Monitoring of actin unfolding by room temperature tryptophan phosphorescence

Slow intramolecular mobility of native and inactivated actin from rabbit skeletal muscle during the process of protein unfolding induced by GdnHCl was studied using tryptophan room temperature phosphorescence (RTP). By this method, the conclusion was confirmed that an essentially unfolded intermediate preceded the formation of inactivated actin [Turoverov et al. Biochemistry (2002) 41, 1014-1019]. It was found that the kinetic intermediate generated at the early stage of protein denaturation has no tryptophan RTP, suggesting the high lability of its structure. Symbate changes of integral intensity and the mean lifetime of RTP during the U* --> I transition suggests a gradual increase of the number of monomers incorporated in the associate (U* --> I(1)... --> I(n)... --> I(15)), which is accompanied by an increase of structural rigidity. The rate of inactivated actin formation (I identical with I(15)) is shown to increase with the increase of protein concentration. It is shown that, no matter what the means of inactivation, actin transition to the inactivated state is accompanied by a significant increase of both integral intensity and the mean lifetime of RTP, suggesting that inactivated actin has a rigid structure.     

A comparison of the fatty acid composition of Gammarus lacustris and its food sources from a freshwater reservoir, Bugach, and the saline Lake Shira in Siberia, Russia

We studied fatty acid (FA) composition in samples from bodies and intestinal contents of the littoral amphipod Gammarus lacustris Sars, from the Bugach freshwater reservoir. Simultaneously, samples of seston and bottom sediments were also collected from the reservoir during early August. There were no differences in FA composition of gut contents, seston and sediments of pebbly bottom. Seston was the main food source of Gammarus but some FAs Gammarus got from sediments. The FA composition of G. lacustris and seston from the Bugach freshwater reservoir were compared with those of the animals from the saltwater Lake Shira (Siberia). While FA composition of the two Gammarus populations differed significantly, those of seston were practically similar: the composition of long-chain unsaturated fatty acids, 20:53, 22:63 and 20:46, were significantly higher in animals from saline Shira Lake, whereas 16:1 and 16:0 were higher in the freshwater populations of amphipods from the Bugach freshwater reservoir. Taking into account the relevant literature data, we hypothesise that this difference in C16 acid might be a distinguishing characteristic of FA composition of freshwater and saltwater crustaceans.     

Analysis of sequences from human brain cDNA gene bank which are functionally active in nervous tissue and tumor cells

Construction of a human cortex cDNA bank is described as well as the isolation from this bank of pBH71 and pBH3 clones with preferential expression in nervous and in tumor cells. The clones can be included into the third class of cDNA according to Sutcliff's classification. The mRNA corresponding to this cDNA class is considered to play the key role in determination of specificity of nervous tissue. Expression of the pBH71 sequence was revealed in human cortex and in tissues of different genesis (from neuroblastoma to uterus myoma), a 2 kb mRNA which corresponds to one and the same cDNA chain having been found in all tissues under analysis. The nucleotide sequence of cDNA insertion into the pBH71 clone of 447 n.p. was determined, and particular features of cDNA nucleotide composition and possible schemes of its translation were analysed. Weak homology was found between the 3'-end of cDNA insertion of the pBH71 clone and the 3'-end region of human proopiomelanocortine. The cDNA of the pBH3 clone hybridizes with the 0.8 kb mRNA revealed in human cortex and neuroendocrine tumors of different nature. No homology was revealed between the cDNA sequence of the pBH3 clone and any genes deciphered.     

