Proteinase activity of nuclei from various tissues towards endogenous histones]

The proteinase activities of nuclei isolated from tissues differing in their mitotic activities (brain, thymus, liver, ascite lymphoma) towards the histones and non-histone acid -- extractable proteins were studied. The sensitivity of different histone fractions to nuclear proteinase depends on temperature and time of nuclei incubation under conditions providing for complete dissociation of chromatin proteins from DNA (2 M NaCl--5 M urea). The proteinase activity in the brain and thymus nuclei is revealed only under prolonged (43 hrs) incubation of the nuclei at 25 degrees C, which is accompanied by partial proteolysis of histone H1. Histone H4 from brain nuclei and histone H2a from thymus nuclei are preferably degraded. In the nuclei isolated from the mice ascite cell lymphoma NK/ly and from rat liver the enzyme activity is revealed mainly towards the arginine-enriched histones H3 and H4. The proteolysis of the arginine-enriched histones in tumour cell nuclei is more complete. A high sensitivity to proteolysis was observed for non-histone acid-extractable proteins with low electrophoretic mobility, which were found in brain and tumour cell nuclei.     

A recombineering-based gene tagging system for Arabidopsis

One of the most information-rich aspects of gene functional studies is characterization of gene expression profiles at cellular resolution, and subcellular localization of the corresponding proteins. These studies require visualization of the endogenous gene products using specific antibodies, or, more commonly, generation of whole-gene translational fusions with a reporter gene such as a fluorescent protein. To facilitate the generation of such translational fusions and to ensure that all cis-regulatory sequences are included, we have used a bacterial homologous recombination system (recombineering) to insert fluorescent protein tags into genes of interest harbored by transformation-competent bacterial artificial chromosomes (TACs). This approach has several advantages compared to other classical strategies. First, the researcher does not have to guess what the regulatory sequences of a gene are, as tens of thousands of base pairs flanking the gene of interest can be included in the construct. Second, because the genes of interest are not amplified by PCR, there are practically no limits to the size of a gene that can be tagged. Third, there are no restrictions on the location in which the fluorescent protein can be inserted, as the position is determined by sequence homology with the recombination primers. Finally, all of the required strains and TAC clones are publically available, and the experimental procedures described here are simple and robust. Thus, we suggest that recombineering-based gene tagging should be the gold standard for gene expression studies in Arabidopsis.     

Red and near-infrared light induces intracellular Ca2+ flux via the activation of glutamate N-methyl-D-aspartate receptors

Red and near-infrared (NIR) light effect on Ca²⁺ ions flux through the influence on N-methyl-D-aspartate receptors (NMDARs) and their functioning in HeLa cells was studied in vitro. Cells were irradiated by 650 and 808 nm laser light at different power densities and doses and the obtained effect was compared with that caused by the pharmacological agents. The laser light was found to elevate Ca²⁺ influx into cell cytoplasm in a dose-dependent manner without changes of the NMDAR functioning. Furthermore, the light of both wavelengths demonstrated the ability to elevate Ca²⁺ influx under the pharmacological blockade of NMDARs and also might partially abolish the blockade enhancing Ca²⁺ influx after selective stimulation of the receptors with NMDA. Simultaneously, the light at moderate doses demonstrated a photobiostimulating effect on cells. Based on our experiments and data reported in the literature, we suggest that the low-power visible and NIR light can instigate a cell membrane depolarization via nonthermal activation, resulting in the fast induction of Ca²⁺ influx into cells. The obtained results also demonstrate that NIR light can be used for nonthermal and nonpharmacological stimulation of NMDARs in cancer cells.     

CFTR Gene Variants and Genotypes in Russian Patients with CBAVD Syndrome

Russian Journal of Genetics - CBAVD syndrome is one of the common genetic causes of male infertility, associated with obstructive azoospermia, commonly resulting from pathogenic CFTR gene variants....     

A Nanocapsule Based on Natural Mineral Clinoptilolite Surrounded by a Lecithin Envelope

Biophysics - A technology for obtaining nanoparticles of natural clinoptilolite, a mineral from the zeolite family, under laboratory conditions has been developed. The size of zeolite particles was...     

