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.     

Metabolic changes associated with bud break induced by thidiazuron

Bud break in apple (Golden Delicious,Malus domestica Borkh) was induced by thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-ylurea). In control and thidiazuron-treated shoots, higher amounts of soluble carbohydrates (sorbitol, fructose, glucose, sucrose) and galacturonic acid were found in the phloem, but higher amounts of starch and cell wall polysaccharides, including cellulose and xylose, were found in the xylem. A decrease in soluble carbohydrates and starch in both phloem and xylem was associated with induction of bud break by thidiazuron. However, little change in cell wall polysaccharides was found. Total carbohydrates were higher in the upper than in the lower portion of shoots. The breaking of dormancy by thidiazuron was also associated with an increase in organic acid content and respiration in buds. KCN inhibited bud respiration during all stages of development. Organic acid content was inversely related to carbohydrate content in developing buds. Axes contained more carbohydrates and organic acids than did scales.     

Expression of HLA-DR antigens on tumour cells does not contribute to skin reactivity to autologous cholesteryl hemisuccinate (CHS)-treated tumour cells in patients with metastatic melanoma

Summary Skin tests with autologous cholesteryl hemisuccinate (CHS)-treated and untreated cells were performed in ten metastatic melanoma patients. In the majority of cases evident reaction was noted with CHS-treated cells (9/10) while the reaction with untreated cells was mostly negative (7/10). Tumour cell suspensions used for skin tests were characterized for reactivity with monoclonal antibody TAL 1B5 detecting the HLA-DR alpha chain. There were no differences between CHS-treated and untreated cells with respect to HLA-DR expression and no correlation was found between grade of skin reaction to CHS-treated cells and the proportion of HLA-DR positive cells in the injected cell sample.     

Nuclear magnetic resonance studies of isolated structural domains of yeast phosphoglycerate kinase

The structural integrity and substrate binding properties of the two genetically engineered domains of yeast phosphoglycerate kinase were investigated using one- and two-dimensional nuclear magnetic resonance techniques. Both domains were found to fold with regions of native-like structure, with the N-domain showing greater conformational flexibility than the C-domain. The 'basic patch' region of the N-domain is, however, clearly perturbed by removal of the C-domain. This is most likely due to the absence of stabilizing interactions between the C-terminal peptide (including alpha-helices XIII and XIV) and the N-domain. The C-domain is able to bind nucleotide with an affinity only three times less than that of the native protein.     

Flexibility and folding of phosphoglycerate kinase

Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.     

Flavonoids as superoxide scavengers and antioxidants

The superoxide anions scavenging activity and antioxidation of seven flavonoids--quercetin, rutin, morin, acacetin, hispidulin, hesperidin, and naringin--were studied. The superoxide anions were generated in a phenazin methosulphate-NADH system and were assayed by reduction of nitroblue tetrazolium. The scavenging activity ranked: rutin was the strongest, and quercetin and naringin the second, while morin and hispidulin were very weak. The concentration values yielding 50% inhibition of lipid peroxidation in mouse liver homogenate were in order of 10(-6) M for quercetin, rutin, and morin; and of 10(-5) M for acacetin and hispidulin, while naringin and hesperidin had no antioxidative action. In comparison with the antioxidative and scavenging activities of flavonoids, there are no correlations.     

Thiosulfate production from cystine by the keratinolytic prokaryote Streptomyces fradiae

In cultures of Streptomyces fradiae on wool as the only source of nutrition inorganic thiosulfate (in amounts up to 0.5 mg of Na₂S₂O₃·5 H₂O/ml) was formed as the final product of metabolization of sulfur from cystine of keratin proteins. The presence of thiosulfate was proved by qualitative tests and thin-layer chromatography and estimated quantitatively by spectrophotometry, titrimetry, and capillary isotachophoresis. Metabolization of organic sulfur to thiosulfate excreted into the medium is a process not yet described in microorganisms.