• Volume 10, Issue 3

      September 1986,   pages  283-422

    • Alteration in the metabolism of free radicals in wheat seedlings grown in the presence of 4-chloro-5-dimethylamino-2-phenyl-3(2H) pyridazinone

      R Mannar Mannan Salil Bose

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      When wheat seedlings were grown in the presence of 62.5-500μM 4 chloro-5-dimethylamino-2-phenyl-3(2H) pyridazinone, an inhibitor of photosystem II electron transport, there was a marked inhibition of in vivo photosystem II electron transport as revealed by the analysis of fast chlorophyll a fluorescence transients in intact leaves and by the inhibition (95% at 500μM) of net photosynthesis in intact leaves Accompanying this inhibition of photosystem II electron transport, there was a decrease in the content of photosynthetic pigments. The extent of lipid peroxidation, measured in terms of malondialdehyde content was not increased; rather it was found decreased. An analysis of in vitro lipid peroxidation of the thylakoid membranes of control and 4-chloro-5-dimethylamino-2-phenyl-3(2H) pyridazinone treated plants in the presence of a sensitizer dye (toluidine blue) showed a similar rate both in the control and treated samples suggesting that the availability of unsaturated fatty acids as a substrate for lipid peroxidation was not limiting even though it decreased in the treated plants. On the other hand, it appears that the availability of the free radicals for lipid peroxidation was decreased byenhanced activity of the enzyme systems involved in the metabolism of free radicals. Measurements of the activities of enzymes involved in the metabolism of free radicals showed an increase in the activities of NADPH-glutathione reductase (6–8 fold) and catalase (15–30%) and a decrease in the activity of superoxide dismutase (30–45%) in the treated plants.

    • Mitochondrial functions during experimentally induced cardiac hypertrophy

      C Thirunavukkarasu C Rajamanickam

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      When the hearts of albino rats are subjected to pressure-induced stress through constriction of ascending aorta, changes in the mitochondrial functions are observed as early as 24 h after the imposition of the stress. These include the abolition of oxidative phosphorylation, decrease in the energy dependent [45Ca]-uptake and decrease in the rate of energized swelling. A large influx of calcium ions and an increase in the fluidity of mitochondrial membranes also occur in this period. At later stages of hypertrophy (17, 28, 40%), these mitochondrial functions gradually return to normal levels.

    • Iodide transport in isolated cells of mouse submaxillary gland

      R K Banerjee A K Bose T K Chakraborty P K De A G Datta

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      A method has been developed to isolate cells from the submaxillary gland of mouse by treatment with pronase. Three fractions of cells have been isolated having almost equal iodide concentrating activity. The isolated cells show time dependent uphill transport of iodide. The transport is substrate-saturable, having aKm value of 0.3 μM for iodide. The transport is sensitive to antithyroid drugs, metabolic inhibitors and to some extent to ouabain. Pseudohalide such as thiocyanate competes with the transport of iodide. Thyroid hormones or thyroid stimulating hormone have no significant effect on the iodide transport in these cells.

    • Hydrogen peroxide formation and lipid peroxidation in rat uterus—effect of hormones and vitamin E

      Shyamali Mukherjee Mihir Nag Tultul Nayyar Indrani Maitra Parul Chakrabarti Pramod R Dasgupta

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      The effect of estradiol-17β and progesterone given separately as well as in combination on the rate of hydrogen peroxide formation and lipid peroxidation in the uteri of ovariectomized rats was studied. Estradiol in 3μg dose per day per animal elicited maximum stimulatory response and progesterone (100μg), on the other hand, was without any such effect. However, progesterone given along with estradiol completely prevented the effect due to the latter. In the same way, vitamin E, a well known antioxidant was found to be extremelv effective in protecting the uterus from the highly peroxidative action of estradiol-17β.

    • Ontogeny of human fetal catalase superoxide dismutase and lipid peroxidation: A comparative study

      Kathakali De(Addya) Diptis Sengupta

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      A comparative study on the activity profile of catalase and superoxide dismutase, the two scavenging enzymes, as well as the developmental profile of lipid peroxidation in the human fetal brain, liver and kidney have been done for gestation periods ranging from 12 weeks to 28 weeks and beyond. The activity of the scavenging enzymes increase gradually inall the tissues with the advancement of pregnancy. Brain is an exception in case of catalase where the activity remains more or less same throughout the developmental period except in the case of fetuses, 28 weeks and above where significant decrease in the catalase activity is observed. A high level of lipid peroxidation is observed during early stages of development which declines thereafter.

