Ion permeation through a voltage- sensitive gating pore in brain sodium channels having voltage sensor mutations

Voltage-gated sodium channels activate in response to depolarization, but it is unknown whether the voltage-sensing arginines in their S4 segments pivot across the lipid bilayer as voltage sensor paddles or move through the protein in a gating pore. Here we report that mutation of pairs of arginine gating charges to glutamine induces cation permeation through a gating pore in domain II of the Na(V)1.2a channel. Mutation of R850 and R853 induces a K(+)-selective inward cationic current in the resting state that is blocked by activation. Remarkably, mutation of R853 and R856 causes an outward cationic current with the opposite gating polarity. These results support a model in which the IIS4 gating charges move through a narrow constriction in a gating pore in the sodium channel protein during gating. Paired substitutions of glutamine allow cation movement through the constriction when appropriately positioned by the gating movements of the S4 segment.     

Evenly tritium-labeled peptides and their in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.     

Expression of an expanded polyglutamine domain in yeast causes death with apoptotic markers

Huntington's disease is caused by specific mutations in huntingtin protein. Expansion of a polyglutamine (polyQ) repeat of huntingtin leads to protein aggregation in neurons followed by cell death with apoptotic markers. The connection between the aggregation and the degeneration of neurons is poorly understood. Here, we show that the physiological consequences of expanded polyQ domain expression in yeast are similar to those in neurons. In particular, expression of expanded polyQ in yeast causes apoptotic changes in mitochondria, caspase activation, nuclear DNA fragmentation and death. Similar to neurons, at the late stages of expression the expanded polyQ accumulates in the nuclei and seems to affect the cell cycle of yeast. Interestingly, nuclear localization of the aggregates is dependent on functional caspase Yca1. We speculate that the aggregates in the nuclei disturb the cell cycle and thus contribute to the development of the cell death process in both systems. Our data show that expression of the polyQ construct in yeast can be used to model patho-physiological effects of polyQ expansion in neurons.     

Antitumor activity of L-asparaginase from Erwinia carotovora against different human and animal leukemic and solid tumor cell lines

The dose- and time-dependent antitumor and cytotoxic effects of L-asparaginases from Erwinia carotovora (ECAR LANS) and Escherichia coli (MEDAC) have been investigated using human leukemic cells and human and animal solid tumor cells. These included human T-cell acute lymphoblastic leukemia cell lines (Jurkat, Jurkat/A4, Molt-4), human chronic myeloid leukemia K562 cells, human promyelocytic leukemia HL-60, and also human solid tumor cells (prostate carcinoma LnCap, breast adenocarcinoma MCF7, ovarian adenocarcinoma SCOV-3 and carcinoma CaOV, hepatocarcinoma Hep G2, fibrosarcoma HT-1080) and animal solid tumor cells (rat Gasser??s ganglion neurinoma cells GGNC-1, mouse glioblastoma EPNT-5). We investigated sensitivity of tumor cells (seeded at different density) to L-asparaginases, as well the effect of L-asparaginases on cell growth rate, protein and DNA synthesis in the presence of various cytostatics. Cell cycle analysis by flow cytofluorimetry and detection of apoptotic cells before and after treatment with L-asparaginases indicate that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division. The cytofluorometric study of solid and leukemic cells revealed that except HL-60 cells the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. Combined treatment of cells using a combination of L-asparaginase and doxorubicin significantly increased the number of apoptotic cells up to 60% (MCF-7 cells), 40% (Jurkat cells) and even 99% (HL-60). High levels of DNA and protein synthesis rates in asparaginase-treated tumor cells suggest lack of massive entry of tumor cells to apoptosis. This conclusion is based on the fact of sensitivity of multi-resistant Jurkat/A4 cells to L-asparaginases (it is nearly impossible to induce apoptosis in these cells). Since ECAR LANS did not influence growth of normal human fibroblasts it appears that the enzyme cytotoxicity is associated only asparagine deficiency.     

