Balance between anionic and cationic extracellular peroxidase activities in Sedum album leaves after ozone exposure. Analysis by high-performance liquid chromatography

Peroxidase activity was assayed with different electron donors (guaiacol, ascorbate, syringaldazine) in the intercellular fluid of Sedum album L. leaves after ozone exposure. Anionic and cationic peroxidases were separated and purified by high performance ion-exchange and gel permeation chromatography. Both isoperoxidases were tested as regards their molecular weight and apparent kinetic constants with different substrates. Ascorbate peroxidase activity was rapidly stimulated after ozone exposure, whereas syringaldazine peroxidase activity reached its maximum 24 h later. Increases in ascorbate and syringaldazine peroxidase activities occurred simultaneously with increases in cationic and anionic peroxidase activities, respectively. Apparent K_m values indicate a high affinity of cationic peroxidases for ascorbate and of anionic peroxidases for syringaldazine. The metabolic role of this balance between cationic and anionic peroxidases after ozone exposure is discussed.     

An auxiliary protein for DNA polymerase-delta from fetal calf thymus

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.     

Ammonium repression of cephalosporin production by Streptomyces clavuligerus

Production of beta-lactam antibiotics took place during growth of Streptomyces clavulgerus in chemically defined medium. The specific activities of isopenicillin N synthetase ("cyclase"), isopenicillin N epimerase, and deacetoxycephalosporin C synthetase ("expandase") increased during the exponential phase of growth. Specific cephalosporin productivity during fermentation followed a similar pattern, reaching a maximum near the end of the growth phase and decaying rapidly in the stationary phase. Ammonium chloride depressed cephalosporin production, presumably as a result of repression of cyclase and expandase formation, but not of epimerase. No inhibitory effects on enzyme activity by ammonium were found. Addition of tribasic magnesium phosphate [Mg3(PO4)2 X 8H2O] prevented the repression of cyclase and markedly stimulated cephalosporin production. Cephamycin C and, in smaller amounts, O-carbamoyldeacetylcephalosporin C were the only cephalosporins detected. Growth with ammonium resulted in lower titers of both compounds, and did not change the relative proportion of each. The correlation found between cephalosporin productivity and cyclase specific activity in different media suggests that formation of this enzyme may be the rate-limiting step in the pathway.     

Effect of Ca2+ on the ouabain-insensitive, active Na+ uptake in inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells

The ouabain-insensitive, active Na+ uptake of inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can be increased by the presence of micromolar concentrations of Ca2+ in the assay medium. The concomitant ATP hydrolysis associated with the Na+ uptake is also increased by the presence of Ca2+. The Na+ uptake and the concomitant ATP hydrolysis are inhibited by 2 mM furosemide. The effect of Ca2+ is not due to the activity of an Na+-Ca2+ exchanger. The present results are in accordance with our previous model (Proverbio, F., Proverbio, T. and Marín, R. (1982) Biochim. Biophys. Acta 688, 757-763) in which we proposed that Ca2+ seems to modulate the activity of the ouabain-insensitive Na+ pump, in two different ways: (1) in a strong association with the membranes in which Ca2+ (stable component) is essential for the pump activity and (2) in a weak association with the membranes in which Ca2+ (labile component) can be quickly and easily removed by reducing the free Ca2+ concentration of the assay medium to values lower than 1 microM. The Ka for Ca2+ (for the labile component) is around 5 microM. The Ca2+ modulation of the ouabain-insensitive Na+ pump is an indication that Ca2+ could regulate the magnitude of the Na+ extrusion accompanied by Cl- and water present in rat kidney proximal tubular cells.     

Nodular fasciitis initially diagnosed by aspiration cytology

A case of nodular fasciitis diagnosed by fine needle aspiration cytology is described. The cytologic findings included fusiform cells, mitoses, macrophages, multinucleated giant cells and mesenchymal elements in a characteristic granular background substance. The cytopathologic diagnosis was subsequently confirmed by the histopathologic study of the tumor and by electron microscopy.     

Effects of glutathione depletion on gluconeogenesis in isolated hepatocytes

Glutathione-depleted hepatocytes, by incubation with diethylmaleate (DEM) or phorone (2,6-dimethyl-2,5-heptadiene-4-one), i.e., substrates of the GSH S-transferases (EC 2.5.1.18), showed rates of gluconeogenesis from various precursors significantly lower than controls; however the rate of glucose synthesis from fructose was similar to that of controls. Isolated hepatocytes from rats pretreated with those substrates 1 h before isolation to deplete hepatic glutathione (GSH) also showed a decrease of the rate of gluconeogenesis from lactate plus pyruvate. Incubation of hepatocytes with L-buthionine sulfoximine, a specific inhibitor of gamma-glutamyl-cysteine synthetase (EC 6.3.2.2), resulted in a decreased rate of gluconeogenesis from lactate plus pyruvate only when GSH values were lower than 1 mumol/g cells. Freeze-clamped livers from GSH-depleted rats showed a higher concentration of malate and glycerol 3-phosphate, indicating that GSH depletion probably affects phosphoenolpyruvate carboxykinase and glycerol-3-phosphate dehydrogenase activities. Several indicators of cell viability, such as lactate dehydrogenase leakage, malondialdehyde accumulation, ATP concentration, or urea synthesis from different precursors, were not affected by GSH depletion under the experimental conditions used here. Besides, the GSH/GSSG ratio remained unchanged in all cases.     

