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Applied Biochemistry and Microbiology (v.41, #2)
Effect of water stress on the photosynthetic apparatus of plants and the protective role of cytokinins: A review by I. I. Chernyad’ev (pp. 115-128).
Characteristics of photosynthetic apparatus (the pool of pigments and proteins; the activity of photosystems; the intensities of in vivo photoassimilation of carbon dioxide and in vitro activity of enzymes of carbon metabolism; leaf structure; chloroplast structure), undergoing changes under the conditions of water deficiency, have been reviewed. The protective role of cytokinins is due to their regulatory effects on the renewal of disrupted cellular structures, the condition of the stomata, and de novo synthesis and activation of proteins that are required for increasing plant resistance to water stress.
Activation of peroxidase-catalyzed oxidation of chromogenic substrates by tetrazole and its 5-substituted derivatives by E. I. Karasyova; P. N. Gaponik; D. I. Metelitza (pp. 129-138).
Peroxidase-catalyzed oxidation of 2,2-azino-di(3-ethyl-benzthiazolydine-6-sulfonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) is activated by tetrazole and its 5-substituted derivatives—5-amino-(AmT), 5-methyl-(MeT), 5-phenyl-(PhT), and 5-CF3-(CF3-T) tetrazoles. In phosphate-citrate or phosphate buffer (pH 6.4 or 7.2; 20°C), the activating effect of tetrazoles on TMB and ABTS oxidation decreased in the series AmT > MeT > T > PhT > CF3-T and T > AmT > MeT > PhT, respectively. The coefficient (degree) of activation (α), expressed in M−1, determined for both substrates and all activators, depended on substrate type, buffer nature, and pH (it increased as pH increased from 6.4 to 7.2). For TMB oxidation, good correlation between logα and the Hammet constants σmeta for m-substituents in the benzene series NH2, CH3, C6H5, and CF3 was found. It is suggested that AmT, MeT, and T can be used as activators of peroxidase-catalyzed oxidation of TMB and ABTS in enzyme immunoassay and designing peroxidase-based biosensors.
Purification and properties of serine proteinases from European catfish Silurus glanis L. pancreas by N. N. Ulitina; V. V. Khablyuk; M. T. Proskuryakov (pp. 139-144).
Three trypsin isoforms (designated as T1, T2, and T3), three chymotrypsin isoforms (Kh1, Kh2, and Kh3), and two elastase isoforms (E1 and E2) were isolated from the pancreas of European catfish Silurus glanis L. by salting out with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion exchange chromatography on DEAE cellulose. Isoelectric points of the enzymes, determined by isoelectric focusing, amounted to 4.42 for T1, 5.64 for T2, 6.90 for T3, 4.93 for Kh1, 5.23 for Kh2, 6.18 for Kh3, 6.17 for E1, and 8.48 for E2. Molecular weights of proteinases within each group were close and amounted to 30,100 Da for trypsins, 39,800 Da for chymotrypsins, and 24,000 Da for elastases. The enzymes isolated displayed maximal activities at alkaline pH values. Inhibitor analysis demonstrated that all the proteinases isolated from European catfish pancreas belonged to the serine type.
Thermostabilities of plant phenol oxidase and peroxidase determining the technology of their use in the food industry by N. I. Mchedlishvili; N. T. Omiadze; L. K. Gulua; T. A. Sadunishvili; R. K. Zamtaradze; M. O. Abutidze; E. G. Bendeliani; G. I. Kvesitadze (pp. 145-149).
