Collagen degradation in ischaemic rat hearts.

Myocardial extracellular matrix is organized into a complex arrangement of intercellular and pericellular fibres and fibrils that serves as a supporting framework for contracting cells. Recent evidence suggests that changes in ventricular shape and function occurring after ischaemic injury may be related to alterations of this matrix. In this report we describe the rapid and extensive loss of collagen in myocardial infarction produced by ligating the left anterior descending coronary artery of the rat for 1-3 h. The total collagen content in the myocardial infarct zones after 1, 2 and 3 h of ligation was 75 +/- 8%, 65 +/- 7% and 50 +/- 10% respectively (mean +/- S.D.) of that of either the non-infarcted tissue controls or of the same regions in sex- and age-matched normal left ventricles. A marked decrease also occurred in the residual collagens which were not extractable with 6 M-guanidine hydrochloride, suggesting that rapid degradation of insoluble collagen fibres may also occur. The decreased collagen content in the 3 h myocardial infarct coincided with the appearance of several enzyme activities. Collagenase, other neutral proteinase and presumed lysosomal serine proteinase activities were increased by 3, 3 and 2 times the control values respectively. These results suggest that the increased activities of collagenase and other neutral proteinases may be responsible for the rapid degradation of extracellular matrix collagen in myocardial infarct.     

Effect of Sorbinil on myo-Inositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Levels

Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.     

Effect of Increased Glucose Levels on Na+/K+-Pump Activity in Cultured Neuroblastoma Cells

Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.     

Nitrate assimilation in Neurospora crassa: Enzymatic and immunoblot analysis of wild-type and nit mutant protein products in nitrate-induced and glutamine-repressed cultures

Summary The nitrate assimilatory pathway in Neurospora crassa is composed of two enzymes, nitrate reductase and nitrite reductase. Both are ₂type homodimers. Enzymebound prosthetic groups mediate the electron transfer reactions which reduce inorganic nitrate to an organically utilizable form, ammonium. One, a molybdenum-containing cofactor, is required by nitrate reductase for both enzyme activity and holoenzyme assembly. Three modes of regulation are imposed on the expression of nitrate assimilation, namely: nitrogen metabolite repression, nitrate induction and autogenous regulation by nitrate reductase. In this study, nitrocellulose blots of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved proteins from crude extracts of the wild type and specific nitrate-nonutilizing (nit) mutants were examined for material cross-reactive with antibodies against nitrate reductase and nitrite reductase. The polyclonal antibody preparations used were rendered monospecific by reverse affinity chromatography. Growth conditions which alter the regulatory response of the organism were selected such that new insight could be made into the complex nature of the regulation imposed on this pathway. The results indicate that although nitrate reductase and nitrite reductase are coordinately expressed under specific nutritional conditions, the enzymes are differentially responsive to the regulatory signals.     

Five genetic loci involved in the synthesis of acidic exopolysaccharides are closely linked in the genome of Rhizobium sp strain NGR234

Summary R-prime plasmids were constructed from a derivative of Rhizobium strain NGR234 (ANU280) and were shown to contain overlapping genomic DNA segments involved in biosynthesis of exopolysaccharides (EPS). The R-primes originally constructed carried the mutant allele from Tn5-induced EPS-deficient (Exo^–) mutant ANU2811. This plasmid-located mutant allele was dominant to the corresponding wild-type allele as merodiploid strains were Exo^–. Exo⁺ revertants occurred at a low rate (1×10^-7) and these were shown to result from double reciprocal recombination events, which led to the isolation of R-prime plasmids carrying functional wild-type exo alleles. R-prime plasmids that carry overlapping segments of DNA from parental strain ANU280 complemented 28 of the 30 group 2 Exo^– mutants of strain ANU280. Complementation of these Exo^– mutants also restored their symbiotic abilities of effective nodulation. Subsequent in vivo recombination between the wild-type alleles located on the R-prime and the corresponding mutated allele on the genome, was used to generate a new family of R-primes, which carried mutations in the exo genes. The 30 group 2 Exo^– mutants were classified into 7 distinct genetic groups based upon complementation and physical mapping data. Five of the seven exo loci were gentically linked and located on a 15-kb region of DNA. Mutations at two loci were dominant only when the mutations were R-prime plasmid-located while a mutation at a second locus was cis-dominant to two other exo loci. At least five genes involved in the synthesis of acidic exopolysaccharide synthesis have been identified.     

