Blunted IgE-mediated activation of mast cells in mice lacking the Ca2+-activated K+ channel KCa3.1

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.     

Decrease in the intensity of the cellular immune response and nonspecific inflammation upon exposure to extremely high frequency electromagnetic radiation

The effect of low-intensity extremely high-frequency electromagnetic radiation (EHF EMR, 42.0 GHz, 0.1 mW/cm2, 20 min daily) on cell-mediated immunity and nonspecific inflammatory response in mice was studied. The intensity of cell-mediated immune response in the reaction of delayed-type hypersensitivity and nonspecific inflammation was estimated by a relative increase in the thickness of foot pad after immunization of animals by sheep red blood cells or zymosan. It was shown for the first time that the radiation reduces both immune and nonspecific inflammatory responses. It was shown with the use of models of acute inflammation and full-thickness skin wounds that EHF EMR suppresses the nonspecific inflammatory response but does not influence the duration of the pathological process. We suppose that the basis of the effects revealed is the modification of functional activity of phagocytic cells under the influence of EHF EMR. The results suggest that some therapeutic effects of EHF EMR can be realized via the inhibition of inflammatory processes.     

Structural Analysis of Glutaredoxin Domain of Mus musculus Thioredoxin Glutathione Reductase

Thioredoxin glutathione reductase (TGR) is a member of the mammalian thioredoxin reductase family that has a monothiol glutaredoxin (Grx) domain attached to the thioredoxin reductase module. Here, we report a structure of the Grx domain of mouse TGR, determined through high resolution NMR spectroscopy to the final backbone RMSD value of 0.48±0.10 Å. The structure represents a sandwich-like molecule composed of a four stranded β-sheet flanked by five α–helixes, with the CxxS active motif located on the catalytic loop. We structurally characterized the glutathione-binding site in the protein and describe sequence and structural relationships of the domain with glutaredoxins. The structure illuminates a key functional center that evolved in mammalian TGRs to act in thiol-disulfide reactions. Our study allows us to hypothesize that Cys105 might be functionally relevant for TGR catalysis. In addition, the data suggest that the N-terminus of Grx acts as a possible regulatory signal also protecting the protein active site from unwanted interactions in cellular cytosol.     

Inhibition of voltage-gated K+ channels in dendritic cells by rapamycin

Rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), is a widely used immunosuppressive drug. Rapamycin affects the function of dendritic cells (DCs), antigen-presenting cells participating in the initiation of primary immune responses and the establishment of immunological memory. Voltage-gated K(+) (Kv) channels are expressed in and impact on the function of DCs. The present study explored whether rapamycin influences Kv channels in DCs. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by whole cell patch clamp. To more directly analyze an effect of mTOR on Kv channel activity, Kv1.3 and Kv1.5 were expressed in Xenopus oocytes with or without the additional expression of mTOR and voltage-gated currents were determined by dual-electrode voltage clamp. As a result, preincubation with rapamycin (0-50 nM) led to a gradual decline of Kv currents in DCs, reaching statistical significance within 6 h and 50 nM of rapamycin. Rapamycin accelerated Kv channel inactivation. Coexpression of mTOR upregulated Kv1.3 and Kv1.5 currents in Xenopus oocytes. Furthermore, mTOR accelerated Kv1.3 channel activation and slowed down Kv1.3 channel inactivation. In conclusion, mTOR stimulates Kv channels, an effect contributing to the immunomodulating properties of rapamycin in DCs.     

Genome sequencing of mouse induced pluripotent stem cells reveals retroelement stability and infrequent DNA rearrangement during reprogramming

The biomedical utility of induced pluripotent stem cells (iPSCs) will be diminished if most iPSC lines harbor deleterious genetic mutations. Recent microarray studies have shown that human iPSCs carry elevated levels of DNA copy number variation compared with those in embryonic stem cells, suggesting that these and other classes of genomic structural variation (SV), including inversions, smaller duplications and deletions, complex rearrangements, and retroelement transpositions, may frequently arise as a consequence of reprogramming. Here we employ whole-genome paired-end DNA sequencing and sensitive mapping algorithms to identify all classes of SV in three fully pluripotent mouse iPSC lines. Despite the improved scope and resolution of this study, we find few spontaneous mutations per line (one or two) and no evidence for?endogenous retroelement transposition. These results show that genome stability can persist throughout reprogramming, and argue that it is possible to generate iPSCs lacking gene-disrupting mutations using current reprogramming methods.     

Multiplication of the late slow fluctuation of induced potential to light under conditions of aminazin administration]

A study was made of the effect of chlorpromazine on the multiplication of late slow component of evoked potential to the flash-light (1 sec.) in experiments on nonanesthetized rabbits with electrodes chronically implanted in different brain structures. Electro-skin pain stimulation of the pore was applied simultaneously in some experiments. Chlorpromazine was found to decrease the multiplication of the late slow component to the flash-light alone, and to double this component in response to light when it was combined with pain stimulation. A supposition was made on the role of adrenergic structures in the formation of the acceptor of the action results of a defensive character, serving as one of the most important mechanisms of the functional system.     

Sodium channel activity in leukemia cells is directly controlled by actin polymerization

The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in non-excitable cells. However, the molecular mechanisms underlying the changes in channel function remain to be defined. In the present work, inside-out patch experiments were employed to elucidate the role of submembranous actin dynamics in the control of sodium channels in human myeloid leukemia K562 cells. We found that the application of cytochalasin D to the cytoplasmic surface of membrane fragments resulted in activation of non-voltage-gated sodium channels of 12 picosiemens conductance. Similar effects could be evoked by addition of the actin-severing protein gelsolin to the bath cytosol-like solution containing 1 microm [Ca(2+)](i). The sodium channel activity induced by disassembly of submembranous microfilaments with cytochalasin D or gelsolin could be abolished by intact actin added to the bath cytosol-like solution in the presence of 1 mm MgCl(2) to induce actin polymerization. In the absence of MgCl(2), addition of intact actin did not abolish the channel activity. Moreover, the sodium currents were unaffected by heat-inactivated actin or by actin whose polymerizability was strongly reduced by cleavage with specific Escherichia coli A2 protease ECP32. Thus, the inhibitory effect of actin on channel activity was observed only under conditions promoting rapid polymerization. Taken together, our data show that sodium channels are directly controlled by dynamic assembly and disassembly of submembranous F-actin.     

Transformation of natural genetic variation into Haemophilus influenzae genomes

Many bacteria are able to efficiently bind and take up double-stranded DNA fragments, and the resulting natural transformation shapes bacterial genomes, transmits antibiotic resistance, and allows escape from immune surveillance. The genomes of many competent pathogens show evidence of extensive historical recombination between lineages, but the actual recombination events have not been well characterized. We used DNA from a clinical isolate of Haemophilus influenzae to transform competent cells of a laboratory strain. To identify which of the ~40,000 polymorphic differences had recombined into the genomes of four transformed clones, their genomes and their donor and recipient parents were deep sequenced to high coverage. Each clone was found to contain ~1000 donor polymorphisms in 3-6 contiguous runs (8.1±4.5 kb in length) that collectively comprised ~1-3% of each transformed chromosome. Seven donor-specific insertions and deletions were also acquired as parts of larger donor segments, but the presence of other structural variation flanking 12 of 32 recombination breakpoints suggested that these often disrupt the progress of recombination events. This is the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, connecting experimental studies of transformation with the high levels of natural genetic variation found in isolates of the same species.