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Applied Biochemistry and Microbiology (v.48, #6)
Analysis of functional properties of biologically active substances using eukaryotic cell models (review) by K. V. Lisitskaya; I. V. Nikolaev; A. A. Torkova; V. O. Popov; O. V. Koroleva (pp. 525-540).
This work presents an analysis of current data on the investigation into the functional properties of biologically active substances in model systems based on cultivated human cells. The knowledge regarding the practical application of cell cultures for the analysis of functional properties of bioactive substances is summarized, including antioxidant, immunomodulating, pro- and prebiotics, and chemoprevention properties. The most promising directions in cell culture model development for the investigation of functional properties, including three-dimensional models, are discussed.
Obtaining homogenous preparations of succinate dehydrogenase isoforms from the D-507 strain of Sphaerotilus natans by A. T. Eprintsev; T. L. Wu; N. V. Selivanova; A. Khasan Khamad (pp. 541-545).
Enzymatic preparations of two isoforms of succinate dehydrogenase (SDG) with specific activity of 22.00 E/mg and 14.75 E/mg of protein were obtained from the colorless sulfur bacterium Sphaerotilus natans D-507 cultured organotrophically. Both SDG forms were shown to be heterotetromers with subunit molecular masses of 70.8, 35.0, 31.8, and 16.2 kDa. The K m values for the first and the second forms of SDG were evaluated as 0.615 and 0.531 mM, respectively, with an optimal pH value of 7.2. It was found that the Cl− ion has an activating effect on the SDG activity that can be explained by the specific chemical modification of the enzyme molecule. The results suggest that the isolated enzyme forms are included in different multienzyme complexes, which provide the functioning of the tricarboxylic acid cycle, and SDG preparations can be used for the investigation of other enzyme systems or in vitro modeling of supramolecular cellular structures.
Purification and characterization of methanol dehydrogenase of Methylobacterium nodulans rhizosphere phytosymbionts by T. A. Kuznetsova; A. P. Beschastny; O. N. Ponamoreva; Yu. A. Trotsenko (pp. 546-551).
Methanol dehydrogenase (MDH) of the facultative methylotrophic phytosymbiont Methylobacterium nodulans has been purified for the first time to an electrophoretically homogeneous state and characterized. The native protein with a molecular mass of 70 kDa consists of large (60 kDa) and small (6.5 kDa) subunits. The purified protein displayed a spectrum identical to that of pyrroloquinoline quinone (PQQ)-containing MDH, pI 8.7, pH optimum in the range 9–10. The enzyme was inactive in the absence of ammonium or methylamine and exhibited a wide substrate specificity with regard to C1–C5 alcohols with the high-est affinity to methanol (K M = 70 μM), but it did not oxidize benzyl and secondary alcohols. The apparent K M values to primary alcohols increased with the length of the carbon chain. The enzyme was characterized by a high stability level even in the absence of a substrate. An immobilized enzyme was used for amperometric methanol detection.
Catalase activity of hydrocarbon-oxidizing bacteria by O. A. Gogoleva; N. V. Nemtseva; O. V. Bukharin (pp. 552-556).
The dynamics of catalase activity of the hydrocarbon-oxidizing bacteria Gordona terrae, Rhodococcus rubropertinctus, and Rhodococcus erythropolis during petroleum product destruction has been studied. A direct relationship between decreasing catalase activity of hydrocarbon-oxidizing microorganisms and the intensity of petroleum product destruction has been established experimentally. The revealed dependence allows one to consider the catalase activity of bacteria as an indicator of the initial stage of petroleum product oxidation and may be used for choosing destructor strains to construct biopreparations suitable for natural ecosystem remediation.
Characteristics and identification of bacteriocins produced by Lactococcus lactis subsp. lactis 194-K by E. A. Ustyugova; A. V. Timofeeva; L. G. Stoyanova; A. I. Netrusov; G. S. Katrukha (pp. 557-563).
The Lactococcus lactis subsp. lactis 194-K strain has been established to be able to produce two bacteriocins, one of which was identified as the known lantibiotic nisin A, and the other 194-D bacteriocin represents a polypeptide with a 2589-Da molecular mass and comprises 20 amino acid residues. Both bacteriocins were produced in varying proportions in all of the studied culture media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was 380- to 1123-fold lower in the 194-K stain culture broth than that of the 194-D peptide. In comparision to nisin A Bacteriocin 194-D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for 194-D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis of bacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture broth was observed at14–20 h of the strain’s growth.
Degradation of the EDTA and EDTA complexes with metals by immobilized cells of Chelativorans oligotrophicus LPM-4 bacteria by T. N. Kuvichkina; E. N. Kaparullina; N. V. Doronina; Yu. A. Trotsenko; A. N. Reshetilov (pp. 564-568).
Enzymatic oxidative degradation of EDTA and EDTA complexes with metals has been investigated using immobilized cells of Chelativorans oligotrophicus LPM-4. A polarographic method, which makes it possible to register oxygen consumption by cells, has been used. For the first time, it has been indicated that the Cd-EDTA and Ni-EDTA complexes undergo degradation by the bacteria under study.
Production of surfactants by Acinetobacter calcoaceticus K-4 grown on ethanol with organic acids by T. P. Pirog; T. A. Shevchuk; A. D. Konon; E. Yu. Dolotenko (pp. 569-576).
