyridamole killed hepatoma cells with an IC50 of 20 microM. Dipyridamole markedly depressed the pools of ATP, GTP, CTP and UTP; in combination chemotherapy with acivicin, an anti-glutamine agent, synergistic action was observed on the pools of nucleotides in hepatoma 3924A in vivo. These investigations emphasize the importance of the capacity to utilize precursors by the salvage enzymes and may explain, in part at least, the failure of inhibitors of the de novo pathways to yield lasting chemotherapeutic results. Combination chemotherapy of inhibitors of the de novo pathways with an inhibitor of the salvage pathways (dipyridamole) should impact on our understanding of the contribution of salvage pathways and provide a rational basis for successful combination chemotherapy of neoplastic diseases.

Animals↗
PubMedDate not supplied

Reciprocal alterations of GMP reductase and IMP dehydrogenase activities during differentiation in HL-60 leukemia cells.

The study was undertaken to elucidate the regulatory roles of GMP reductase (GMPR) and IMP dehydrogenase (IMPDH) on purine interconversion during differentiation. Treatment of HL-60 cells with retinoic acid (1 microM) induced granulocytic differentiation which was accompanied with a 2.4-fold increase in GMPR and 55% decrease in IMPDH activities. Maturation induced by 12-O-tetradecanoylphorbol 13-acetate or dimethylsulfoxide was also associated with similar reciprocal alterations. Incubation with guanosine (200 microM), which expands the guanine nucleotide pool, elevated GMPR (1.9-fold) and decreased IMPDH (73%) activities. The synchronous and opposing alterations in GMPR and IMPDH activities should amplify the metabolic response due to differentiation or guanylate pool expansion.

Adenine Nucleotides↗