Myoglobin: cytochrome b5 interactions and the kinetic mechanism of metmyoglobin reductase

Metmyoglobin (metMb) reduction by metMb reductase from heart muscle requires cytochrome b5 as electron-transfer mediator. The existence of a metMb-ferrous cytochrome b5 complex is demonstrated by mutual perturbation of the proteins' respective electrophoretic titration curves between pH 4 and 7. The same technique shows a preferential binding of cytochrome b5 over metMb by the enzyme. The paramagnetic hyperfine shifts in the cytochrome b5 1H NMR spectrum are perturbed by metMb, indicating the formation of a specific bimolecular complex with a 1:1 stoichiometry and a binding constant estimated to be less than 10 microM. The resonances assigned to the cytochrome b5 heme 6-propionate methylene group exhibit the largest complexation shifts. Computer modeling implicates lysines 47, 50, and 98 of metMb as contact points with cytochrome b5 carboxylate residues 43, 44, 60, and heme 6-propionate. The mechanism of the enzymatic reduction establishes metMb reductase as an NADH-cytochrome b5 oxidoreductase. Cytochrome b5 is reduced at near diffusion-controlled rates by the enzyme with a turnover number of 1000 min-1 X Km for the cytochrome is 0.9 microM versus 100 microM reported for the erythrocyte enzyme. Ferrous cytochrome b5 then reduces metMb nonenzymatically with an apparent rate constant of 4.9 X 10(4) M-1 min-1 X Acetylation of metMb, which does not affect its oxygen affinity or chemical reduction, renders it a poor substrate for enzymatic reduction. This study suggests a function for the three exterior lysine residues conserved in all mammalian myoglobin sequences: they are contact points for complexation with cytochrome b5.     

Cloning in Escherichia coli and molecular analysis of the sucrose system of the Salmonella plasmid SCR-53

Summary The sucrose utilization system of the conjugative plasmid scr-53 originating from a sucrose-fermenting Salmonella strain has been cloned in Escherichia coli K12 using pBR325 as a vector. Bacteria harboring a recombinant plasmid with a 4.9 kilobase PstI-insert were able to grow in media containing sucrose as the sole carbon source. A gene that directs the synthesis of a -d-fructofuranosyl-fructohydrolase enzyme was located on a 2.6 kilobase SalI-EcoRI DNA fragment. Three polypeptides of 60,000, 39,000 and 25,000 daltons were detected by a maxicell system. The advantage of using the resulting plasmids for industrial applications is discussed.     

Dielectrophoretic behavior of yeast cells: effect of growth sources and cell wall and a comparison with fungal spores.

Saccharomyces cerevisiae showed different dielectrophoretic behavior depending on the source of carbon for growth. Growth on fermentable carbon sources produced a dielectrophoretic response that decreased according to the amount of sugar present in the culture medium. Growth on nonfermentable carbon sources produced a constant dielectrophoretic yield, independent of the amount and source of carbon present in the medium. The dielectrophoretic yield, however, was independent of the nitrogen source. The yield spectrum for S. cerevisiae protoplasts was similar to that for the cells, although a decrease in the absolute value was observed. This decrease could be explained by the reduction in cell size and by assuming that the cell wall contributes a negative net charge to the yield. Fungal spores responded to the nonuniform electric field in the same range of frequencies as assayed for yeast cells.     

Effects of beta-pinene on yeast membrane functions.

The effects of beta-pinene on yeast cells were studied. This terpene inhibited respiration with glucose or ethanol as the substrate. The inhibition depended on the ratio of the terpene to the amount of yeast cells; for a fixed concentration of pinene, inhibition decreased as the amount of yeast cells increased. Pinene also inhibited the pumping of protons and K+ transport, but this inhibition was more marked with with ethanol than with glucose as the substrate, indicating the mitochondrial localization of the inhibition. The studies on isolated mitochondria showed a series of effects, starting with the disappearance of the respiratory control and deenergization of the organelles and followed by an inhibition of respiration at higher concentrations of the terpene. The effect on respiration could be localized to the cytochrome b region of the electron transport chain. No effect could be detected on the activity of ATPase. The effects can be ascribed to a localization of pinene on membranes which was also accompanied by a decrease in the fluorescence polarization of diphenyl hexatriene, probably meaning an increase in the fluidity of the membrane, localized preferentially to the mitochondria.