Effect of lactic acid on growth and butanediol production byKlebsiella oxytoca

Summary Production of 2,3-butanediol byKlebsiella oxytoca was enhanced in the presence of low levels (<8 g/l) of added sodium lactate. Cell growth was inhibited, however, and essentially stopped above 15 g/l added lactate. Levels of by-products (acetic acid and ethanol) were also higher. With 3 g/l lactate and an initial glucose level of 98 g/l, butanediol concentration and productivity increased 164% with 98% utilization of glucose. With high glucose concentration (219 g/l), addition of 2.64 g/l lactate after the growth phase resulted in 81 g/l butanediol, with a productivity of 0.65 g/l/h and 71% glucose utilization.     

The importance of mast cells for the neutrophil influx in immune complex-induced peritonitis in mice

The role of mast cells in polymorphonuclear leukocyte (PMN) influx in Ag-antibody complex-induced peritonitis was evaluated in mast cell-deficient WBB6F1-W/Wv (W/Wv) mice and their normal littermates, WBB6F1-+/+ (+/+). Peritoneal cell influx was evaluated after i.p. injection of preformed immune complexes. The first significant elevation in the PMN count over PBS-treated controls in +/+ mice was observed 2 h after stimulation. During the period of maximum leukocyte concentrations (6 to 10 h), the increase in total cell count was 5-fold and in PMN 25-fold. In W/Wv mice the PMN influx started 2 h later than in the +/+ mice, and the maximum response (8 to 10 h) was only 50% of that in controls. Reconstitution of mast cells in W/Wv mice for 2 wk or more restored the PMN response to immune complexes. Mast cell release due to AG-antibody complexes was evaluated by measuring fluorescence intensity after berberine sulfate staining for heparin in mast cells from unstimulated as well as stimulated +/+ mice. There was a significant decrease in fluorescence intensity as early as 15 min after stimulation. By 30 min the fluorescence intensity had declined by 65%. This indicates extensive mast cell release that started before PMN mobilization. These experiments demonstrate that mast cells make a significant contribution to immune complex-induced inflammation.     

Evidence for a plasmid conferring salt-tolerance in the plant-root associated,diazotrophKlebsiella sp. NIAB-I

Summary Using analytical and preparative methods, we demonstrated the presence of an indigenous plasmid (pNIAB-I) in a diazotroph,Klebsiella sp. NIAB-I isolated, from the roots of Kallar grass, growing on saline lands in Pakistan. The plasmid is approximately 50 kilobase (kb) in size. Transformation experiments indicated that non-halophilic bacteria such asE. coli K12 strain (MV10) andK. pneumoniae M5AI on acquiring this plasmid become tolerant to high salt (NaCl) and alkaline pH.     

Characterization of a high-affinity binding site for a DNA-binding protein from sea urchin embryo mitochondria.

Based on electrophoretic mobility shift assays, DNase I footprinting and modification interference analyses we have identified a sequence-specific DNA-binding protein in blastula stage mitochondria of the sea urchin Strongylocentrotus purpuratus, which interacts with a binding site around the major pause site for DNA replication. This region straddles the boundary of the genes for ATP synthase subunit 6 and cytochrome c oxidase subunit III, and contains also a prominent origin of lagging-strand synthesis. The protein is thermostable, and its natural high-affinity binding site comprises the sequence 5'-AGCCT(N7)AGCAT-3'. Binding studies have demonstrated that two copies of the imperfect repeat, as well as the 7 bp spacing between them, are essential for tight binding. Based on the location of its binding site, we tentatively designate the protein mitochondrial pause-region binding protein (mtPBP) 1.     

Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment

Spring wheat (Triticum aestivum L.) zygotic embryos were successfully cryopreserved, without the addition of exogenous cryoprotectants, using only an abscisic acid (ABA) pretreatment. Optimum survival was obtained when embryos were cultured in vitro for 10 days on semisolid Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L (±) ABA prior to cryopreservation. The embryos resumed growth within three days when returned to MS medium devoid of ABA but containing 2mg/L 2,4-dichlorophenoxyacetic acid. The embryogenic calli produced from these embryos exhibited normal plant regeneration on auxin-free media. Changes in dw/fw ratio, as well as the esterified fatty acid and sucrose concentrations correlated positively with the development of tolerance to cryopreservation.NRCC Publication No. 33519     

L-histidine induced changes in adenosine 3':5'-monophosphate levels in rat brain and amounts of apo-, holo-a and holo-b fatty acid synthetases in rat liver.

