Effect of calcium overload on the phosphoinositide breakdown in the rat left ventricular papillary muscle

We investigated the effect of Ca²⁺ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca²⁺ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca²⁺-containing medium following either Ca²⁺-containing or Ca²⁺-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [³H]inositol phosphates ([³H]IPs) in the tissues prelabeled with [³H]inositol. Ca²⁺ repletion following Ca²⁺-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [³H]IPs accumulations. Treatment with ouabain following Ca²⁺-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [³H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [³H]IPs were significantly potentiated by prior Ca²⁺-free superfusion instead of Ca²⁺-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [³H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca²⁺-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca²⁺, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.Abbreviations [³H]IPs [³H]Inositol Phosphates - IP Inositol Phosphate - IP₂ Inositol Bisphosphate - IP₃ Inositol Trisphosphate - PI Phosphatidylinositol - PI-4-P Phosphatidylinositol-4-phosphate - PI-4,5-P₂ Phosphatidylinositol 4,5-bisphosphate - PRZ Prazosin - ATR Atropine - INDO Indomethacin - min Minutes     

The effect of oxidants on biomembranes and cellular metabolism

During the reductive process in the tissues, the aerobes generate a number of oxidants. Unless these oxidants are reduced, oxidative damage and cell death would occur. Oxidation of plasma membrane lipids leads to autocatalytic chain reactions which eventually alter the permeability of the cell. The role of oxidative damage in the pathophysiology of diabetic complications and ischemic reperfusion injury of myocardium, especially the changes in the channel activity which may lead to arrhythmia have been studied. Hyperglycemia activates aldose reductase which could efficiently reduce glucose to sorbitol in the presence of NADPH. Since NADPH is also aldose required by glutathione reductase for reducing oxidants, its diversion would lead to membrane lipid oxidation and permeability changes which are probably responsible for diabetic complications such as cataractogenesis, retinopathy, neuropathy etc. Antioxidants such as butylated hydroxy toluene (BHT) and also reductase inhibitors prevent or delay some of these complications. By using patch-clamp technique in isolated frog myocytes, we have shown that hydroxy radicals generated by ferrous sulfate and ascorbate as well as lipid peroxides such as t-butyl hydroperoxide facilitate the entry of Na⁺ by oxidizing Na⁺-channels. Increased intracellular Na⁺ leads to an increase in Na⁺/Ca²⁺ exchange. The increased Na⁺ concentration by itself may produce electrical disturbance which would result in arrhythmia. Increased Ca²⁺ may affect proteases and may help in the conversion of xanthine dehydrogenase to xanthine oxidase, consequently increased production of super oxide radicals. Increased membrane lipid peroxidation and other oxygen free-radical associated membrane damage in myocytes has been demonstrated.     

Microbial transformation of rifamycin B: A new extracellular oxidase fromCurvularia lunata

Summary A highly active extracellular rifamycin oxidase was isolated fromCurvularia lunata var.aeri. The enzyme has a pH optimum of 6.5 and temperature optimum of 50°C.     

Actinomycetes inducing phytotoxic or fungistatic activity in a Douglas-fir Forest and in an adjacent area of repeated regeneration failure in Southwestern Oregon

Actinomycetes were isolated from the upper 1 - 3 cm of the soil layer in a well-developed forest and in an adjacent clearcut area where Douglas-fir [Pseudotsuga menziesii (MIRB.) Franco] regeneration had been impaired for two decades. The population density in the clearcut area was two times as high as that in the forested area. The percentage of actinomycetes that inhibited seed germination of the test plants was significantly higher in isolates obtained from the clearcut area than in those obtained from the forested area, and isolates from the clearcut showed five times the phytotoxic effect of those from the forest. Some actinomycete isolates, 4 % from the clearcut and 2.6 % from the forest, significantly reduced in vitro growth of two common ectomycorrhizal fungi of Douglas-fir,Laccaria laccata andHebeloma ovstuliniforme. Two actinomycete isolates from the clearcut reduced fungal growth by 40 % and 73 %. Reduction of the nutrient in the growth medium did not affect the antifungal activity of the actinomycetes. The data support the idea that, along with other factors, phytotoxic and antifungal actinomycetes may suppress natural regeneration or establishment of planted seedlings - either directly or. indirectly - through inhibition of seed germination or of mycorrhizal fungi.     

Reversible modulation of the mitochondrial ATP synthase with energy demand in cultured rat cardiomyocytes

The ATP synthase capacity of rat heart myocytes can be measured in sonicates of cultured cardiomyocytes. In these cells, transitions in ATP synthase capacity occur on changing to the anoxic or uncoupled state (drop in ATP synthase capacity of over 40%) or on electrically stimulating the cells to contract (rise of 70%). These changes occur rapidly (half time less than 1 min) and are completely reversed on returning to the original conditions. It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase. The naturally occurring inhibitor protein from mitochondria may be responsible for this regulation.     

Functional cysteinyl residues in human placental aldose reductase

Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 M urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species.     

Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA.

The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative.     

Glucagon and p21 ras enhance the phosphorylation of the same 38-kilodalton membrane protein from rat liver cells.

We had reported earlier the enhanced phosphorylation of a 38-kilodalton protein (p38) in rat liver plasma membrane by ras proteins. Now we show that glucagon increased the phosphorylation of the same protein. The nature and site(s) of phosphorylation were the same as those for the ras proteins. Both ATP and GTP could donate phosphate for the phosphorylation of p38. The stimulation of p38 phosphorylation by glucagon was guanine nucleotide dependent. This observation, together with our data on the stimulation of p38 phosphorylation by AIF4-, suggest the involvement of G proteins in the reaction. We also showed that glucagon stimulates the phosphorylation of p38 in vivo.     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.