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PubMed1981

Acute changes of myocardial norepinephrine and glycogen phosphorylase in ischemic and non-ischemic areas after coronary ligation in dogs.

The myocardial norepinephrine (NE) content and glycogen phosphorylase activity were measured before and after coronary artery ligation in dogs anesthetized with pentobarbital. Coronary artery ligation was performed by ligating a branch of the left anterior descending coronary artery (LAD). Before coronary artery ligation, the NE content in the circumflex area (778.05 +/- 37.83 ng.g-1 wet tissue) was significantly higher than that in the LAD area (630.57 +/- 35.75 ng.g-1 wet tissue). The NE content in the circumflex area (non-ischemic area) decreased significantly 1.5, 3, 30. 60, and 420 min after coronary artery ligation, while that in the LAD area (ischemic area) did not decrease significantly except for the NE level obtained after 420 min of coronary artery ligation. The glycogen phosphorylase activity increased significantly 1.5 min after coronary artery ligation in both non-ischemic and ischemic areas. From the foregoing results, it is suggested that NE release occurs in both ischemic and non-ischemic areas within at least 1.5 min after coronary artery ligation, and that within 60 min after coronary artery ligation the NE content decreases in the non-ischemic area but not in the ischemic area probably because of very limited washout of the released NE in the ischemic area.

Animals↗
PubMed1997

Cloning of purine nucleoside phosphorylase II gene from Bacillus stearothermophilus TH 6-2 and characterization of its gene product.

Bacillus stearothermophilus TH 6-2 has two kinds of purine nucleoside phosphorylases (Pu-NPases). The type I enzyme (Pu-NPase I) is a functional and structural homolog of eukaryotic purine nucleoside phosphorylases that catalyze the phosphorolysis of inosine, guanosine, and their derivatives. The type II enzyme (Pu-NPase II) is a minor enzyme that efficiently phosphorolyzes adenosine and its derivatives rather than other purine nucleosides like Escherichia coli Pu-NPase. The gene coding for Pu-NPase II (punB gene) has been cloned and sequenced from TH 6-2 strain. The deduced amino acid sequence of Pu-NPase II shared 54% identity with that of E. coli enzyme, while it had no significant homology to that of Pu-NPase I or eukaryotic enzymes. By producing the Pu-NPase II in E. coli cells, the Pu-NPase II has been purified and characterized.

Amino Acid Sequence↗
PubMed1999

The activity of thymidine phosphorylase correlates with tumor size and lymph nodes status in breast carcinoma.

The platelet-derived endothelial cell growth factor (PD-ECGF) is one of the potent angiogenic factors. Recently, its homology with thymidine phosphorylase (dThdPase), an enzyme involved in pyrimidine nucleoside metabolism, has been shown. In the present study, dThdPase activity was evaluated spectrophotometrically in 43 breast carcinomas and in 19 cases of non-neoplastic breast tissues. The mean dThdPase activity in breast cancer was almost six fold higher than in normal, non-neoplastic breast tissues (1.92 and 0.29 mumol thymine (T) x mg prot.-1 x h-1 respectively). The enzyme activity significantly correlated with axillary lymph node status (p = 0.0076) and with tumor size (p = 0.0099). Besides, the intratumoral microvessel density (MD) was evaluated using the CD 31 mouse anti-human monoclonal antibody, and there was no correlation between the level of enzymatic activity and a number of microvessels. The positive significant correlation of thymidine phosphorylase activity with prognostic factors in breast cancer patients with no relation to the number of microvessels needs further examination to confirm the prognostic significance of the level of dThdPase.

Analysis of Variance↗
PubMed2001

Glycogen phosphorylase inhibitors for treatment of type 2 diabetes mellitus.

