Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites

The zinc-deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13-ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1-anilinonaphthalene 8-sulphonic acid is bound with comparable affinity. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc-deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase. 2,2'-Bipyridine, which chelates the active-site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc-depleted enzyme. The stopped-flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc-depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His-67, bound to the zinc ion, or the zinc-liganding water molecule in the case of the native enzyme (pK close to 9), or the free His-67 residue in the case of the zinc-deficient enzyme (pK about 8).     

Biosynthesis of monensins a and b: the role of isoleucine

Isoleucine added to the cultivation medium of Streptomyces cinnamonensis C-100-5 induced a relative increase of the production of monensin B at the expense of monensin A. U-14C-Isoleucine was found not to be a specific monensin B precursor. The incorporation of 1-13C-2-methylbutyrate into monensins A and B showed the label to be evenly incorporated in both products at carbon atoms originating from C(1) of propionate. In regulatory mutants insensitive to 2-amino-3-chlorobutyrate isoleucine influenced the production of monensins only slightly but strains resistant to 2-aminobutyrate and norleucine decreased their total production by 2-12% in the presence of isoleucine which was associated with a decrease of monensin A content by 14-52%. The inhibitory effect of isoleucine on the biosynthesis of valine, a specific precursor of the butyrate unit of monensin A, is discussed.     

Maintenance of aPaecilomyces variotii culture in the frozen and Freeze-dried state and in distilled water

The highest number of viable.elements ofPaecilomyces variotii, forming colonies after a 1-year maintenance, was detected in frozen sample. Decreased viability of the frozen culture and culture maintained in distilled water was usually statistically significant after 1 year and decreased further with increasing age of the culture used for sample preparation. Freeze-drying also significantly decreased the strain viability, depending on culture age. In the freeze-dried culture stored in a refrigerator the relative number of viable elements was substantially higher than after storage at room temperature. After a 3-year storage of freeze-dried P.variotii in a refrigerator 14-44% of culture elements survived, as compared with the number detected immediately after freeze-drying.     

Benzothiazolium salts—relationships between their structure,toxicity and effect on the plastid system ofEuglena gracilis

The effects of 41 benzothiazolium salts on Euglena gracilis were characterized with regard to the influence on growth and on chlorophyll synthesis, and to their ability to induce permanent loss of chloroplasts. Some salts induced white mutants of E. gracilis (the first benzothiazole derivatives with this activity). The relationship between the biological effect and chemical structure was confirmed and expressed quantitatively by means of Free - Wilson and Fujita - Ban analysis.     

Degradation of cellulose by thermophilic bacteria

Degradation of 1—.10% crystalline cellulose and concentration of:free reducing sugars in the medium, were studied during cultivation of a wild coculture of obligately thermphilic bacteria in 3-L fermentors at 60^°C and pH 7.0 under anaerobic conditions. The coculture was composed of five different species ofBacillus and a single cellulolytic species lof Clostridium. The proportion of degraded substrate was inversely proportional to the initial concentration of cellulose. The higher the initial substrate concentration the lower the proportion of its.degradation. Cellulose at 1 — 2 % concentration is best degraded (98 % in:5.d). The fermentation time increases with increasing cellulose concentration, the level of reducing saccharides increases together with the initial rate of substrate degradation. In the presence of 10 %) cellulose the rate of degradation within a period of a 1-d fermentation is close toV, being 0.455 g L^-1 h^-1withK _m of 12.5 g/L. However, during further cultivation (1—3 d) the rate of degradation of 4—10 % cellulose decreases, probably due to the effect of accumulated reducing saccharides whose levels reach 55—60 mg/L.     

Characteristics of the saprophytic reference strain FA ofClaviceps paspali

Growth properties and morphological features of the saprophytic collection cultureClaviceps paspali strain FA were investigated. The strain was characterized by a range of utilization of 13 carbon sources in a basic synthetic and a peptone medium. A temperature of 23 °C was more favourable for growth than 28 °C. Stages of changes of the culture cultivated for a long time on Sabouraud’s medium were detected by electron microscopy. The white culture was characterized by true septated mycelium with different types of terminal parts of hyphae. Arthrospores occurred after a 14-d cultivation, small spherical conidia released individually from hyphae were quite rare. As compared with other strains of the same species, strain FA did not form spherical clusters of conidia. On the other hand, a spontaneous rupture of the surface cell wall in different parts of hyphae and release of the cytoplasm were observed. In corn-steep containing media the formation of individual, pair and chain-like forms of arthritic conidia was stimulated. Destructive autolytic changes of hyphae were detected in the medium with potato extract.     

