Molecular identification of filterable bacteria and archaea in the water of acidic lakes of northern Russia

Wetland ecosystems are the natural centers of freshwater formation in northern Russia lowland landscapes. The humic acidic waters formed in bogs feed the numerous lakes of the northern regions. One milliliter of the water in these lakes contains up to 10⁴ ultrasmall microbial cells that pass through “bacterial” filters with a pore size of 0.22 μm. The vast majority of these cells do not grow on nutrient media and cannot be identified by routine cultivation-based approaches. Their identification was performed by analysis of clone libraries obtained by PCR amplification of archaeal and bacterial 16S rRNA genes from the fraction of cells collected from water filtrates of acidic lakes. Most of the obtained bacterial 16S rRNA gene sequences represented the class Betaproteobacteria and exhibited the highest homology of (94–99%) with 16S rRNA genes of representatives of the genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia. The archaeal 16S rRNA gene clone library comprised genes of Euryarchaeota representatives. One-third of these genes exhibited 97–99% homology to the 16S rRNA genes of taxonomically described organisms of the orders Methanobacteriales and Methanosarcinales. The rest of the cloned archaeal 16S rRNA genes were only distantly related (71–74% homology) to those in all earlier characterized archaea.     

Status of the reproductive system of bony fish from the Teterev River and the Kiev Reservoir 20 years after the Chernobyl accident

The status of the reproductive system is investigated in five species of Teleostei from the Teterev River and the Kiev Reservoir (Ukraine), descendants of F₄2–F₇ generations of fish exposed to radiation from the accident at the Chernobyl nuclear power plant. Twenty years after the accident, a wide range of morphofuinctional anomalies of this system is present in fish. The most significant of them are: asymmetry and anomalous morphology of gonads, proliferation of connective tissue—sterilization, mass destruction of follicular and sex cells of various developmental stages, and hermaphroditism. Among the investigated species, disturbances of reproductive glands were preset to the highest extent in pike Esox lucius and crucian carp Carassius carassius. In the series of investigated generations the maximum frequency of occurrence of gonad anomalies was noted in F₄ and F₅, due to prolonged mutagenesis (Dubinin, 1986).     

Interconnection of physico-chemical characteristics of organic solvents and their denaturing ability in relation to proteins]

Reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was studied. The regularities of this process were analyzed, employing both experimental and literary data based on the results of kinetic and spectroscopic measurements. In all the systems under study denaturation proceeded in a threshold manner, i. e., an abrupt change in the catalytic and/or spectroscopic properties of the dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of reversible protein denaturation by organic solvents was proposed. This model is based on the widely accepted viewpoint that the undisturbed water shell around the protein globule is necessary for maintaining the dissolved protein in the native state. Quantitative analysis of the model led to an equation establishing a relationship between the threshold concentration of an organic solvent and its physico-chemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation fits well in the experimental data for all the proteins tested. Based on the above thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents (termed as the denaturation capacity or DC) was proposed. Different organic solvents arranged according to their DC values form the DC scale of organic solvents which permits to predict theoretically the threshold concentration of any organic solvent for a given protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactate dehydrogenase isoenzyme spectrum in various areas of the reticular formation of the brain in rats: the effect of emotional stress

The changes in lactate dehydrogenase (LDG) isoenzyme content in the various brain areas were studied in intact Wistar rats and upon immobilization stress. LDG fraction levels were compared to BP changes during immobilization. The proportion of "anaerobic" LDG fractions was higher and the proportion of "aerobic" fractions lower in the dorsal area of midbrain substantia reticularis than in medulla oblongata reticular formation. The changes in LDG fraction content related to BP alterations during immobilization were observed in dorsal and ventral areas of midbrain (but not medulla oblongata) substantia reticularis. The proportion of anaerobic LDG4 fraction in the dorsal area of midbrain substantia reticularis was higher in rats with hypertensive responses, than in hypotensive animals. The changes in LDG5 fraction content were opposite. In the ventral area of midbrain reticular formation BP reduction was accompanied by a significant rise in "anaerobic" and a decrease in "aerobic" LDG fraction levels. The data obtained indicate certain differences in the intensity of aerobic and anaerobic processes of carbohydrate degeneration in various areas of substantia reticularis in control rats, as well as the correlation of changes in energy metabolism in the brain with BP alterations during emotional stress.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.      

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.