The effect of fumarate (C4-dicarboxylic acid, a gluconeogenesis precursor) and citrate (a lipid synthesis regulator) on the production of surfactants by Acinetobacter calcoaceticus K-4 grown on ethanol has been studied. Simultaneous addition of fumarate and citrate to concentrations of 0.01–0.02% at the end of the exponential phase of K-4 growth in a medium with 2 vol % ethanol increases the conditional surfactant concentration by 45–55% in comparison with indices in medium without organic acids. The increased level of surfactant production in the presence of fumarate and citrate is determined by the increase in the activities of enzymes involved in the production of glycolipids (phosphoenolpyruvate synthase and trehalose phosphate synthase) and aminolipids (NADP+-dependent glutamate dehydrogenase) in of 1.7–7 times, as well as by the simultaneous functioning of two anaplerotic pathways: the glyoxylate cycle and the reaction catalyzed by phosphoenolpyruvate carboxylase.
Oxidation of sulfur-containing substrates by aboriginal and experimentally designed microbial communities by T. A. Pivovarova; A. G. Bulaev; P. V. Roshchupko; A. V. Belyi; T. F. Kondrat’eva (pp. 577-582).
Aboriginal and experimental (constructed of pure microbial cultures) communities of acidophilic chemolithotrophs have been studied. The oxidation of elemental sulfur, sodium thiosulfate, and potassium tetrathionate as sole sources of energy has been monitored. The oxidation rate of the experimental community is higher as compared to the aboriginal community isolated from a flotation concentrate of pyrrhotine-containing pyrite-arsenopyrite gold-arsenic sulfide ore. The degree of oxidation of the mentioned S substrates amounts to 17.91, 68.30, and 93.94% for the experimental microbial community and to 10.71, 56.03, and 79.50% for the aboriginal community, respectively. The degree of oxidation of sulfur sulfide forms in the ore flotation concentrate is 59.15% by the aboriginal microbial community and 49.40% by the experimental microbial community. Despite a higher rate of oxidation of S substrates as a sole source of energy by the experimental microbial community, the aboriginal community oxidizes S substrates at a higher rate in the flotation concentrate of pyrrhotine-containing pyrite-arsenopyrite gold-arsenic sulfide ore, from which it was isolated. Bacterial-chemical oxidation of the flotation concentrate by the aboriginal microbial community allows for the extraction of an additional 32.3% of gold from sulfide minerals, which is by 5.7% larger compared to the yield obtained by the experimental microbial community.
Purification, characterisation and coal depolymerisation activity of lignin peroxidase from Lenzitus betulina MTCC-1183 by M. Yadav; S. K. Singh; S. Yadava (pp. 583-589).
Lignin peroxidase from the culture filtrate of Lenzitus betulina MTCC-1183 has been purified to homogeneity using concentration by ultrafiltration and anion exchange chromatography on DEAE cellulose. The molecular weight of the purified lignin peroxidase using SDS-PAGE analysis was 43 kDa. Specific activity of the enzyme was 29.58 IU/mg. The K m values for veratryl alcohol and H2O2 for the purified enzyme were 54 and 81 μM, respectively. The k cat value of the purified enzyme was 2.3 s−1 using 3,4-dimethoxybenzyl alcohol as the substrate. The optimal conditions for the lignin peroxidase assay were detected at pH 2.4 and 22°C. Thermal stability of the purified enzyme has also been studied and its activation energy for deactivation was 287 kJ/mol. The purified lignin peroxidase depolymerised humic acid in presence of H2O2. Depolymerisation of coal by the L. betulina MTCC-1183 has been demonstrated using humic acid as a model of coal.
Development of immunochromatographic test system for rapid detection of the lipopolysaccharide antigen and cells of the causative agent of bovine brucellosis by N. A. Byzova; A. V. Zherdev; S. Z. Eskendirova; K. K. Baltin; G. B. Unysheva; K. K. Mukanov; E. M. Ramankulov; B. B. Dzantiev (pp. 590-597).
A rapid method for detection of the surface lipopolysaccharide antigen and the cells of the causative agent of bovine brucellosis was developed. The method represents a sandwich format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid movement along the membrane components of the test strip, immunochemical reactions, and the formation of colored bands. The novel method requires 10 minutes to determine the lipopolysaccharide antigen of the cell wall of the brucellosis causative agent at concentrations down to 10 ng/mL and the Brucella abortus cells at concentrations down to 106 cells/mL (5 × 104 cells in the sample). The specificity of the immunodetection was confirmed. The designed test system can be used for the rapid field diagnosis of brucellosis in cattle.
Prolonged release of chlorambucil and etoposide from poly-3-oxybutyrate-based microspheres by E. V. Filatova; S. G. Yakovlev; A. P. Bonartsev; T. K. Makhina; V. L. Myshkina; G. A. Bonartseva (pp. 598-602).
Microspheres were obtained on the basis of poly(3-oxibutyrate) (POB) with the inclusion of the Chlorambucil and Etoposide cytostatic drugs in a polymer matrix, and the morphology, kinetics of drug release from microspheres, and the interaction between microspheres and tumor cells in vitro were studied. Data on the kinetics of drug release suggests that a prolonged release occurs by drug diffusion from the polymer matrix at the initial stage and at the expense of hydrolytic degradation of the polymer at a later stage. A study of the biocompatibility and biological activity of biopolymeric microspheres showed that chlorambucil operates actively and strongly inhibits the growth of cultured cells for a short time (24 h). Etoposide acts weaker (the percentage of cell growth suppression during 48 h does not exceed 50%), but subsequently it has a basis for the creation of new dosage forms with prolonged action of Etoposide and chlorambucil for cancer therapy.
Scanning assay of β-galactosidase activity by W. Li; X. Zhao; S. Zou; Y. Ma; K. Zhang; M. Zhang (pp. 603-607).
β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.