Intracellular distribution of NADP-linked isocitrate dehydrogenase,fumarase and citrate synthase in bovine heart muscle

By fractional extraction of minced bovine heart muscle with iso-osmotic sucrose and phosphate buffer solutions, it is shown that less than 4% of the total citrate synthase in the tissue is in the cytosol. Using citrate synthase as a marker for broken mitochondria, two methods of fractionation of 750 × g supernatants from homogenates of bovine heart muscle show that 10% of the total fumarase and NADP-linked isocitrate dehydrogenase activities are present in the cytoplasm. Homogenates prepared by sonication and osmotic shock and by sand-grinding gave closely similar results as regards enzyme distributions and extent of mitochondrial breakage. The results are compared with those reported for other tissues.     

Purification and partial characterization of citrate synthase from Phaseolus vulgaris mitochondria

Citrate synthase (E.C. 4.1.3.7) has been isolated from bean mitochondria by an improved procedure. The purified enzyme had a specific activity of 50. In most respects (e.g. sedimentation constant, K_ms, pH sensitivity and ionic strength inhibition) the enzyme is similar to that prepared from mammalian sources. The feature distinguishing the plant enzyme from the others was its inhibition by several sulfhydryl reagents. The substrates conferred either complete protection (acetyl coenzyme A) or partial protection (oxalacetic acid) against the inhibition. Dithiothreitol (DTT) was capable of partially reversing the inhibition. The efficacy of DTT varied with the sulfhydryl reagent and was inversely related to the period of incubation of the enzyme with the reagent.     

A tailed primers protocol to identify the association of eNOS gene variable number of tandem repeats polymorphism with ischemic stroke in Chinese Han population by capillary electrophoresis

Endothelial nitric oxide synthase (eNOS) plays an important role in mediating endothelium-dependent vasodilatation and antithrombotic action and is thus involved in the development of ischemic stroke (IS). Controversial results regarding the association of eNOS gene variable number of tandem repeats (VNTR) polymorphism with IS have been reported by conventional PCR-polyacrylamide gel electrophoresis methods. We aimed to identify any common association of eNOS gene VNTR polymorphism with IS in Chinese Han population by capillary electrophoresis (CE). The VNTR polymorphism of 27 bp within the eNOS intron-4 was determined by CE with specially designed tailed primers in Chinese Han patients with IS (n = 457) and matched elderly controls without IS (n = 457). Significant differences in BMI, WHR, hypertension, diabetes, smoking, TG, HDL, LDL, LDL, and FBG were observed between cases and controls. The distributions of eNOS VNTR polymorphism were not significantly associated with IS after adjustment for cardiovascular risk factors (OR = 1.18, 95% CI: 0.82–1.69). This finding was consistent with the further meta-analysis in Asians. The meta-analysis in Americans demonstrated that 4a/4b + 4a/4a genotype was significantly associated with IS risk with an OR of 1.54 (95% CI, 1.09–2.17) compared with the 4b/4b genotype. Our data suggests that BMI, WHR, hypertension, diabetes, smoking, TG, LDL, and FBG may increase the risk of IS. However, eNOS VNTR polymorphism may be not an independent major contributor for IS in Chinese Han population. The VNTR polymorphism might be associated with IS in Americans based on meta-analysis.     

Field investigations of ammonia exchange between barley plants and the atmosphere. II. Nitrogen realiocation, free ammonium content and activities of ammonium-assimilating enzymes in different leaves

The activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) in different leaves of field-grown spring barley were measured during the reproductive growth phase in 2 consecutive years. Concurrently, the contents of soluble ammonium ions and free amides in the leaves were determined. The studies were carried out to investigate the relationship between variations in these parameters and emission of NH3 from the plant foliage. GS and GOGAT activities declined very rapidly with leafage. The decline in enzyme activities was followed by an increase in soluble ammonium ions and amides in the leaf tissues. During the same period, about 75% of leaf and stem nitrogen was reallocated to the developing ear. The amount of NH₃ volatilized from the foliage during the reproductive growth phase amounted to about 1% of the reallocated nitrogen. The experimental years were characterized by very favourable conditions for grain dry matter formation and for re-utilization of nitrogen mobilized from leaves and stems. Ammonia volatilization occurring under conditions with declining GS and GOGAT activities and increasing tissue concentrations of NH₄⁺ may be useful in protecting the plant from accumulation of toxic NH₃ and NH₄⁺ concentrations in the tissues.     