Association of attenuated mutants of Salmonella enterica serovar Enteritidis with porcine peripheral blood leukocytes

In this study, we were interested in the association of attenuated mutants of Salmonella enterica serovar Enteritidis with subpopulations of porcine white blood cells (WBC). The mutants included those with inactivated aroA, phoP, rfaL, rfaG, rfaC and fliC genes and a mutant with five major pathogenicity islands removed (ΔSPI1-5 mutant). Using flow cytometry, we did not observe any difference in the interactions of the wild-type S. Enteritidis, aroA and phoP mutants with WBC. ΔSPI1-5 and fliC mutants had a minor defect in their association with granulocytes and monocytes, but not with T- or B-lymphocytes. All three rfa mutants associated with granulocytes, monocytes and B-lymphocytes more than the wild-type S. Enteritidis did. Electron microscopy confirmed that the association correlated with the intracellular presence of S. Enteritidis and that the Salmonella-containing vacuole in the WBC infected with the rfa mutants, unlike all other strains, did not develop into a spacious phagosome. Intact lipopolysaccharide, but not the type III secretion system encoded by SPI-1, SPI-2 or the flagellar operon, is important for the initial interaction of S. Enteritidis with porcine leukocytes. This information can be used for the design of live Salmonella vaccines preferentially targeting particular cell types including cancer or tumor cells.     

The influence of insulin on contamination of the common carp Cyprinus carpio by the monogenetic fluke Dactylogyrus vastator

The influence of insulin on the contamination of the carp Cyprinus carpio with the monogenetic fluke Dactylogyrus vastator is studied. Fishes reacted to the introduction of the hormon by the decrease in the degree of intensity of infection and by the increase in the number of lifeless parasites on gills, by contrast to control fishes and fishes processed with stress hormones (adrenaline and cortisol). We assume that the decrease in the abundance index in live monogenetic fluke and the increase in the number of lifeless oness is caused by the deficiency of nutrients accessible for the growth and development caused by activation of hormone-induced anabolic processes and stimulation of mechanisms of immune protection.     

Effects of temperature on the p53-DNA binding interactions and their dynamical behavior: comparing the wild type to the R248Q mutant

Background

The protein p53 plays an active role in the regulation of cell cycle. In about half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain. Interestingly, a number of these mutations possess temperature-induced DNA-binding characteristics. A striking example is the mutation of Arg248 into glutamine or tryptophan. These mutants are defective for binding to DNA at 310 K although they have been shown to bind specifically to several p53 response elements at sub-physiological temperatures (298–306 K).

Methodology/Principal Findings

This important experimental finding motivated us to examine the effects of temperature on the structure and configuration of R248Q mutant and compare it to the wild type protein. Our aim is to determine how and where structural changes of mutant variants take place due to temperature changes. To answer these questions, we compared the mutant to the wild-type proteins from two different aspects. First, we investigated the systems at the atomistic level through their DNA-binding affinity, hydrogen bond networks and spatial distribution of water molecules. Next, we assessed changes in their long-lived conformational motions at the coarse-grained level through the collective dynamics of their side-chain and backbone atoms separately.

Conclusions

The experimentally observed effect of temperature on the DNA-binding properties of p53 is reproduced. Analysis of atomistic and coarse-grained data reveal that changes in binding are determined by a few key residues and provide a rationale for the mutant-loss of binding at physiological temperatures. The findings can potentially enable a rescue strategy for the mutant structure.     

Some radiobiological effects in higher plants growing on the territory of the East Ural Radioactive Trace

The spontaneous level of cytogenetic damage in three plant species (Achyrophorus maculatus (Scop.) L.; Plantago lanceolata L.; and Plantago media L.) growing on the territory of the East Ural Radioactive Trace is studied. The radiation resistance of plants from radioactive and control nonpolluted sites is determined. The effects of additional fractionated irradiation at different doses and the role of antioxidant systems in the formation of the radioprotector effect are examined. It is shown that the level of the mutation process in the plant population growing at the radiation polluted sites is increased compared to the control populations from nonpolluted territories. Additional acute γ irradiation of seeds collected from polluted and nonpolluted territories demonstrates improved radiation resistance of the plants from the polluted territory. In the control population of A. maculatus in the versions with a one-hour interval between fractions, the radiation effect follows the additivity principle; at the same time, at a one-day interval between fractions, a highly significant radioprotective effect is manifested clearly in the experimental population. For higher plants, the enhanced effectiveness of the functioning of the antioxidant systems in plants growing on territories contaminated with radiation is shown for the first time. Thus, the radioprotector mechanisms of low-dose chronic and preliminary irradiation are similar and one of these mechanisms is activation of the antioxidant systems in plants growing under conditions of chronic low-intensity irradiation for long periods of time.     

Mutation p.E92K is the primary cause of cystic fibrosis in Chuvashes

Molecular genetic study of the CFTR gene in cystic fibrosis patients from the Chuvash Republic is presented. We found linkage disequilibrium of the disease with 22-7-16-13 haplotype using intragenic markers. Major mutation p.E92K was revealed in chromosomes carrying this haplotype. The frequency of this mutation in Chuvash patients was 66.6%. Population study of the distribution of two mutations (p.E92K and F508del) of the CFTR gene revealed that their population frequency in heterozygous carriers was one per 37 subjects while calculated cystic fibrosis frequency in Chuvashia is one per 5420 newborns.