External factors involved in the regulation of an extracellular proteinase synthesis in Bacillus megaterium

Summary A mathematical model was formulated to describe the kinetics and stoichiometry of growth and proteinase production in Bacillus megaterium. Synthesis of the extracellular proteinase in a batch culture is repressed by amino acids. The specific rate of formation of the enzyme (r _E) can be described by the formula {ie373-1}, where k ₂ and k ₃ stand for the non-repressible and repressible part of enzyme synthesis respectively, k _{S 2} is a repression coefficient and S ₂ indicates the concentration of amono acids; the values of k ₂ and k _{S 2} depend on the composition of the mixture of amino acids. Even in a high concentration, a single amino acid is less effective than a mixture of amino acids. The dependence of the proteinase repression on the concentration of an external amino acid (leucine) follows the same course as its rate of incorporation into proteins, approaching saturation at concentrations higher than 50 M (half saturation approximately 10 M). However, the total uptake of leucine did not exhibit any saturation even at 500 M external concentration.Symbols X biomass concentration, g/l - E proteinase concentration, unit/l - t time, h - S ₁ concentration of glucose, g/l - S ₂ concentration of amino acids, g/l - specific growth rate, l/h - rE specific rate of enzyme production, unit/g/h - k ₁ growth kinetic constant, l/h - k ₂ product formation kinetic constant (for non-repressible part of enzyme synthesis), unit/g - k ₃ product formation kinetic constant (for repressible portion of enzyme synthesis), unit/g - k _{S 1} saturation constant, g/l - k _{S 2} repression coefficient for a certain amino acid or amino acids mixture, g/l     

Physiological activity of immobilized cells of Claviceps fusiformis during long-term semicontinuous cultivation

Summary The 550-day semicontinuous cultivation of Claviceps fusiformis immobilized in calcium alginate is documented. The vegetative mycelium from seed or from early-production submerged culture is the best choice for immobilization. No extracellular glucans are produced by immobilized cells. Immobilized spores give low yields of clavine alkaloids. Alginate concentrations in a range of 2%–4% do not influence yield and spectrum of alkaloids. The cytoplasm of the immobilized cells becomes condensed (after 3 days), polysaccharides disappear, and centres of lipid synthesis are formed in the cytoplasm. After 60 days the cells harbour a great number of lipid particles, mitochondria are diminishing and their cristae partly disappear, indicating a decreased respiration capacity. After 350–500 days the volume of most cells is increased many times and the cells are filled with large oval bodies of electrondense material. Chloramphenicol protects immobilized cultures against bacterial contamination.     

Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.     

Effect of bacterial growth-inhibiting ingredients on the Ames mutagenicity of medicinal herbs

A solvent fractionation method was introduced to screen for mutagenicity in 10 medicinal herbs being consumed in Korea. The Ames mutagenicity test result of Scutellariae and Rhei was significantly increased by eliminating growth-inhibiting substances through solvent fractionation of the crude extract. It is suggested that a physicochemical pretreatment should reduce the false-negative results which are caused by the presence of growth-inhibiting substances in complex mixtures.     

Characterization of the Stimulation of Ethylene Production by Galactose in Tomato (Lycopersicon esculentum Mill.) Fruit

We have characterized the stimulation of ethylene production by galactose in tomatoes (Lycopersicon esculentum Mill.). The effect of concentration was studied by infiltrating 0, 4, 40, 100, 200, 400, or 800 micrograms galactose for each gram of fresh fruit weight into mature green `Rutgers' fruit. Both 400 and 800 micrograms per gram fresh weight consistently stimulated a transient increase in ethylene approximately 25 hours after infiltration; the lower concentrations did not. Carbon dioxide evolution of fruit infiltrated with 400 to 800 micrograms per gram fresh weight was greater than that of lower concentrations. The ripening mutants, rin and nor, also showed the transient increase in ethylene and elevated CO₂ evolution by 400 micrograms per gram fresh weight galactose. 1-Aminocyclopropane-1-carboxylic acid (ACC) content and ACC-synthase activity increased concurrently with ethylene production. However, galactose did not stimulate ACC-synthase activity in vitro. The infiltrated galactose in pericarp tissue was rapidly metabolized, decreasing to endogenous levels within 50 hours. Infiltrated galacturonic acid, dulcitol, and mannose stimulated transient increases in ethylene production similar to that of galactose. The following sugars produced no response: sucrose, fructose, glucose, rhamnose, arabinose, xylose, raffinose, lactose, and sorbitol.     

Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis

We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.     

Recalibration of the Pseudomonas aeruginosa Strain Pao Chromosome Map in Time Units Using High-Frequency-of-Recombination Donors

High-frequency-of-recombination donors of P. aeruginosa strain PAO were generated using a temperature-sensitive, replication mutant of the IncP-1 plasmid R68, loaded with the transposon Tn2521. Fourteen donors so isolated mobilized the chromosome in a polarized manner from a number of different transfer origins. The donors were used to construct a time of entry map of the entire chromosome and this was achieved by determining the time of entry of 32 randomly dispersed markers in crosses using nalidixic acid to interrupt chromosome transfer. Analysis of the time of entry data enabled the recalibration of the chromosome map to 75 min.     

The nifA gene of Rhizobium meliloti is oxygen regulated.

Experiments using plasmid-borne gene fusions and direct RNA measurements have revealed that expression from the nifA gene is induced in Rhizobium meliloti when the external oxygen concentration is reduced to microaerobic levels. Induction occurs in the absence of alfalfa and in the presence of fixed nitrogen and does not require ntrC. The production of functional nifA gene product (NifA) can be demonstrated by its ability to activate the nitrogenase promoter P1. Aerobic induction of nifA can also occur during nitrogen starvation at low pH, but in this case induction is dependent on ntrC and does not lead to P1 activation. The data indicate that reduced oxygen tension is potentially a major trigger for symbiotic activation of nitrogen fixation in Rhizobium species.     

Regulation of protein kinase C activity by gangliosides

The activity of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the presence of phosphatidylserine and its physiological regulator, diacylglycerol, could be suppressed by a mixture of brain gangliosides. Half-maximal inhibition was observed at 30 microM and was nearly complete at 100 microM. Inhibition was observed at all concentrations of Ca2+ between 10(-8) and 10(-4) M. Inhibition of protein kinase C activity could not be reversed by increasing the concentration of diacylglycerol or the substrate, histone. Inhibition was also observed when myelin basic protein or a synthetic myelin basic protein peptide was used as substrate. Among the individual gangliosides, the rank order of potency was GT1b greater than GD1a = GD1b greater than GM3 = GM1. Our results suggest that gangliosides may regulate the responsiveness of protein kinase C to diacylglycerol.