    • Purification and characterization of tubulin from the catfishHeteropneustes fossilis

      Anuradha Chaudhuri

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      Tubulin was purified from the brain of the catfishHeteropneustes fossilis by cycles of temperature-dependent assembly and disassembly. Fish tubulin assembles into microtubules in the absence of high molecular weight microtubule associated proteins. Its subunits comigrate with goat brain α andβ tubulin subunits and is composed of 4 major α andβ tubulins each as analyzed by isoelectric focusing and two dimensional gel electrophoresis. Peptide mapping showed it to be very similar to goat brain tubulin. Polymerization of catfish brain tubulin occurs optimally between 18–37°C and the critical protein concentrations of assembly at 18°C and 37°C are the same, as opposed to mammalian brain tubulins.

    • Effect of supplementation with exogenous fatty acid on the biological properties of a fatty acid requiring auxotroph ofSalmonella typhimurium

      J K Deb S K Biswas P Chakrabati M Chakravorty

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      The effects of changes in fatty acid composition of the cell membrane on different biological functions ofSalmonella typhimurium have been studied with the help of a temperature sensitive fatty acid auxotroph which cannot synthesise unsaturated fatty acids at high temperature. On being shifted to nonpermissive temperature the cells continue growing for another one and half to two generations. The rates of protein and DNA syntheses run parallel to the growth rate but the rate of RNA synthesis is reduced. Further, there is a gradual reduction in the rate of transport of exogenous uridine and thymidine into the soluble pool. The transport process can be restored by supplementing the growth medium with cis-unsaturated fatty acids but not trans-unsaturated ones although the growth of the cells is resumed by supplementation with eithercis or trans-unsaturated fatty acids. However, supplementation withtrans, trans-unsaturated fatty acids leads to only partial recovery of the transport process. The rate of oxygen uptake is also affected in cells grown in the presence of thetrans-unsaturated fatty acids, elaidic acid and palmitelaidic acid. Analysis of cells grown under different fatty acid supplementation indicate that fatty acid composition of the cell membrane, especially the ratio of unsaturated to saturated fatty acids varies with temperature shift and supplementation of the growth media with fatty acids.

    • Use of epoxysepharose for protein immobilisation

      G S Murthy N R Moudgal

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      Epoxy Sepharose, an activated affinity matrix which has been used for immobilisation of carbohydrates has been tried for immobilisation of proteins. Under normal conditions of coupling at neutral or alkaline pH proteins do not couple to epoxy Sepharose. However, a very high salt concentration during coupling allows the binding of proteins to epoxy Sepharose at a pH as low as 8.5. Increasing ionic strength and/or pH facilitates the binding. The bioactivity of the proteins is not destroyed by the immobilisation. This matrix, unlike cyanogen bromide-Sepharose, retains its ability to bind albumin by 80–90% even after 60 days of storage in aqueous suspension at 4°C. Its capacity to bind proteins is comparable to that of cyanogen bromide-Sepharose.

    • Interaction of proteins with detergents: Binding of cationic detergents with lysozyme

      M Subramanian B S Sheshadri M P Venkatappa

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      Binding studies of cationic detergents such as cetyl trimethylammonium bromide, Cetylpyridinium bromide and dodecyl trimethylammonium bromide with lysozyme were carried out by equilibrium dialysis, ultraviolet difference and circular dichroism techniques at 25 C. Binding isotherms at pH 5·0, 7·0 and 9·0 show cooperative binding at all concentrations of detergents and the number of available binding sites in lysozyme increases with pH. Gibbs free energy of binding calculated on the basis of Wymans’ binding potential concept increases with pH indicating increased binding strength at higher pH. The ultraviolet difference spectra of the detergent complexes with lysozyme at pH 7·0 and 9·0 in the region of 250–300 nm indicate the involvement of aromatic amino acid residues as probable binding sites and also the carboxylate groups since the binding is cooperative. The circular dichroism spectra also indicate the involvement of aromatic amino acid residues in the binding of these detergents. This is substantiated by the decrease in the intensity of the aromatic positive bands in the near ultraviolet region. The increase in the magnitude of [θ]222 nm values in the far ultraviolet region with the increase in the concentration of the detergent in the complex indicates conformational changes resulting in an increase of α-helical content producing a more ordered structure of lysozyme.These binding studies show that at pH 7·0 and 9·0, hydrophobic interactions play a major role, while at pH 5·0 only electrostatic interactions play prominent role in the binding of these detergents.