Type 2 diabetes mellitus is a severe disease with large economic consequences, which is significantly under-diagnosed and incompletely treated in the general population. Control of blood glucose levels is a key objective in treating diabetic patients, who are most often prescribed one or more oral hypoglycaemic agents in addition to diet and exercise modification as well as insulin. In spite of the availability of different classes of hypoglycaemic drugs, treatment regimens are often unable to achieve an intensive degree of glucose control known to most effectively reduce the incidence and severity of diabetic complications. Hepatic glucose output is elevated in type 2 diabetic patients and current evidence indicates that glycogenolysis (release of monomeric glucose from the glycogen polymer storage form) is an important contributor to the abnormally high production of glucose by the liver. Glycogen phosphorylase is the enzyme that catalyses this release and recent advances in new inhibitors of this structurally and kinetically well studied enzyme have enabled work which further delineate the pharmacological and physiological consequences of inhibiting glucose production by this pathway. Most notably, these agents lower glucose in diabetic animal models, both acutely and chronically, appear to affect both gluconeogenic and glycogenolytic pathways and demonstrate potential for a beneficial effect on cardiovascular risk factors. Cumulatively, this information has bolstered interest and promise in glycogen phosphorylase inhibitors (GPIs) as potential new hypoglycaemic agents for treatment of type 2 diabetes mellitus.

Animals↗
PubMed2004

Pharmacokinetic modeling of species-dependent enhanced bioavailability of trifluorothymidine by thymidine phosphorylase inhibitor.

TAS-102, a new oral drug, is composed of an antitumor drug, alpha,alpha,alpha-trifluorothymidine (FTD), and its metabolic inhibitor, 5-chloro-6-(2-iminopyrrolidine-1-yl)methyl-2,4(1H,3H)-pyrimidinedione hydrochloride (TPI). It has been reported that the oral administration of TAS-102 increases the AUC of FTD in rodents and monkeys in different manners. In this study, a pharmacokinetic model was developed, in an attempt to evaluate the bioavailability of FTD in these animals after the co-administration of TPI. Since TPI inhibits FTD metabolism competitively, a time-dependent as well as concentration-dependent model for the hepatic intrinsic clearance of FTD was developed including the time courses of both FTD and TPI. Based on this modeling, we were able to quantitatively explain the TPI dose-dependent enhancement of AUC of FTD in monkeys, while little increase was observed in rats. These results are consistent to observations that thymidine phosphorylase (TPase) is predominantly expressed in monkeys; while uridine phosphorylase (UPase) is superior to TPase in rats. Since TPase is also predominantly expressed in humans, the pharmacokinetic model developed in this study can be used to explain the bioavailability of TAS-102 in humans.

Administration, Oral↗
PubMed1990

Three-dimensional structure of thymidine phosphorylase from Escherichia coli at 2.8 A resolution.

The three-dimensional structure of thymidine phosphorylase from Escherichia coli has been determined at 2.8 A resolution using multiple-isomorphous-replacement techniques. The amino acid sequence deduced from the deoA DNA sequence is also reported. Thymidine phosphorylase exists in the crystal as an S-shaped dimer in which the subunits are related by a crystallographic 2-fold axis. Each subunit is composed of a small alpha-helical domain of six helices and a large alpha/beta domain. The alpha/beta domain includes a six-stranded mixed beta-sheet and a four-stranded antiparallel beta-sheet. The active site has been identified by difference Fourier analyses of the binding of thymine and thymidine and lies in a cavity between the small and large domains. The central beta-sheet is splayed open to accommodate a putative phosphate-binding site which is probably occupied by a sulfate ion in the crystal.

Amino Acid Sequence↗
PubMed1999

Phosphorylase kinase: the complexity of its regulation is reflected in the complexity of its structure.