Carbon and nitrogen partitioning in young nodulated pea (wild type and nitrate reductase-deficient mutant) plants exposed to NH4NO3

Carbon and nitrogen partitioning was examined in a wild-type and a nitrate reductase-deficient mutant (A317) of Pisum sativum L. (ev. Juneau), effectively inoculated with two strains of Rhizobium leguminosarum (128C23 and 128C54) and grown hydroponically in medium without nitrogen for 21 days, followed by a further 7 days in medium without and with 5 mM NH₄NO₃. In wild-type symbioses the application of NH₄NO₃ significantly reduced nodule growth, nitrogenase (EC 1.7.99.2) activity, nodule carbohydrates (soluble sugars and starch) and allocation of [¹⁴C]-labelled (NO₃⁻, NH₄⁺, amino acids) in roots. In nodules, there was a decline in amino acids together with an increase in inorganic nitrogen concentration. In contrast, symbioses involving A317 exhibited no change in nitrogenase activity or nodule carbohydrates, and the concentrations of all nitrogenous solutes measured (including asparagine) in roots and nodules were enhanced. Photosynthate allocation to the nodule was reduced in the 128C23 symbiosis. Nitrite accumulation was not detected in any case. These data cannot be wholly explained by either the carbohydrate deprivation hypothesis or the nitrite hypothesis for the inhibition of symbiotic nitrogen fixation by combined nitrogen. Our result with A317 also provided evidence against the hypothesis that NO₃⁻ and NH₄⁺ or its assimilation products exert a direct effect on nitrogenase activity. It is concluded that more than one legume host and Rhizobium strain must be studied before generalizations about Rhizobium /legume interactions are made.     

Characterisation of Non-Uniform Photosynthesis Induced by Abscisic Acid in Leaves Having Different Mesophyll Anatomies

The effects of abscisic acid (ABA) on photosynthesis in leavesof Helianthus annuus L. were compared with those in leaves ofVicia faba L. After the ABA treatment, the response of photosyntheticCO₂ assimilation rate, A, to calculated intercellular partialpressure of CO₂, P_i, (A(p_i) relationship) was markedly depressedin H. annuus. A less marked depression was also observed inV.faba. However, when the abaxial epidermes were removed fromthese leaves, neither the maximum rate nor the CO₂ responseof photosynthetic oxygen evolution was affected by the applicationof ABA. Starch-iodine tests revealed that photosynthesis was not uniformover the leaves of H. annuus treated with ABA. The starch contentwas diffferent in each bundle sheath extension compartment (thesmallest subdivision of mesophyll by veins with bundle sheathextensions, having an area of ca. 0.25 mm² and ca. 50 stomata).In some compartments, no starch was detected. The distributionof open stomata, examined using the silicone rubber impressiontechniques, was similar to the pattern of starch accumulation.In V.faba leaves, which lack bundle sheath extensions, distributionof starch was more homogeneous. These results indicate that the apparent non-stomatal inhibitionof photosynthesis by ABA deduced from the depression of A(pⁱ)relationship is an artifact which can be attributed to the non-uniformdistribution of transpiration and photosynthesis over the leaf.Intercellular gaseous environment in the ABA-treated leavesis discussed in relation to mesophyll anatomy. ¹ Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received September 30, 1987; Accepted January 13, 1988)     

Initial Studies on the Regulation of Toluene Degradation by Pseudomonas Putida F1

Pseudomonas putida Fl oxidizes toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is the substrate for “meta” fission of the aromatic nucleus. Kinetic and induction experiments indicate that the genes encoding enzymes for these reactions are part of an operon, designated the tod operon, that is coordinately induced and regulated. Strains unable to utilize toluene as a growth substrate were isolated at high frequencies by using screening procedures that utilize the redox dye, 2,3,5-triphenyl-2H-tetrazolium chloride. Biochemical characterization of strains with mutations in the structural genes of the tod operon showed that toluene induces the first four enzymes in toluene degradation by P. putida Fl. The isolation and characterization of pleiotropicnegative mutants together with mutants altered in terms of their expression of tod genes suggests that the tod operon may be under the control of a positive regulatory element.     

Formation of Hydroxyl Radicals in Biological Systems. Does Myoglobin Stimulate Hydroxyl Radical Formation from Hydrogen Peroxide?

Incubation of horse-heart oxymyoglobin or metmyoglobin with excess H₂O₂ causes formation of myoglobin(IV), followed by haem degradation. At the time when haem degradation is observed, hydroxyl radicals (.OH) can be detected in the reaction mixture by their ability to degrade the sugar deoxyribose. Detection of hydroxyl radicals can be decreased by transferrin or by OH scavengers (mannitol, arginine, phenylalanine) but not by urea. Neither transferrin nor any of these scavengers inhibit the haem degradation. It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H₂O₂ to form OH detectable by deoxyribose, but that H₂O₂ eventually leads to release of iron ions from the proteins. These released iron ions can react to form OH outside the protein or close to its surface. Salicylate and the iron chelator desferrioxamine stabilize myoglobin and prevent haem degradation. The biological importance of OH generated using iron ions released from myoglobin by H₂O₂ is discussed in relation to myocardial reoxygenation injury.