Creatine kinase activity of urinary bladder and skeletal muscle from control and streptozotocin-diabetic rats

The urinary bladder depends on intracellular ATP for the support of a number of essential intracellular processes including contraction. The concentration of ATP is maintained constant primarily via the rapid transfer of a phosphate from creatine phosphate (CP) to ADP catalyzed by the enzyme creatine kinase (CK). Since muscular pathologies associated with diabetes are in part related to intracellular alterations in metabolism, we have characterized the CK activity in both skeletal muscle and urinary bladder from control and streptozotocin-diabetic rats.The following is a summary of the results: 1) Bladder tissue from control rats showed linear kinetics with a V_max = 390 nmoles/mg protein/min, and a K_m = 275 µM. 2) Urinary bladder tissue isolated from diabetic rats displayed biphasic kinetics with V_max = 65 and 324 nmoles/mg protein/min, and K_m's = 10 µM and 190 µM respectively. 3) Skeletal muscle isolated from control rats showed linear kinetics with an approximate V_max of 800 nmoles/mg protein/min and a K_m of 280 µM CP. 4) Homogenates of skeletal muscle from diabetic rats showed complex kinetics not separable into distict component forms. 5) The K_m for ADP for both skeletal muscle and bladder was approximately 10 µM.These studies demonstrate that whereas bladders isolated from both control and diabetic rats possess a low-affinity isomer(s) of CK with similar maximum enzymatic activity, there is a high affinity isomer present within the urinary bladder muscle of diabetic rats that is not present in bladder tissue isolated from control rats. Skeletal muscle isolated from both diabetic and control rats exhibited a maximal activity 2 to 3 times higher than that of the bladder.     

Inhibitory effect of high oxygen pressure on potassium- induced activation of pyruvate dehydrogenase and glucose metabolism in rat brain slices

The effects of high oxygen pressure on pyruvate dehydrogenase (pyruvate: lipoate oxidoreductase (decarboxylating and acceptor-acylating), EC 1.2.4.1) activity, tissue concentration of ATP, and CO2 production from glucose were studied in rat brain cortical slices. The increase in pyruvate dehydrogenase activity and the lowering of cellular ATP, occurring during potassium-induced depolarization at 1 atm of oxygen, were reversed by increasing the oxygen pressure to 5 atm. When brain slices were incubated at 1 atm oxygen with [U-14C]glucose, a high potassium medium approximately doubled the production of 14CO2. Oxygen at 5 atm abolished this potassium-dependent increase in 14CO2 production with no significant effect on glucose oxidation in normal Krebs-Ringer phosphate medium. Adding 4 atm helium to 1 atm oxygen did not interfere with the ability of potassium ions to activate pyruvate dehydrogenase, lower ATP, or increase glucose oxidation. The results show that toxic effects of hyperbaric oxygen, not manifest in "resting" tissue, may be revealed during stress such as potassium depolarization. The site of the toxic effects of oxygen is probably the cell membrane where excess oxygen appears to interfere with the action of the sodium pump, calcium transport or other processes stimulated by increased concentrations of extracellular potassium.     

Inhibitory effect of high oxygen pressure on potassiuminduced activation of pyruvate dehydrogenase and glucose metabolism in rat brain slices

The effects of high oxygen pressure on pyruvate dehydrogenase (pyruvate: lipoate oxidoreductase (decarboxylating and acceptor-acylating), EC 1.2.4.1) activity, tissue concentration of ATP, and CO₂ production from glucose were studied in rat brain cortical slices. The increase in pyruvate dehydrogenase activity and the lowering of cellular ATP, occurring during potassium-induced depolarization at 1 atm of oxygen, were reversed by increasing the oxygen pressure to 5 atm. When brain slices were incubated at 1 atm oxygen with [U-¹⁴C]glucose, a high potassium medium approximately doubled the production of ¹⁴CO₂. Oxygen at 5 atm abolished this potassium-dependent increase in ¹⁴CO₂ production with no significant effect on glucose oxidation in normal Krebs-Ringer phosphate medium. Adding 4 atm helium to 1 atm oxygen did not interfere with the ability of potassium ions to activate pyruvate dehydrogenase, lower ATP, or increase glucose oxidation. The results show that toxic effects of hyperbaric oxygen, not manifest in “resting” tissue, may be revealed during stress such as potassium depolarization. The site of the toxic effects of oxygen is probably the cell membrane where excess oxygen appears to interfere with the action of the sodium pump, calcium transport or other processes stimulated by increased concentrations of extracellular potassium.     