Stabilities of phenol oxidase and peroxidase from tea plant (Camellia sinensis L.) clone Kolkhida leaves, apple (Mallus domestica L.) cultivar Kekhura fruits, walnut (Juglans regia L.) green pericarp, and horseradish (Armoracia lapathifolia Gilib) roots were studied using different storage temperature modes and storage duration. It was demonstrated that both enzymes retained residual activities (∼10%) upon 20-min incubation at 8°C. Phenol oxidases from tea, walnut, and especially apple, as well as tea peroxidase, were stable during storage. A technology for the treatment of plant oxidases was proposed, based on the use of a natural inhibitor of phenol oxidase and peroxidase, isolated from tea leaves, which solves the problem of residual activities of these enzymes that arises during pasteurization and storage of beverages and juices. It was demonstrated that browning of apple juice during pasteurization and beer turbidity during storage could be efficiently prevented using the natural inhibitor of these enzymes.
Cross-effects of extracellular factors of adaptation to stress in Luteococcus casei and Saccharomyces cerevisiae by L. I. Vorob’eva; E. Yu. Khodzhaev; G. M. Ponomareva (pp. 150-153).
Saccharomyces cerevisiae yeasts (lower eukaryotes) were shown to produce a protein exometabolite with reactivation activity. We demonstrated cross-effects of extracellular protein factors of adaptation to stress (heat and UV irradiation) in yeasts and Luteococcus casei bacteria. The possibility for isolation and partial purification of protein exometabolites from the culture liquid of yeasts and bacteria by similar methods, as well as the similarity of elution profiles for the active proteins in high-performance liquid chromatography, suggests that the proteins (or fragments thereof) of the organisms studied are homologous.
Application of molecular systematics to study of bacterial cultures consuming volatile organic compounds by V. G. Khomenkov; A. B. Shevelev; V. G. Zhukov; A. E. Kurlovich; N. A. Zagustina; V. O. Popov (pp. 154-161).
A range of species of four mixed bacterial cultures was studied by molecular systematics methods with the use of 16S rRNA genes. The cultures had been developed for application in minireactors, to degrade volatile organic compounds (VOCs): ethyl benzene, m-xylene, styrene, and o-xylene. A sample of 30 plasmid rDNA clones was obtained for each of the mixed cultures. The clones were analyzed by RFLP according to two restriction sites. Major variants of the 16S-rDNA sequences, corresponding to the most abundant species, were determined for each association. Sequencing of four clones of predominant 16S-rDNAs showed that the culture consuming ethyl benzene was dominated by Pseudomonas fluorescens; o-xylene, by Achromobacter xylosoxydans; styrene, by Pseudomonas veronii; and m-xylene, by Delftia acidovorans. Minor components of all four cultures were generally similar. They included species of the genera Sphingobacter, Rhizobium, Mesorhizobium, Pedobacter, and Paenibacillus. Sampling sequencing of genes for 16S rRNA cloned from total genomic DNA allowed quantitative determination of the composition of actual bacterial associations consuming VOCs in minireactors.
Isolation and study of azobenzene-transforming soil bacteria by N. D. Wackerow-Kouzova (pp. 162-164).
Heterotrophic bacteria were isolated from soil and glass slides and classified as Bacillus cereus SNK12, Paenibacillus polymyxa SNK2, Azotobacter chroococcum ANKII, and Ochrobactrum intermedium ANKI. Their cultures could degrade azobenzene under the conditions of co-metabolism. A rapid test for the ability of bacteria to transform azobenzenes is proposed.
Increase in tylosin production by a commercial strain of Streptomyces fradiae by D. G. Lyutskanova; M. M. Stoilova-Disheva; V. T. Peltekova (pp. 165-168).
Conventional mutagenesis (UV irradiation and exposure to nitrosoguanidine) as well as protoplast formation and regeneration were used to improve the antibiotic activity of a Streptomyces fradiae strain producing tylosin. Variants exceeding the activity of the initial producer strain by 0.5–28.3% were obtained. The most active variants were produced by a combined exposure to UV and nitrosoguanidine, as well as upon regeneration of protoplasts formed from the cells of clones produced by UV irradiation. Unstable inheritance of the trait of increased tylosin production was demonstrated.
Polysaccharides of Ganoderma lucidum: Factors affecting their production by V. G. Babitskaya; V. V. Shcherba; T. A. Puchkova; D. A. Smirnov (pp. 169-173).