Intracellular glycogen stores are used to maintain blood-glucose homeostasis during fasting, are a source of energy for muscle contraction, and are used to support a broad range of cellular activities in most tissues. A diversity of signals accelerate glycogen degradation that are mediated by phosphorylase b kinase (Phk), which phosphorylates and thereby activates glycogen phosphorylase. Phk is among the most complex of the protein kinases so far elucidated. It has one catalytic (gamma) subunit and three different regulatory (alpha, beta, and delta) subunits, a molecular mass of 1.3 X 106 daltons, and each holoenzyme molecule is presumed to contain four molecules of each subunit. The three regulatory subunits inhibit the phosphotransferase activity of the gamma subunit. Ca2+ relieves inhibition via the delta subunit, which is identical to calmodulin but remains an integral component of the holoenzyme even when the [Ca2+] is lowered to nanomolar levels. Phosphorylation of the alpha and beta subunits by the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) also relieves inhibition of the gamma subunit and thereby activates the enzyme. The stimulatory effects of Ca2+ and phosphorylation appear to be structurally coupled and are cooperative. In addition, Phk is activated in vitro by autophosphorylation, limited proteolysis of the regulatory subunits, and various allosteric effectors and these may also be mechanisms of physiological importance. The molecular mechanisms of regulation are currently poorly understood, but new insights are beginning to emerge. This review discusses current knowledge and concepts of the structure, function and regulation of Phk.

Amino Acid Sequence↗
PubMed2002

Prognostic and predictive value of thymidine phosphorylase activity in early-stage breast cancer patients.

The antitumor effects of 5-fluorouracil (5-FU) and its derivatives depend upon the activity of nucleoside metabolic enzymes in tumor tissues. Thymidine phosphorylase (TP) converts 5'-deoxy-5-fluorouridine (5'-DFUR), an intermediate metabolite of capecitabine, to 5-FU. The relationship between TP expression in tumor tissues and patient survival was retrospectively examined in early-stage breast cancer patients treated with either oral 5'-DFUR administered for 6 months or surgery alone in a prospective randomized controlled trial. Thymidine phosphorylase expression in tumor cells and tumor-associated stromal (TAS) cells was examined by immunohistochemistry in 650 tissue samples from patients in this trial (n = 1217). Eight-year follow-up data showed that high TP expression in tumor cells was a significant prognostic indicator of a favorable outcome only for the patients in the 5'-DFUR group. Thus, TP expression was shown to be a predictive factor of 5'-DFUR efficacy. Conversely, a low TP expression in TAS cells was also a potent favorable prognostic indicator. These results on TP status in 2 tumor cell types could provide novel information for predicting prognosis for a patient subgroup, which would receive a probable therapeutic effect from 5'-DFUR, and presumably, from adjuvant therapy of capecitabine in early-stage breast cancer. Determination of TP status might also identify a patient subgroup whose prognosis is quite favorable even without adjuvant therapy. Further investigations on prognostic and predictive implications of TP activity in a clinical setting are warranted.

Administration, Oral↗
PubMedDate not supplied

The significance of thymidine phosphorylase expression in colorectal cancer.

We measured thymidine phosphorylase activity in colorectal cancer tissue and conducted immunostaining to investigate enzyme expression in the tumor tissue. The results showed a correlation between staining ratio of thymidine phosphorylase and cancer progression as well as a correlation between enzyme activity and staining ratios of cancer cells and stromal cells. Since enzyme activity levels can be judged by staining ratios, this method may be useful for assessing cancer malignancy.

Adenocarcinoma↗
PubMed1983

Anergy, zinc deficiency, and decreased nucleoside phosphorylase activity in patients with sickle cell anemia.

Cell-mediated immunity was evaluated in 26 patients with sickle cell anemia using skin tests for delayed hypersensitivity reactions. Patients with impaired delayed hypersensitivity reactions had lower zinc levels in plasma, erythrocytes, and neutrophils than patients with normal delayed hypersensitivity reactions or controls. The activity of nucleoside phosphorylase, an enzyme essential for T-lymphocyte function, was significantly lower in zinc-deficient patients with anergic sickle cell anemia. We conclude that zinc deficiency in patients with sickle cell anemia is associated with impaired delayed hypersensitivity. Decreased activity of nucleoside phosphorylase may be partially responsible for lymphocyte dysfunction in these patients.

Adult↗
PubMed1998

[A new inhibitor of pyrimidine phosphorylase].