Pyrimidine biosynthesis in Pseudomonas oleovorans

AIMS: To investigate the regulation of de novo pyrimidine biosynthesis in the polyhydroxyalkanoate-producing bacterium Pseudomonas oleovorans at the level of enzyme synthesis and at the level of aspartate transcarbamoylase activity. METHODS AND RESULTS: The effect of pyrimidine supplementation on the pyrimidine biosynthetic pathway enzyme activities was analysed relative to carbon source. Two uracil auxotrophs of P. oleovorans were isolated that were deficient for aspartate transcarbamoylase or dihydroorotase activity. Pyrimidine limitation of these auxotrophs increased the de novo pathway activities to varying degrees depending on the pathway mutation and the carbon source utilized. At the level of aspartate transcarbamoylase activity, pyrophosphate and uridine ribonucleotides were found to be strongly inhibitory of the Ps. oleovorans enzyme. CONCLUSIONS: Pyrimidine biosynthesis is regulated in Ps. oleovorans. Taxonomically, the regulation of the pyrimidine biosynthetic pathway appeared dissimilar from previously studied Pseudomonas species. SIGNIFICANCE AND IMPACT OF THE STUDY: New insights regarding the regulation of nucleic acid metabolism are provided that could prove significant during the genetic manipulation of Ps. oleovorans to increase the synthesis of polyhydroxyalkanoates.     

Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics

The kinome of the human malaria parasite Plasmodium falciparum includes two genes encoding mitogen-activated protein kinase (MAPK) homologues, pfmap-1 and pfmap-2, but no clear orthologue of the MAPK kinase (MAPKK) family, raising the question of the mode of activation and function of the plasmodial MAPKs. Functional studies in the rodent malaria model Plasmodium berghei recently showed the map-2 gene to be dispensable for asexual growth and gametocytogenesis, but essential for male gametogenesis in the mosquito vector. Here, we demonstrate by using a reverse genetics approach that the map-2 gene is essential for completion of the asexual cycle of P. falciparum, an unexpected result in view of the non-essentiality of the orthologous gene for P. berghei erythrocytic schizogony. This validates Pfmap-2 as a potential target for chemotherapeutic intervention. In contrast, the other P. falciparum MAPK, Pfmap-1, is required neither for in vitro schizogony and gametocytogenesis in erythrocytes, nor for gametogenesis and sporogony in the mosquito vector. However, Pfmap-2 protein levels are elevated in pfmap-1(-) parasites, suggesting that Pfmap-1 fulfils an important function in asexual parasites that necessitates compensatory adaptation in parasites lacking this enzyme.     

Trypanosoma brucei: a survey of pyrimidine transport activities

Purine uptake has been studied in many protozoan parasites in the last few years, and several of the purine transporters have been cloned. In contrast, very little is known about the salvage of preformed pyrimidines by protozoa, and no pyrimidine transporters have been cloned, yet chemotherapy based on pyrimidine nucleobases and nucleosides has been as effective as purine antimetabolites in the treatment of infectious and neoplastic disease. Here, we surveyed the presence of pyrimidine transporters in Trypanosoma brucei brucei. We could not detect any mediated uptake of thymine, thymidine or cytidine, but identified a very high-affinity transporter for cytosine, designated C1, with a K(m) value of 0.048+/-0.009 microM. We also confirmed the presence of the previously reported U1 uracil transporter and found it capable of mediating uridine uptake as well, with a K(m) of 33+/-5 microM. A higher-affinity U2 uridine transporter (K(m)=4.1+/-2.1 microM) was also identified, but efficiency of the C1 and U2-mediated transport was low. Pyrimidine antimetabolites were tested as potential trypanocidal agents and only 5-fluorouracil was found to be effective. This drug was efficiently taken up by bloodstream forms of T. b. brucei.     