The conditions of polysaccharide production by the fungus Ganoderma lucidum were optimized. The maximal yield of endopolysaccharides and exopolysaccharides was observed at 25–30°C, initial pH of culture medium 4.0–6.0, and at a C:N ratio of ∼18:1 and 25:1, respectively. The greatest yield of mycelium was reached at a more intensive aeration, and the maximal yield of polysaccharides was observed at a less intensive aeration. The optimal ratio between fungus growth and polysaccharide production was observed at 100 rpm and an aeration of 1.0– 1.5 1/1 medium·min.
Effect of culture medium composition on the activity of extracellular lectins of Lentinus edodes by O. M. Tsivileva; V. E. Nikitina; L. V. Garibova (pp. 174-176).
The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. The lectin activity in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C:N ratio, (9.5–12):1) on day 15–18 of culturing at pH 8.0–9.0.
Selection and properties of mutant yeast Pichia guilliermondii strains resistant to chromium (VI) by L. Ya. Babyak; G. P. Ksheminskaya; M. V. Gonchar; D. V. Yanovich; D. V. Fedorovich (pp. 177-181).
Yeast Pichia guilliermondii strains L3 and L2, exposed to UV mutagenesis, produced over 80 mutants capable of growing on media containing 1.5 mM bichromate (Cr(VI)). The mutations making the strains resistant to Cr(VI) were dominant or semidominant. The mutants varied in Cr(VI) resistance, the degree of chromium accumulation in the cells (from 0.1 to 11.6 mg/g dry cells), and the degree of Cr(VI) reduction (from 50% to complete disappearance of bichromate from the culture liquid). Chromium accumulation in mutant cells depended on medium composition, Cr(VI) concentration, and the time of exposure to Cr(VI). The resistance to bichromate can be caused by various reasons: decrease in chromium absorption, altered ability to reduce Cr(VI), or damage of sulfate transport mechanisms.
Studies of hydrolytic activity of enzyme preparations of Penicillium and Trychoderma fungi by A. A. Skomarovsky; A. V. Gusakov; O. N. Okunev; I. V. Solov’eva; T. V. Bubnova; E. G. Kondrat’eva; A. P. Sinitsyn (pp. 182-184).
Enzyme preparations were isolated from the culture liquid of five mutant strains of the cellulase producer Penicillium verruculosum. The hydrolytic activities of these preparations against unbleached eucalypt cellulose was compared to that of commercial preparations of Trichoderma reesei (T. longibrachiatum). In the majority of cases, P. verruculosum enzymes provided higher yields of reducing sugars (RSs) and glucose. A correlation was found between the yield of RSs and the avicelase activity of the preparations in the reaction mixture.
Polysaccharides of diatoms occurring in Lake Baikal by S. A. Alekseeva; N. M. Shevchenko; M. I. Kusaykin; L. P. Ponomorenko; V. V. Isakov; T. N. Zvyagintseva; E. V. Likhoshvai (pp. 185-191).
Polysaccharide composition of neutral, acid- and alkali-soluble fractions of the diatoms Stephanodiscus meyerii Genkal et Popovsk and Aulacoseira baicalensis (K. Meyer) Simonsen of Lake Baikal has been studied. Neutral polysaccharides were represented by chrysolaminarans (1→3;1→6-β-D-glucans). The chrysolaminaran from S. meyerii consists of the high- and low-molecular-weight fractions (40 and 2–5 kDa, respectively) and contains a large number of β-1→6-bound glucose residues. The chrysolaminaran from A. baicalensis is a low-molecular-weight 1→3;1→6-β-D-glucan containing a small number of β-1→6-bonds, with mannitol being attached to the reducing unit of its chain. Acid- and alkali-soluble polysaccharide fractions are practically absent in S. meyerii. The alkali-soluble fraction from A. baicalensis is a low-molecular-weight (2-kDa) glycoprotein, the carbohydrate moiety of which is represented by a heteropolysaccharide.