Saprophytic production of Clavine alkaloids and activity of Hydroxymethylglutaryl-CoA reductase

In submerged Claviceps cultures the activity of hydroxymethylglutaryl-CoA reductase preceded the increase of alkaloid production and of sterol content. During the first alkaloid phase, cell mevalonate was involved in the biosynthesis of both alkaloids and steroids. In the second production phase, it was predominantly used for alkaloid synthesis. Hydroxymethylglutaryl-CoA reductase appears to be a suitable target for physiological manipulation to increase clavine alkaloid yields.     

The adhesin structures involved in the adherence of group B streptococci to human vaginal cells

The adherence of group B streptococci (GBS) of serotypes Ia, II and III to human vaginal cells was studied in vitro. The adherence was not dependent on the viability of bacteria; killing of GBS by UV irradiation or glutaraldehyde treatment did not inhibit the adherence. Killing of GBS by heating to 56 degrees C for 1 h led to a pronounced decrease of adherence, demonstrating the thermosensitivity of the GBS structures involved. The protein nature of these structures was proved by a significant reduction of adherence after pretreatment of GBS with trypsin or pepsin. Pretreatment of GBS with sialidase had no influence on the adherence. Such a pretreatment of vaginal cells caused an increase of adherence showing that the receptors on epithelial cells may be partly masked by sialic acid.     

Evolution of digestibility by Hind III: an analysis by light and electron microscopy

Digestion of Chinese hamster metaphase chromosomes from the Don cell line by Hind III restriction endonuclease followed by Giemsa staining were analysed by light and electron microscopy. The evolution of digestibility was studied and four digestion stages were characterized by different levels of chromosome structure. Three different condensation stages were established according to morphological criteria of length, width and separation among chromatids. It was observed that there are statistically significant differences in the digestion progress at the three condensation stages previously defined.     

Investigation of Homologous Crossing over and Sister Chromatid Exchange in the Wheat Nor-B2 Locus Coding for Rrna and Gli-B2 Locus Coding for Gliadins

Recombination was investigated within the Nor-B2 locus of wheat chromosome 6B that contains several thousand of the 18S-5.8S-26S rRNA (rDNA) repeated units. Additionally, recombination was assessed for several chromosome regions, in arm 6Bq between the centromere and the B2 locus (awn suppressor) and in arm 6Bp between the centromere and Nor-B2, between Nor-B2 and a distal C-band and between Nor-B2 and Gli-B2 coding for gliadins. The experimental design permitted the distinction between crossing over between homologous chromosomes and exchange between sister chromatids. No homologous crossing over within the Nor-B2 locus was found in a sample of 446 chromosomes, but one exchange with the attributes of unequal sister chromatid exchange was identified. The molecular characteristics of this presumed sister chromatid exchange indicate that the spacer variants present in the Nor-B2 locus are clustered. No homologous recombination was detected within the distal Gli-B2 locus containing repeated genes coding for gliadin seed-storage proteins. Both arms of chromosome 6B showed low crossing-over frequency in the proximal regions. The distance from the centromere to Nor-B2 was only from 0.3 to 2.2 cM although it accounts for about two-thirds of the metaphase chromosome arm, which shows a great distortion of the metaphase map of the arm. The level of homologous recombination within the Nor-B2 locus is lower than in the chromosome region immediately distal to it. Whether it is comparable to that in the chromosome region proximal to it could not be determined. Recombination frequencies of different pairs of chromosome 6B in all but one interval paralleled the frequencies of their metaphase I pairing: Lower pairing at metaphase I was paralleled by lower crossing-over frequency. This relationship indicated that reduced metaphase I pairing between 6B chromosomes from different populations is due to impaired crossing-over and not due to precocious chiasma terminalization.