5,5-Bis(hydroxymethyl)-2-oxo-[1-(2-trifluoromethyl)-3,3,3- trifluoropropionamido)-1-trifluoromethyl-2,2,2-trifluoroethyl- 1,3,2-dioxaphosphan (CA-423) is an in vitro inhibitor of the Escherichia coli uridine and thymidine phosphorylases. Unlike widely studied nucleoside analogues, this compound binds to the enzymes irreversibly. Its LD50 in mice was 40 mg/kg. Due to the involvement of pyrimidine phosphorylases in carcinogenesis and the relatively low toxicity of CA-423, it is promising for anticancer therapy.

Animals↗
PubMed1978

Adenosine deaminase and purine nucleoside phosphorylase activities in lymphocytes from patients with lung cancer.

The activity of adenosine deaminase (EC 3.5.4.4.) was significantly lower in lymphocytes from patients with lung cancer than in those from healthy subjects, whereas the activity of purine nucleoside phosphorylase (EC 2.4.2.1.) was significantly higher in lymphocytes from the patients than in those from normal controls. When the one activity was plotted against the other, the plots for patients with lung cancer were all outside the frame formed by the lower and higher limits of the standard deviation of the mean of normal activities of the two enzymes. The ratio of adenosine deaminase activity to purine nucleoside phosphorylase activity was lower in patients with lung cancer than in controls. The possible effect of this ratio on the function of lymphocytes was briefly discussed. These enzyme activities were suggested to be useful measures of the immune responsiveness of patients with lung cancer.

Adenosine Deaminase↗
PubMed1999

Regulation of glycogen synthesis in rat skeletal muscle after glycogen-depleting contractile activity: effects of adrenaline on glycogen synthesis and activation of glycogen synthase and glycogen phosphorylase.

We investigated the effects of insulin and adrenaline on the rate of glycogen synthesis in skeletal muscles after electrical stimulation in vitro. The contractile activity decreased the glycogen concentration by 62%. After contractile activity, the glycogen stores were fully replenished at a constant and high rate for 3 h when 10 m-i.u./ml insulin was present. In the absence of insulin, only 65% of the initial glycogen stores was replenished. Adrenaline decreased insulin-stimulated glycogen synthesis. Surprisingly, adrenaline did not inhibit glycogen synthesis stimulated by glycogen-depleting contractile activity. In agreement with this, the fractional activity of glycogen synthase was high when adrenaline was present after exercise, whereas adrenaline decreased the fractional activity of glycogen synthase to a low level during stimulation with insulin. Furthermore, adrenaline activated glycogen phosphorylase almost completely during stimulation with insulin, whereas a much lower activation of glycogen phosphorylase was observed after contractile activity. Thus adrenaline does not inhibit contraction-stimulated glycogen synthesis.

Animals↗
PubMed2000

Angiogenesis, thymidine phosphorylase, and resistance of squamous cell head and neck cancer to cytotoxic and radiation therapy.

Thymidine phosphorylase (TP), an enzyme involved in the thymidine synthesis and degradation, has been shown to promote tumor angiogenesis. Both TP expression and tumor vascularization are putative postoperative prognostic markers of cancer. Because of its bifunctional role, TP may have interactions with cytotoxic drugs or radiation via pathways requiring thymidine or prodrug activation. The microvessel score and TP expression were examined immunohistochemically on paraffin-embedded bioptical material from 94 locally advanced squamous cell head and neck carcinomas. All patients were treated with conventionally fractionated radiotherapy combined with induction (platinum- and 5-fluorouracil-based) or concurrent platinum chemotherapy. The follow-up of patients ranged from 6 to 108 months (median, 48 months). Nuclear TP expression was significantly associated with increased microvessel score (P < 0.0001, r = 0.45). A low percentage of cancer cells with nuclear TP expression in pretreatment biopsies was associated with a high rate of CR after combined chemoradiotherapy (P = 0.006) and induction chemotherapy (0.01). A better local relapse-free and overall survival was also observed in these patients (P = 0.001 and P = 0.0005, respectively). Biospies on the day after the delivery of 20 Gy of conventionally fractionated radiotherapy showed residual cancer cell nests, frequently of high vascularization and of intense nuclear TP reactivity. It is concluded that thymidine phosphorylase is associated with angiogenesis, with resistance to radiotherapy and cytotoxic therapy, and with poorer survival in squamous cell head and neck cancer. A strong rationale is provided for subsequent clinical trials of concurrent radiotherapy and chemotherapy with antiangiogenic agents or with specific TP inhibitors.

