Construction of DNA-probes and their immunochemical detection using nonradioactive hybridization tests

Simple and efficient chemical approaches to preparation of DNA probes carrying 2,4-dinitrophenyl, dansyl or biotin residues were developed. The residues were introduced using following DNA derivatization procedures: a) transamination of cytidine residues with O-(4-aminobutyl)hydroxylamine; b) mercuration of pyrimidine residues followed by beta-mercaptoethanol modification. It was shown that 2,4-dinitrophenyl-containing DNA probes can be used for nonradioactive hybridization detection of nucleic acids. DNP-DNA: DNA complexes were detected using mouse antibodies specific to 2,4-dinitrophenyl groups, which were developed with peroxidase-conjugated antimouse immunoglobulins. Peroxidase-catalyzed chemoluminescent reaction of luminol oxidation with hydrogen peroxide allowed to detect 10 picograms of the dinitrophenylated single-stranded DNA probe.     

Various methodological aspects of biochemistry

Certain methodological problems of present-day biochemistry are considered. Comprehension of the mechanisms of biochemical processes is shown to be possible on the basis of the structural and functional analysis at the molecular level. Ways of development and use of structural and functional approach to studies of supermolecular structures, cells and organisms on the whole are discussed.     

Dictyosphaerium-like morphotype in terrestrial algae: what is Xerochlorella (Trebouxiophyceae,Chlorophyta)?1

Several strains of terrestrial algae isolated from biological soil crusts in Germany and Ukraine were identified by morphological methods as the widely distributed species Dictyosphaerium minutum (=Dictyosphaerium chlorelloides). Investigation of the phylogeny showed their position unexpectedly outside of Chlorellaceae (Trebouxiophyceae) and distantly from Chlorella chlorelloides, to which this taxon was attributed after revision of the genus Chlorella based on an integrative approach. SSU rRNA phylogeny determined the position of our strains inside a clade recently described as a new genus of the cryptic alga Xerochlorella olmiae isolated from desert biological soil crusts in the United States. Investigation of the morphology of the authentic strain of X. olmiae showed Dictyosphaerium-like morphology, as well as some other characters, common for our strains and morphospecies D. minutum. The latter alga was described as terrestrial and subsequently united with the earlier described aquatic representative D. chlorelloides because of their similar morphology. The revision of Chlorella mentioned above provided only one aquatic strain (D. chlorelloides), which determined its position in the genus. But terrestrial strains of the morphospecies were not investigated phylogenetically. Our study showed that the terrestrial D. minutum is not related to the morphologically similar D. chlorelloides (=Chlorella chlorelloides, Chlorellaceae), and instead represented a separate lineage in the Trebouxiophyceae, recently described as genus Xerochlorella. Therefore, revision of Xerochlorella is proposed, including nomenclatural combinations, epitypifications, and emendations of two species: X. minuta and X. dichotoma. New characters of the genus based on investigation of morphology and ultrastructure were determined.     

Double-exponential kinetics of binding and redistribution of the fluorescent dyes in cell membranes witness for the existence of lipid microdomains

New technique of detecting lateral heterogeneity of the plasma membrane of living cells by means of membrane-binding fluorescent dyes is proposed. The kinetics of dye incorporation into the membrane or its lateral diffusion inside the membrane is measured and decomposed into exponential components by means of the Maximum Entropy Method. Two distinct exponential components are obtained consistently in all cases for several fluorescent dyes, two different cell lines and in different types of experiments including spectroscopy, flow cytometry and fluorescence recovery after photobleaching. These components are attributed to the liquid-ordered and disordered phases in the plasma membrane of studied cells in their dynamic equilibrium.     

Work with youth--the basis of educational programs aimed at the prevention of HIV infection

During the recent 10 years, 1987-1997, syphilis morbidity was found to increase 60-fold in Omsk Province. In 1997 adolescents constituted 6.0% in the structure of persons who contacted the disease. In this year more than 500 adolescent using drugs were registered in the region. The introduction of drugs by injection was practiced by 9.6% of the adolescents. The program aimed mainly at changing the behavior of young people with orientation on the choice of a healthy mode of life and theirs information about risk factors associated with sex transmitted diseases particularly HIV infection is discussed.     

A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family

Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.     

Recognition between flexible protein molecules: induced and assisted folding

This review focuses on a very important but little understood type of molecular recognition--the recognition between highly flexible molecular structures. The formation of a specific complex in this case is a dynamic process that can occur through sequential steps of mutual conformational adaptation. This allows modulation of specificity and affinity of interaction in extremely broad ranges. The interacting partners can interact together to form a complex with entirely new properties and produce conformational signal transduction at substantial distance. We show that this type of recognition is frequent in formation of different protein-protein and protein-nucleic acid complexes. It is also characteristic for self-assembly of protein molecules from their unfolded fragments as well as for interaction of molecular chaperones with their substrates and it can be the origin of 'protein misfolding' diseases. Thermodynamic and kinetic features of this type of dynamic recognition and the principles underlying their modeling and analysis are discussed.     

Cerebral blood flow modulates hyperbaric oxygen induced neurotoxicity by neuronal and endothelial nitric oxide

The goal of work was to reveal changes in microcirculation of the rat brain and the role of nitric oxide (NO) in development of seizures at hyperbaric oxygen exposure. The Wistar rats with implanted paired platinum electrodes in left and right striatum were used for experiments. The latency of seizures was defined by the EEG, the cerebral blood flow (CBF) was measured by hydrogen clearance. One group of animals was exposed to a 5-ata oxygen, while the others before oxygen treatment were injected with: Nw-nitro-L-arginine methyl ester (L-NAME), blockator of constitutive NO synthase; 7-nitroindozol (7NI), specific inhibitor of neural NO synthase. The latency of seizures was 41 +/- 1.9 min at 5 ata oxygen exposure. CBF was decreased to 10-14% but before seizures it increased to 23 +/- 9%. L-NAME and 7NI prevented development of hyperoxygen hyperemia and onset of seizures. The results indicate occurrence of hyperbaric oxygen changes of the CBF that modulate neurotoxic effects of NO in neurons as well as in cerebral vessels.     

Institute of Biochemistry and the development of molecular biophyics in the Ukraine

The development of molecular biophysics research in Ukraine is reviewed with historical perspective. Vladimir Belitser and his school were among the pioneers in the studies of protein denaturation and assembly of intermolecular structures. New methods of research of protein conformational transitions and dynamics at equilibrium have been developed at the Palladin Institute of Biochemistry. Presently, the biophysics aspects in different areas of protein research are actively explored. The studies of protein folding in both experimental and theoretical aspects promise especially good prospects.