When fasted rats were fed a chow or fat-free diet supplemented 5% with L-histidine for three days, the brain adenosine 3′:5′-monophosphate (cAMP) level increased. A 50% increase occurred in rats fed a chow diet and 20% increase in rats fed a fat-free diet. Purification of liver fatty acid synthetase and the isolation of liver apo-, holo-$\text{a}$ and holo-$\text{b}$ fatty acid synthetases demonstrated that L-histidine feeding caused changes in the relative amounts of these enzymes. Apo- and holo-$\text{b}$ fatty acid synthetases increased while the holo-$\text{a}$ form simultaneously decreased. This effect was observed in rats fed either chow or fat-free diets supplemented with L-histidine.     

L-histidine-induced suppression of lipogenic enzymes.

Diets supplemented 5% with L-histidine produce hypercholesterolemia and increase cholesterol biosynthesis in rat liver. We now report an $\text{inhibitory}$ effect of L-histidine on lipogenic enzymes in the liver. In this study, L-histidine was added to chow and fat-free diets and fed to rats for 18 days. After two days of fasting, the rats were refed the same diet for three days prior to sacrifice. L-histidine decreased fatty acid synthetase activity by 51% when it was added to the chow diet and by 26% when it was added to the fat-free diet. Isocitrate dehydrogenase activity was not altered significantly in rats fed diets supplemented with L-histidine.     

Mutants of Salmonella typhimurium responding to cysteine or methionine: their nature and possible role in the regulation of cysteine biosynthesis.

Nineteen mutants of Salmonella typhimurium responding to either cysteine or methionine (cym) have been identified amongst cysteine (cys) and methionine (met) auxotrophs. Their growth responses to known intermediates in the related pathways of cysteine and methionine biosynthesis and complementation patterns in abortive transduction tests divided the mutants into six groups. Results of conjugation, cotransduction and deletion mapping experiments substantiated these groups, each of which carried a lesion within known cys genes. Enzyme assays on cym mutants from five of the six groups confirmed their cys gene deficiencies. Growth response and enzyme assay data were not consistent with mutants being leaky cys mutants (spared by methionine). None of eight cym mutants tested were able to convert [35S]methionine into [35S]cysteine. Selenate specifically inhibits the early enzymes of cysteine synthesis. In cym mutants this inhibition was relieved by cysteine but not by methionine, indicating that cym mutants require active cys enzymes for growth on methionine. There was evidence that methionine stimulated in vivo activity of cys enzymes in a cym mutant. Resistance to inhibition by 1,2,4-triazole results in reduced levels of the O-acetyl serine sulphydrylase. In cym mutants triazole resistance gave unstable suppression of the cym phenotype. Cym mutants may result from mutation in regulatory regions common to each of the cys genes, with the precise role of methionine as yet unknown.     

Circulating soluble CD4 directly prevents host resistance and delayed-type hypersensitivity response to Cryptococcus neoformans in mice

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed-type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane-bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti-CD4 mAb restored the attenuated host defense. Second, in CD4 gene-disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene-transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock-transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti-CD4 mAb. Finally, serum levels of IFN-gamma, IL-12 and IL-18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL-4 and IL-10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1-type cytokines.     

Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 was mutagenized using UV-C irradiation to produce yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol. UV-C irradiation potentially produces large numbers of random mutations broadly and uniformly over the whole genome to generate unique strains. Wild-type cultures of S. stipitis NRRL Y-7124 were subjected to UV-C (234 nm) irradiation targeted at approximately 40% cell survival. When surviving cells were selected in sufficient numbers via automated plating strategies and cultured anaerobically on xylose medium for 5 months at 28°C, five novel mutagenized S. stipitis strains were obtained. Variable number tandem repeat analysis revealed that mutations had occurred in the genome, which may have produced genes that allowed the anaerobic utilization of xylose. The mutagenized strains were capable of growing anaerobically on xylose/glucose substrate with higher ethanol production during 250- to 500-h growth than a Saccharomyces cerevisiae yeast strain that is the standard for industrial fuel ethanol production. The S. stipitis strains resulting from this intense multigene mutagenesis strategy have potential application in industrial fuel ethanol production from lignocellulosic hydrolysates.