Identification of signature and primers specific to genus <Emphasis Type="Italic">Pseudomonas</Emphasis> using mismatched patterns of 16S rDNA sequences

Background

Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus.

Results

Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment.

Conclusions

The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

     

NRAMP1 gene: structure,function, and human infectious diseases

The human infectious disease become of the great importance for Health Welfare. The infectious diseases mortality rate reaches one third of total mortality among 51 million patients died annually. The genetic factors seem to be most responsible for potency of human body to withstand to infections, caused by a variety of causative agents. The detection of the coincident factors and understanding the mechanisms of formation of susceptibility and resistance to infectious agents appeared to be important aspects for development of the new methods of prevention and treatment the infectious diseases. In inbred mice the natural resistance to infections, caused by Mycobacterium bovis, Mycobacterium avium, Mycobacterium lepraemurium, Leishmania donovani and Salmonella typhimurium is controlled by gene Nrampl (natural resistance associated macrophage protein gene). The gene codes for integral membrane protein, expressed by phagocytes. Protein is localized in the endosomal/lysosomal compartment of the silent macrophage, being recruited to the membrane of the phagosome containing particles under phagocytosis. This function is to transfer bivalent cations of metals from phagosome inward macrophage, that appears to affect negatively on destiny of microbes consumed. The human homologue of the Nrampl gene, denoted as NRAMP1, is situated on human chromosome 2q35. The gene Nrampl consists of 15 exones of different spread disparted by intrones of various sizes. Several polymorphous variants of the gene are described. The experimental presuppositions to more extensive investigation of the role of the gene NRAMPl in development of human pathology are pointed out.     

The earliest atrypides and athyridides (Brachiopoda) from the Ordovician of Kazakhstan

The middle Ordovician brachiopod faunas of Kazakhstan provide one of the most complete records of the evolution and radiation of some of the oldest known spire-bearing brachiopods. By contrast with North American faunas, Kazakhstanian atrypide taxa mostly belong to the suborders Atrypidina and Lissatrypidina, whereas the suborder Anazygidina is completely absent. Kazakhstanian species referred previously to ZygospiraKuzgunia are reassigned to Sulcatospira, which appeared in the Caradoc Diplograptus multidensClimacograptus clingani biozones (Sulcatospira? praecursor and Sulcatospira prima sp. nov.). Primitive, and possibly the oldest known Athyridida also appeared in Kazakhstan sometime during the Caradoc (Kellerella misiusi sp. nov.) and became widespread in brachiopod assemblages developed in carbonate mud mounds. Phylogenetic analysis suggests the early divergence of the Anazygidina, Atrypidina and Athyridida, which probably evolved independently from various primitive smooth Lissatrypidina. The new atrypide subfamily Pectenospirinae and two new atrypide genera (Rozmanospira gen. nov. and Pectenospira gen. nov. with P. pectenata sp. nov. as type species) are erected.     

Neither high-pass filtering nor mathematical differentiation of the EMG signals can considerably reduce cross-talk.

Using mathematical simulation of motor unit potentials (MUPs), detected by a point and rectangular plate electrode, we have shown that the muscle tissue does not act like a low-pass frequency filter on MUPs. Depending on the electrode type and its longitudinal position, the relative weight of the terminal phases (reflecting the excitation extinction) in MUPs and thus of high frequencies in the MUP power spectrum, increase with the MU depth. Therefore, high-pass filtering or differentiating signals detected neither monopolarly nor bipolarly could eliminate the cross-talk produced by high frequency components of MUPs from deep MUs. Such methods could be effective against the main components but not against the MUP leading edge and terminal phases. To reduce the cross-talk, position of the detecting electrodes should correspond to anatomy of muscles producing the cross-talk. Monopolar electrode should be located above the ends of the muscles. Cross-talk of the muscles located beyond the muscle of interest could be higher than that produced above the end-plate of deep muscles. On the contrary, under detection by a longitudinal bipolar electrode, the cross-talk is much smaller above the end-plate region or beyond deep muscles. The cross-talk is the greatest above the ends of the deep muscles.     

Binding of actinomycin D analogues containing some substituents at position 7 of the antibiotic chromophore to DNA

Spectrophotometric methods are used to study the binding to DNA of Actinomycin D (AMD) and its analogues: 7-nitro-AMD; 7-amino-AMD; 7-(Z-Val-Glo-NH)-AMD; 7-(AcO- . +H2-Val-Glo-NH)-AMD; 7-(AcO- . +H2-Val-Glo-Val-Glo-NH)-AMD. The binding constants are calculated from the binding isotherm of AMD and those of the AMD analogues to calf thymus DNA obtained by spectrophotometric titration. Introduction of smaller substituents such as the nitro or amino groups into position 7 of chromophore influences insignificantly the antibiotic binding to DNA, whereas bulky substituents cause a decrease in the affinity of the AMD analogues for DNA, although the spectral characteristics are not affected.     

Cytogenetic analysis of human oocytes and the process of their maturation outside the body

A cytogenetic analysis of human oocytes, after their removal from follicles and during extracorporeal maturation, has been performed. The heterogeneity of follicular oocytes according to the character of nuclear chromatin was established. It is probably related to the rate of reinitiation of meiosis in vivo and to the rate of atretic process. The dynamics of human ovum maturation in vitro was studied. 50.8% oocytes reached metaphase II in 46 hours of cultivation, but only 22.8% of these had no chromosomal aberrations. It is supposed that the high rate of chromosomal anomalies in vitro is a possible consequence of degenerative changes of oocytes that started still in vivo.     

On the mechanism of microbial cell destruction during ballistic disintegration processes

Summary Schizosaccharomyces pombe cells were used to determine the mechanism of cell destruction. The values of breaking point of the cell wall substance at the varied temperatures of destruction had been determined to obtain the necessary results. It was established that the cells are destroyed because of forces resulting from the gradient movement of the medium near the surface of the milling bodies. The destructible part of the cell wall is preliminarily weakened by the influence of high temperature.     

A chlorotetracycline fluorescent probe--an indicator of calcium ion binding with calcium-binding proteins

It has been found in in vitro experiments that fluorescence intensity of deionized solution containing a chlorotetracycline fluorescent probe increases insignificantly at the addition of calmodulin of S-100 proteins. Subsequent introduction of Ca2+ into the medium results in the pronounced fluorescence increase depending on Ca2+ concentration. Addition of specific protein blockers--W7 (calmodulin inhibitor) and antibodies to S-100 brought about a decrease of fluorescence. In in vivo experiments on chlorotetracycline-stained neurons of Helix Pomatia ganglia subesophageal complex it has been shown that bringing of antibodies to S-100 and calmodulin significantly decreases the fluorescence intensity of these cells. These data suggest that the chlorotetracycline probe is an indicator of calcium ions binding with calcium-binding proteins both in in vitro and in vivo systems.