Inhibitory Effect of Aryl Thienyl-Ketones and - Thioketones on Arachidonic Acid-induced Malondialdehyde Formation in Human Platelets: Biological Data and Molecular Modelling

Abstract

A series of anti-thrombotic aryl thienyl-ketones and -thioketones was assayed in vitro for their inhibitory effect on malondialdehyde (MDA) production induced by arachidonic acid in human platelets. For several compounds MDA formation was strongly inhibited indicating that the anti-platelet target was situated on the cyclooxygenase pathway. A comparison between the inhibition constant K₁ and the IC₅₀ values revealed competitive inhibition kinetics. The molecular structure of one active compound was analysed by X-ray diffraction and theoretical calculations to provide information on its electronic and lipophilic properties.     

Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

Mitochondria: Role of citrulline and arginine supplementation in MELAS syndrome

Mitochondria are found in all nucleated human cells and generate most of the cellular energy. Mitochondrial disorders result from dysfunctional mitochondria that are unable to generate sufficient ATP to meet the energy needs of various organs. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a frequent maternally inherited mitochondrial disorder. There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS syndrome and results in impaired blood perfusion that contributes significantly to several complications including stroke-like episodes, myopathy, and lactic acidosis. Both arginine and citrulline act as NO precursors and their administration results in increased NO production and hence can potentially have therapeutic utility in MELAS syndrome. Citrulline raises NO production to a greater extent than arginine, therefore, citrulline may have a better therapeutic effect. Controlled studies assessing the effects of arginine or citrulline supplementation on different clinical aspects of MELAS syndrome are needed.     

The mechanism of the hyperglycaemic action of synthetic peptides related to the C-terminal sequence of human growth hormone

Synthetic part sequences of human pituitary growth hormone (hGH 176–191 and hGH 177–191) corresponding to residues 176–191 or 177–191 of the hormone have been tested for their effects on glycogen and pyruvate metabolism in the rat, both in vivo and in vitro. When injected, the peptides caused transient increases in blood glucose and lactate, while decreasing the activity ratio of glycogen synthase in muscle, adipose tissue and liver and of pyruvate dehydrogenase in muscle and adipose tissue, but not in liver. These decreases were associated with the conversion of the enzymes from their active to their inactive forms, since the peptides did not affect the total amount of either the synthase or the dehydrogenase. The time course of the effect on the enzymes was similar to that for the effect on blood metabolites, and responses for synthase were produced over the range 0.07–7 nmols hGH 177–191/kg body weight. Phosphorylase activity was not affected by the peptides, nor was the capacity to dispose of injected L-lactate. Experiments with adipocytes and hepatocytes showed that the peptides also affected glycogen synthase and pyruvate dehydrogenase activities in vitro. The peptides had no effect on the overall rate of gluconeogenesis from lactate by hepatocytes. However, at times corresponding to those at which glycogen synthase was inactivated, the peptides caused increased incorporation of lactate into free glucose and decreased incorporation into glycogen. It was concluded that the peptides acted directly on their target tissues, and that the observed hyperlactataemia was the result of the inactivation of pyruvate dehydrogenase. The addition lactate increased the flux through the gluconeogenic pathway, and appeared as glucose because the peptide also inactivated glycogen synthase. Thus, the hyperglycaemia produced by hGH 177–199 and related peptides is explicable in terms of a modified Cori Cycle.     

2,3-Bisphosphoglycerate,fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.