Proteomic differences in seed filling between yellow-seeded progeny of <Emphasis Type="Italic">Brassica napus-Sinapis alba</Emphasis> (Brassicaceae) and black-seeded parent <Emphasis Type="Italic">B. napus</Emphasis>

Comparative proteomics of seed filling between yellow-seeded progeny from somatic hybrids Brassica napus-Sinapis alba and black-seeded parent (B. napus) were taken out using two-dimensional electrophoresis (2-DE). The process indicated distinct differences in 2, 3, 4, 5, 6 weeks after fertilization (WAF) and mature seed. A total of 8 out of the 27 discriminate proteins were identified by mass spectrum analysis and MASCOT comparison, including protein kinase, enolase, triosephosphate isomerase, and dioxygenase. PCR primers contrived for the putative genes were applied for further identification of progenies and both parents, which indicated that spot A3-5 might be the novel protein of intergeneric hybrid, i.e., A5-2 derived from S. alba. Applying these specific primers, this study demonstrates that the new yellow-seeded germplasm is different from the existing yellow seed materials of rapeseed.     

<Emphasis Type="Italic">Paralepidapedon variabile</Emphasis> sp. n. (Trematoda,Lepocreadioidea, Lepidapedidae) and other representatives of the genus <Emphasis Type="Italic">Paralepidapedon</Emphasis> from Antarctic fish

In the Ross Sea and Amundsen Sea, four representatives of the genus Paralepidapedon—Paralepidapedon cf. dubium Prudhoe et Bray 1973 sensu Sokolov et Gordeev 2013, P. lepidum (Gaevskaya et Rodyuk 1988), Paralepidapedon sp., and P. variabile sp. n.—were found in demersal fishes Muraenolepis marmorata and Macrourus whitsoni. Paralepidapedon variabile sp. n. is described from Muraenolepis marmorata in the Amundsen Sea. Paralepidapedon variabile sp. n. differs from other species of the genus Paralepidapedon by the position of the anterior border of the vitellarium at the level of the anterior edge of the ventral sucker or genital pore and by the highly variable shape of the testes: from roundish with a smooth edge to sinuate–lobate. Paralepidapedon lepidum was found for the first time in the Antarctic.     

Orienting reflex: "targeting reaction" and "searchlight of attention"

The concept of orienting reflex based on the principle of vector coding of cognitive and executive processes is proposed. The orienting reflex to non-signal and signal stimuli is a set of orienting reactions: motor, autonomic, neuronal, and subjective emphasizing new and significant stimuli. Two basic mechanisms can be identified within the orienting reflex: a "targeting reaction" and a "searchlight of attention". In the visual system the first one consists in a foveation of a target stimulus. The foveation is performed with participation of premotor neurons excited by saccadic command neurons of the superior colliculi. The "searchlight of attention" is based on the resonance of gamma-oscillations in the reticular thalamus selectively enhancing responses of cortical neurons (involuntary attention). The novelty signal is generated in novelty neurons of the hippocampus, which are selectively tuned to a repeatedly presented standard stimulus. The selective tuning is caused by the depression of plastic synapses representing a "neuronal model" of the standard stimulus. A mismatch of the novel stimulus with the established neuronal model gives rise to a "novelty signal" enhancing the novel input. The novelty signal inhibits current conditioned reflexes (external inhibition) contributing to redirecting the behavior. By triggering the expression of early genes the novelty signal initiates the formation of the long-term memory connected with neoneurogenesis.     

Soluble and membrane glycoproteins of the rat brain in radiation injury

The content of total protein, glycoproteins, and N-acetylneuraminic acid in different membrane fractions of rat brain tissues was studied 60 min and 24 h following X-irradiation with a dose of 0.31 C/kg. A slight increase in the amount of glycoproteins was noted upon electrophoresis due to the occurrence of low molecular weight carbohydrate-containing proteins. The derangement of the membrane glycoprotein structure is supposed to be one on the causes of radiation destruction of membranes.     

The effect of x-ray radiation and hypoxia on the proteolytic activity of the normal rat spleen and during tumor growth

The influence of X-radiation on activity of lysosomal enzymes (D, L, H cathepsins) in rat spleen tissue and in inoculated rat sarcoma 45 has been investigated. Intact rats and rats with tumors were subjected to whole-body and sarcoma 45 to local irradiation with doses of 0.155 C/kg and 0.31 C/kg in conditions of breathing gas hypoxic mixture containing 90% of nitrogen and 10% of oxygen (GHM-10). The combined exposure to radiation and GHM-10 was shown to produce a certain protective action (e.g. normalized cathepsin activity) in the spleen. In the tumor tissue the protective effect of GHM-10 was absent.