Investigation of novel oligoelectrolyte polymer carriers for their capacity of DNA delivery into plant cells

Development of modern agriculture and biotechnology is closely connected with the use of novel and effective genetic engineering methods. Presently, non-viral nanoparticle-mediated plant transformation methods gain more attention because of their stability, safety, and convenience of performance. In this work, new polymeric dimethylaminoethyl metacrylate (DMAEM)-based polymers were synthesized and investigated for their properties in gene delivery. Formation of stable complexes between TN 83/6, TN 84/5, DLM-9-DM and LM-8-DM polymers and plasmid DNA, as well as the DNA protection by the PDMAEM polymers against nuclease degradation were confirmed by electrophoresis in agarose gel. In addition, model organisms Allium cepa and Nicotiana tabacum L. were studied to evaluate cytotoxic effect of the PDMAEM carriers. The created PDMAEM-based carriers were effective in delivery of plasmid DNA into moss and tabacco protoplasts (obtaining stable transformants of Ceratodon purpureus moss, as well as in transient expression of the reporter yfp gene product in N. tabacum protoplasts). Thus, novel PDMAEM-based polymers were shown to be promising carriers for delivery of DNA into plant cells, and carriers possess high potential for further applications in this field.     

Fatty acid composition of Cladocera and Copepoda from lakes of contrasting temperature

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Seasonal correlations of elemental and ω3 PUFA composition of seston and dominant phytoplankton species in a eutrophic Siberian Reservoir

The elemental and fatty acid composition of seston was studied for 3 years, from May to October, in a small Reservoir. Under comparatively low C:P ratio, multivariate canonical analysis revealed no straightforward simple correlations between phosphorus and single ω3 PUFA species, but complex significant interaction between elemental composition (stoichiometry) of seston and total sestonic ω3 PUFA as a whole. Since sestonic C, P and N were found to originate mostly from phytoplankton, the contents of particulate elements and PUFA were attributed to single species in periods of their pronounced dominance. Phytoplankton species of genera of Stephanodiscus, Peridinium, Gomphosphaeria, Planktothrix and Anabaena in periods of their pronounced dominance had relatively constant species-specific elemental and PUFA composition. Phytoplankton species significantly differed in their elemental and PUFA composition, as well as in ratios of C:N, N:P, PUFA:P and partly C:P that indicate food quality for zooplankton. Hence, there were no phytoplankton species of clearly high or low nutritional value. All of phytoplankters, or at least detritus, that originated from them, may meet specific elemental and biochemical requirements of specific groups of zooplankton. Dividing phytoplankton on basis of their elemental and biochemical composition, i.e., nutrition quality, into large taxa (cyanobacteria, diatoms, etc.) appeared to be too coarse for assessing nutritional value for zooplankton.     

Plasmid pEC156, a Naturally Occurring Escherichia coli Genetic Element That Carries Genes of the EcoVIII Restriction-Modification System,Is Mobilizable among Enterobacteria

Type II restriction-modification systems are ubiquitous in prokaryotes. Some of them are present in naturally occurring plasmids, which may facilitate the spread of these systems in bacterial populations by horizontal gene transfer. However, little is known about the routes of their dissemination. As a model to study this, we have chosen an Escherichia coli natural plasmid pEC156 that carries the EcoVIII restriction modification system. The presence of this system as well as the cis-acting cer site involved in resolution of plasmid multimers determines the stable maintenance of pEC156 not only in Escherichia coli but also in other enterobacteria. We have shown that due to the presence of oriT-type F and oriT-type R64 loci it is possible to mobilize pEC156 by conjugative plasmids (F and R64, respectively). The highest mobilization frequency was observed when pEC156-derivatives were transferred between Escherichia coli strains, Enterobacter cloacae and Citrobacter freundii representing coliform bacteria. We found that a pEC156-derivative with a functional EcoVIII restriction-modification system was mobilized in enterobacteria at a frequency lower than a plasmid lacking this system. In addition, we found that bacteria that possess the EcoVIII restriction-modification system can efficiently release plasmid content to the environment. We have shown that E. coli cells can be naturally transformed with pEC156-derivatives, however, with low efficiency. The transformation protocol employed neither involved chemical agents (e.g. CaCl₂) nor temperature shift which could induce plasmid DNA uptake.