    • Purification and characterization of putrescine synthase from cucumber seedlings. A multifunctional enzyme involved in putrescine biosynthesis

      G L Prasad P R Adiga

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      The multifunctional enzyme, putrescine synthase has been purified fromCucumis sativus and characterized. This enzyme harbours agmatine iminohydrolase, ornithine transcarbamylase, putrescine transcarbamylase and carbamate kinase activities, whose concerted action results in agmatine → putrescine conversion. The enzyme resolved into two aggregation forms, enzyme aggregated and enzyme monomer upon electrophoresis at pH 8.3. Evidence has been provided by two-dimensional gel electrophoresis that both enzyme aggregated and enzyme monomer comprise of identical polypeptide chains. Under non-reducing conditions on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the protein moves as a single 150 KDa polypeptide; however, in the presence of 2-mercaptoethanol on sodium dodecyl sulphate-polyacrylamide gel elec trophoresis, it migrates as 3 polypeptides of molecular weight 48,000, 44,000 and 15,000. The enzyme undergoes age-dependentin vivo proteolytic degradation from a 66 KDa polypeptide (primary translational product), through 48 KDa polypeptide to 44 KDa species and finally to small molecular weight peptides.

    • The mode of inhibition of the biosynthesis of phenylalanine ammonia lyase by its product cinnamic acid in aging potato parenchyma tissue

      S G Shirsat P M Nair

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      Excised potato parenchyma tissue upon aging in air and light, showed synthesis of RNA, as assessed by the incorporation of [3H]-uridine, reaching a maximum in 18 h. The increase in RNA synthesis was in parallel with the increase in phenylalanine ammonia lyase synthesis. The treatment of the tissue, initially with actinomycin D at 15 μg/g tissue, or with trans-cinnamate at 3·5 mM, caused 65% and 50% inhibition of RNA synthesis, respectively. The inhibition was reduced to 20% by delaying the treatment of trans-cinnamate to the ninth hour. A comparative study on the nuclear fractions, isolated from trans-cinnamate treated and untreated aged parenchyma tissues showed that trans-cinnamate treatment inhibited thein vitro RNA synthesis by 38%. Studies on thein vivo synthesis of PAL and other proteins showed that trans-cinnamate treatment mainly impaired thede novo synthesis of PAL. The total RNA, isolated from trans-cinnamate treated parenchyma, was 66% less efficient in the translation of PAL, in cell free wheat germ protein synthesizing system. The translation product, purified by affinity chroma tography on phenylalanine conjugated sepharose-4B was found to be homogeneous and showed a single radioactive peak corresponding to protein band on polyacrylamide gel electrophoresis.

    • Solubilization and interaction of α-tocopherol in water-aerosol OT-isooctane systems

      Amarnath Maitra Antaryami Sarangi

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      Solubilization and interaction of α-tocopherol into bis(2-ethylhexyl)sulphosuc cinate sodium salt microemulsion systems have been studied by temperature dependent phase transition, viscosity and nuclear magnetic resonance studies. Tocopherol being an amphiphilic molecule dissolves into the interfacial surfactant monolayer of the microemul sion droplets. The dissolution leads to an enhancement of the rigidity of the surfactant monolayer as studied by the increase in mixing and phase transition temperatures of the microemulsion droplets. Solubilization of tocopherol into microemulsion droplets causes an increase in the effective size of the droplet and as a consequence, the inter-droplet interactions are also increased. The water binding capacity of the surfactant (bis(2-ethylhexyl)sulphosuccinate sodium salt) is reduced due to solubilization of tocopherol as is evidenced from the downfield shifts of water proton magnetic resonances. In the presence of the dissolved electrolytes into the aqueous core, tocopherol is squeezed out of the microemulsion droplets increasing the membrane fluidity and permeability.

    • A method for the isolation of intact Sertoli cell-germ cell associations from rat seminiferous tubules and their further partition into Sertol cell and germ cell fractions

      MeenaKumari S Duraiswami

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      A technique is described for obtaining a Sertoli cell-enriched and a germ cell-enriched fraction from immature rat testes. Sertoli cell-germ cell associations were obtained by incubating washed seminiferous tubule fragments with Collagenase and Pancreatin. They were then manually dissociated into a suspension comprising Sertoli cells as well as the various germ cell types characteristic for a given day of ontogeny. Fractionation into a Sertoli cell-enriched fraction and a germ cell-enriched fraction was effected by centrifugation following layering over a stepwise gradient of Ficoll-400. While the time-span compares favourably with other procedures reported in the literature, it is believed this is the first time a method is described that enables the simultaneous recovery of both the Sertoli cells and the germ cells.

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