Antineoplastic Combined Chemotherapy Protocols↗
PubMedDate not supplied

[Phosphorescent analysis of the intramolecular dynamics of the muscle glycogen phosphorylase b].

Large-scale functionally significant changes in the intramolecular dynamics of muscle glycogen phosphorylase b (EC 2.4.1.1) in solution upon ligand binding, transition from dimeric to tetrameric form, temperature denaturation and aggregation were registered at room temperature using the tryptophan phosphorescence technique. It was shown that binding of glucose-1-phosphate (substrate, 0.25-4 mM) and glucose (competitive inhibitor, 0.5-8 mM) to the active site and temperature-induced protein aggregation decrease large-scale structural fluctuations of the protein matrix at the level of domains and subunits; whereas the transition of glycogen phosphorylase b to tetrameric form (R-conformation) leads to a dramatic increase in the structural flexibility of the peripheral parts of the protein globule.

Adenosine Monophosphate↗
PubMedDate not supplied

[Phosphorylase kinase: mathematic modeling].

A mathematical model of the dynamic behavior of phosphorylase kinase was devised. Based on the results obtained, the function of this protein is discussed. It is suggested that phosphorylase kinase doses in a cAMP-dependent manner additional portions of glucoso-l-phosphate, which the muscle cell receives in response to contraction.

Calcium↗
PubMed2000

Mitochondrial neurogastrointestinal encephalomyopathy: an autosomal recessive disorder due to thymidine phosphorylase mutations.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder defined clinically by severe gastrointestinal dysmotility; cachexia; ptosis, ophthalmoparesis, or both; peripheral neuropathy; leukoencephalopathy; and mitochondrial abnormalities. The disease is caused by mutations in the thymidine phosphorylase (TP) gene. TP protein catalyzes phosphorolysis of thymidine to thymine and deoxyribose 1-phosphate. We identified 21 probands (35 patients) who fulfilled our clinical criteria for MNGIE. MNGIE has clinically homogeneous features but varies in age at onset and rate of progression. Gastrointestinal dysmotility is the most prominent manifestation, with recurrent diarrhea, borborygmi, and intestinal pseudo-obstruction. Patients usually die in early adulthood (mean, 37.6 years; range, 26-58 years). Cerebral leukodystrophy is characteristic. Mitochondrial DNA (mtDNA) has depletion, multiple deletions, or both. We have identified 16 TP mutations. Homozygous or compound heterozygous mutations were present in all patients tested. Leukocyte TP activity was reduced drastically in all patients tested, 0.009 +/- 0.021 micromol/hr/mg (mean +/- SD; n = 16), compared with controls, 0.67 +/- 0.21 micromol/hr/mg (n = 19). MNGIE is a recognizable clinical syndrome caused by mutations in thymidine phosphorylase. Severe reduction of TP activity in leukocytes is diagnostic. Altered mitochondrial nucleoside and nucleotide pools may impair mtDNA replication, repair, or both.

Adult↗
PubMed1975

A study of the mechanism of action of phosphorylase B using isotope-labelled substrates.

We have measured the alpha-deuterium and alpha-tritium secondary kinetic isotope effects on the synthesis and phosphorolysis of glycogen catalyzed by glycogen phosphorylase B (EC 2.4.1.1). The isotope-labeled substrates glucose-1T-1-phosphate and 1C14-glucose-1D-1-phosphate were used in the experiments, together with 6T-glucose-1-phosphate and C14-glucose-1-phosphate as controls. Measurement by the double label technique showed that both effects studied were equal to zero within the limits of experimental error. The possible mechanism of action of phosphorylase B is discussed.

Alkaline Phosphatase↗