Enhanced production of pigment-free pullulan by a morphogenetically arrested Aureobasidium pullulans (ATCC 42023) in a two-stage fermentation with shift from soy bean oil to sucrose

A two-stage fermentation process was established for the production of pigment-free pullulan by the yeast-like fungus Aureobasidium pullulans (ATCC 42023). In the first stage, starting at pH 4.5 with soy bean oil as the carbon source and glutamate as the nitrogen source, a cell mass of about 15 g l^–1 dry cell weight was obtained, the population being restricted mainly to the yeast form of the microorganism (yeast form more than 90% of total cells) and the formation of pigment in the culture being prevented. Small amounts of pullulan (less than 2 g l^–1) are produced at this phase, and the viscosity remained low throughout the entire growth stage. When the oil and glutamate source were nearly exhausted (below 5% of initial amounts), the cells were shifted to a production stage with sucrose as the carbon source with continued nitrogen depletion. Production of pullulan started immediately with no lag period. During 50 h of the production phase more than 35 g l^–1 of pullulan was produced (productivity approx. 0.7 g l^–1), resulting in a large increase in the viscosity of the broth. The production yield of pollulan on the sugar was about 0.6 g g^–1. Morphogenesis from the yeast form of the microorganism to chlamydospores was still restrained and no pigment was formed in the culture during the production stage. A pigment-free polysaccharide, with a molecular mass in the range of 600–750 kDa, was recovered from the supernatant of the broth after solvent precipitation.     

Repression of lipopolysaccharide biosynthesis in Escherichia coli by an antisense RNA of Acetobacter methanolicus phage Acm1

Lysogenic Acetobacter methanolicus strains carrying the prophage Acm1 were found to be unable to synthesize both the capsutar polysaccharide (CPS) and the O-specific side-chain of lipopolysaccharide (LPS) and to represent rough variants of the host bacterium. A 262 bp DNA fragment of phage Acm1, obviously required for interference with LPS biosynthesis, was cloned and expressed in Escherichia coli Independently of the O-type, transformation of various E. coli strains with the recombinant DNA resulted in a suppression of biosynthesis of the O-specific chains. The DNA fragment of phage Acm1 contained three very short open reading frames of 21, 24, and 36 bp. However, attempts to express phage-encoded peptides were not successful. Instead, the Acm1-derived DNA fragment was shown to code for the synthesis of a trans-acting RNA molecule of 97 nucleotides, designated lbi (L PS b iosynthesis-i nterfering) RNA. This RNA contains sequence complementarity to E. coli target RNA sequences and appears to have the ability to form intracellularly RNA hybrid duplexes with mRNA. The data presented in this study support the hypothesis that the phenotypic effect of conversion to rough-type LPS is accompanied by the expression of an antisense RNA of phage Acm1.     

High Resolution Proton NMR Spectroscopy of Multiple Sclerosis Lesions

Abstract: Tissue from postmortem multiple sclerosis and normal control brains was extracted with perchloric acid and analysed using proton NMR spectroscopy. The content of N -acetyl-derived groups (the sum of N -acetylaspartate, acetate, and N -acetylaspartylglutamate) was decreased in multiple sclerosis plaques compared with normal control white matter (mean, 4.36 vs. 6.64 µmol/g wet weight). In normal appearing white matter adjacent to plaques a corresponding decrease was seen, with no change in white matter distant from plaques. A decrease in the content of total creatine was observed in multiple sclerosis plaques in comparison with normal control white matter (mean, 4.64 vs. 6.56 µmol/g wet weight), which correlated strongly with the decrease in N -acetyl-derived groups. No changes in other metabolites such as total choline or myo -inositol were seen. The decreases in content of N -acetyl-derived groups are in agreement with observations from in vivo proton NMR spectroscopy in multiple sclerosis patients. The decrease in total creatine is in contrast to most of the observations made in vivo where total creatine is assumed to be unchanged and metabolite levels are often expressed as a total creatine ratio. The use of a total creatine ratio in vivo could lead to an underestimation of reductions in N -acetylaspartate and an apparent increase in other metabolites in the multiple sclerosis lesion.     

Modulation of K+ channels by hydrogen peroxide.

External application of hydrogen peroxide (H2O2) was found to inhibit the time-dependent fast inactivation process of three cloned voltage-gated K+ channels expressed in Xenopus oocytes: KShIIIC, KShIIID and HukII. As expected from kinetic models where some channels are still opening while a significant fraction of channels is already inactivated there was a large increase in current magnitude concomitant to inactivation block. The channels otherwise functioned normally. The effects of H2O2 were specific (other cloned voltage-gated K+ channels were not affected), and reversible, the currents returned to normal upon removal of the H2O2. H2O2 is produced during normal metabolism; it could act as a modulator of excitability through effects on K+ channels if effective local concentrations are reached in neuronal regions close to the channel. KShIIIC and KShIIID currents are very similar to an O2-sensitive K+ current present in type I cells of the carotid body which is believed to underlie the modulation of excitability of these cells by changes in arterial O2 pressure. H2O2 has been proposed as an intermediary between O2 and cellular response in the carotid body; our results provide support for this model.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bivariate flow cytometry of farm animal chromosomes: a potential tool for gene mapping

Bivariate flow karyotypes of chromosomes from sheep, cattle and pig lymphocytes and from a cattle-mouse somatic cell hybrid line were obtained using a dual laser fluorescence-activated cell sorter (FACS). Pig chromosomes were resolved into 19-20 peaks, indicating that most, if not all, pig chromosomes could be separated by this technique. Sheep chromosomes showed incomplete separation but three clear peaks, presumably representing the three large metacentric chromosomes, plus five other clusters were obtained. Cattle chromosomes showed poor separation but about four peaks could be distinguished, indicating that certain chromosomes could be sorted in this species. The use of cattle-mouse hybrids may enable other individual cattle chromosomes to be obtained. It is concluded that FACS separation will be a useful additional tool for gene mapping.