TOXIC EFFECTS OF OXYGEN ON CEREBRAL METABOLISM

Abstract— The effect of 100% O₂ at one atmosphere on carbohydrate metabolism in cell-free homogenates of rat brain was studied under different experimental conditions. The principal findings were the following:

• 1 Compared to 10% O₂-90% N₂, oxygen at one atmosphere inhibited metabolism of α-oxoglutarate and depressed the net synthesis of ATP. With glucose as substrate, accumulation of ATP was also markedly inhibited but substrate utilization was only slightly affected.

• 2 Glycolysis in brain was relatively resistant to oxygen toxicity, except in the presence of added Cu²⁺.

• 3 With α-oxoglutarate as the substrate, inhibition of the formation of ATP occurred earlier than inhibition of substrate utilization, indicating the particular sensitivity of oxidative phosphorylation to inactivation by oxygen in vitro.

• 4 Cu²⁺ and Fe²⁺ accentuated oxygen toxicity but appeared to act by different mechanisms. Co²⁺ exerted a protective effect.

• 5 The sulphydryl compounds, dithiothreitol and reduced glutathione, strongly diminished the toxic effect of oxygen.

     

Metabolic basis for contractile dysfunction following chronic partial bladder outlet obstruction in rabbits

Our study is designed to correlate nitrite concentration, an index of nitric oxide (NO) release with mast cell peroxidase (MPO), a marker of cardiac mast cell degranulation and cardioprotective effect of ischaemic preconditioning in isolated perfused rat heart subjected to 30 min of global ischaemia and 30 min of reperfusion. Ischaemic preconditioning, comprised of four episodes of 5 min global ischaemia and 5 min of reperfusion, markedly reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent and incidence of ventricular premature beats (VPBs) and ventricular tachycardia and fibrillation (VT/VF) during reperfusion phase. Ischaemia-reperfusion induced release of MPO was markedly reduced in ischaemic preconditioned hearts. Increased release of nitrite was noted during reperfusion phase after sustained ischaemia in preconditioned hearts as compared to control hearts. No alterations in the release of nitrite was observed immediately after ischaemic preconditioning. However, ischaemic preconditioning markedly increased the release of MPO prior to global ischaemia. It is proposed that cardioprotective and antiarrhythmic effect of ischaemic preconditioning may be ascribed to degranulation of cardiac mast cells. Depletion of cytotoxic mediators during ischaemic preconditioning and consequent decreased release of these mediators during sustained ischaemia-reperfusion may be associated with preservation of structures in isolated rat heart responsible for NO release.     

Inhibition of growth and decreased survival of B104 rat neuroblastoma cells after exposure to hyperbaric oxygen

Summary The toxic effects of hyperbaric oxygen (HBO) on growth and survival of B104 rat neuroblastoma cells were investigated. Cells in log phase growth were incubated at 37°C with 10 atm O₂ for 1 to 4 h. After exposure to HBO, cells were monitored for their subsequent growth and survival. Two hours of exposure caused a slowing of growth, which returned to normaly by the end of the 7th d of the postexposure period. Exposures to O₂ of 3 h or longer caused a complete cessation of growth for 4 d after the exposure and very litle or no recovery after this period. Increased hydrostatic pressure for 6 h using helium as the inert gas had no effect on growth. A colony formation assay was used to quantitative the degree of cell death induced by HBO. The resulting survival curve was of the exponential type with a broad shoulder between 0 to 2.5 h of exposure to 10 atm O₂. The curve fell off sharply at 2.5 h with an exponential decrease in survival when the exposure to HBO was extended to 4 h. At 2 h about 50% of cells were killed, but at 4 h only 2% survived the treatment. These results show that the depression of the growth rate by HBO is related to the number of cells that are killed by the exposure. This system provides a model in which the molecular and cellular effects of HBO can be investigated. This work was supported by the National Institutes of Health, Bethesda, MD, Grants AM-21423 and CA-14489, and by grants from the W. W. Smith Foundation and the Philadelphia Foundation. The studies presented here are part of a dissertation submitted by G. J. Gendimenico in partial fulfillment of the requirements for the degree of Ph.D. in pharmacology from the University of Pennsylvania.