Effect of aluminum and iron on lipid metabolism in aquatic invertebrates by T. I. Regerand; Z. A. Nefedova; N. N. Nemova; T. R. Ruokolainen; L. V. Toivonen; L. V. Dubrovina; K.-M. Vuori; L. V. Markova (pp. 192-198).
Methods of thin-layer, gas-liquid, and liquid chromatography were applied to the study of the effect of various concentrations of aluminum and iron salts on the contents of phospholipids, cholesterol, and fatty acids in the aquatic invertebrate Hydropsyche contubernalis L. (Trichoptera). It was found that the effect of the metals under study on lipid contents in living organisms depended on the composition of the aqueous medium and concentrations of the metals. Aluminum and iron altered the value of the cholesterol-to-phospholipid molar ratio. In the absence of lethal effects, this was indicative of attempts to switch adaptational biochemical mechanisms to stabilize cellular structures.
The use of liposomes for detection of the surface lipopolysaccharide antigen, Vibrio cholerae cells, and antibodies against them by S. N. Skopinskaya; S. P. Yarkov; E. N. Chramov (pp. 199-204).
A test system for determination of Vibrio cholerae cells, surface O-antigen, and antibodies against them was developed on the basis of complement-dependent lysis of liposomes sensitized by the lipopolysaccharide antigen from Vibrio cholerae 569B. The factors that affect the function of the liposomal reagent were studied, and the conditions for detecting antibodies and antigenic material were optimized. This system is highly specific and sensitive to use for the determination of anticholeraic antibodies (30–50 times as effective as agglutination tests), lipopolysaccharide antigen (100 ng/ml, which corresponded to 3.0 ng of lipopolysaccharide in the sample studied), and Vibrio cholerae cells (3.3 × 107 m.b./ml, which corresponded to 106 m.b. in sample). It takes 30–40 min to detect the lipopolysaccharide antigen and antibodies and 90 min to detect V. cholerae cells.
Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin by N. G. Titova; V. V. Sviridov (pp. 205-212).
The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin was not detected, and free antibodies accounted for 68 to 98% of the original amount of the antibodies. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80–100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalence of serum antibodies, and monovalence of the toxin epitopes. A new model of the toxin-antibody interaction is proposed.
Growth-regulating activity of N-benzyl- and O-benzyl-containing compounds belonging to a new group of synthetic analogues of natural auxins by R. G. Gafurov; A. A. Makhmutova (pp. 213-218).
We studied the effect of benzylamine, benzyl alcohol, and their derivatives (constituting a new group of synthetic analogues of natural auxins) on rooting of leaf and stem cuttings, rhizogenesis and growth of barley plantlets and tomato seedlings, and tomato plant productivity. These compounds promoted rooting of leaf and stem bean cuttings, increased rhizogenic activity, and stimulated the development of root systems in barley and tomato seeds. The activity of the compounds studied was similar to that of standard substances (3-indoleacetic acid potassium salt and 2-naphthylacetic acid). The benzyl group attached to the oxygen or nitrogen atom was shown to be the smallest molecular structure which provided auxin activity of the compounds. Derivatives of benzyl alcohol containing the quaternary ammonium fragment possessed auxin and anti-gibberellin (retardant) properties. They were selected by chemical synthesis of low-molecular-weight bioregulators with desired properties (a combination of chemical fragments with complementary physiological activity in the molecule). Auxin and anti-gibberellin (retardant) activities produced a synergistic effect. Germination of seeds treated with these compounds was accompanied by a more significant increase in the weight and length of roots (compared to standard auxins). The rate of seedling establishment reached 100%. The development of fruits and accumulation of reserve nutrient substances were synchronized and accelerated after spraying vegetating plants with solutions of studied compounds. The synergistic effect underlay a significant increase in the amount and quality of the crop (e.g., tomatoes).