Inhibition of Neuronal Degenerin/Epithelial Na+ Channels by the Multiple Sclerosis Drug 4-Aminopyridine

The voltage-gated K⁺ (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na⁺ and Ca²⁺ currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na⁺ (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.     

Halotolerant, alkaliphilic urease-producing bacteria from different climate zones and their application for biocementation of sand

Microbially induced calcium carbonate precipitation (MICP) is a phenomenon based on urease activity of halotolerant and alkaliphilic microorganisms that can be used for the soil bioclogging and biocementation in geotechnical engineering. However, enrichment cultures produced from indigenous soil bacteria cannot be used for large-scale MICP because their urease activity decreased with the rate about 5 % per one generation. To ensure stability of urease activity in biocement, halotolerant and alkaliphilic strains of urease-producing bacteria for soil biocementation were isolated from either sandy soil or high salinity water in different climate zones. The strain Bacillus sp. VUK5, isolated from soil in Ukraine (continental climate), was phylogenetically close in identity (99 % of 16S rRNA gene sequence) to the strain of Bacillus sp. VS1 isolated from beach sand in Singapore (tropical rainforest climate), as well as to the strains of Bacillus sp. isolated by other researchers in Ghent, Belgium (maritime temperate climate) and Yogyakarta, Indonesia (tropical rainforest climate). Both strains Bacillus sp. VS1 and VUK5 had maximum specific growth rate of 0.09/h and maximum urease activities of 6.2 and 8.8 mM of hydrolysed urea/min, respectively. The halotolerant and alkaliphilic strain of urease-producing bacteria isolated from water of the saline lake Dead Sea in Jordan was presented by Gram-positive cocci close to the species Staphylococcus succinus. However, the strains of this species could be hemolytic and toxigenic, therefore only representatives of alkaliphilic Bacillus sp. were used for the biocementation studies. Unconfined compressive strengths for dry biocemented sand samples after six batch treatments with strains VS1and VUK5 were 765 and 845 kPa, respectively. The content of precipitated calcium and the strength of dry biocemented sand at permeability equals to 1 % of initial value were 12.4 g Ca/kg of dry sand and 454 kPa, respectively, in case of biocementation by the strain VS1. So, halotolerant, alkaliphilic, urease-producing bacteria isolated from different climate zones have similar properties and can be used for biocementation of soil.     

S-nitrosoglutathione-induced toxicity in Drosophila melanogaster: Delayed pupation and induced mild oxidative/nitrosative stress in eclosed flies

The toxicity of the nitric oxide donor S-nitrosoglutathione (GSNO) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with GSNO at concentrations of 1.0, 1.5 or 4.0 mM. Food supplementation with GSNO caused a developmental delay in the flies. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies that emerged from control larvae and larvae fed on food supplemented with GSNO. Larval exposure to GSNO resulted in lower activities of aconitase in both sexes and also lower activities of catalase and isocitrate dehydrogenase in adult males relative to the control cohort. Larval treatment with GSNO resulted in higher carbonyl protein content and higher activities of glucose-6-phosphate dehydrogenase in males and higher activities of superoxide dismutase and glutathione-S-transferase in both sexes. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by GSNO.     

Peptide Mimetic of the S100A4 Protein Modulates Peripheral Nerve Regeneration and Attenuates the Progression of Neuropathy in Myelin Protein P0 Null Mice

We recently found that S100A4, a member of the multifunctional S100 protein family, protects neurons in the injured brain and identified two sequence motifs in S100A4 mediating its neurotrophic effect. Synthetic peptides encompassing these motifs stimulated neuritogenesis and survival in vitro and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration and survival of myelinated axons. H3 accelerated electrophysiological, behavioral and morphological recovery after sciatic nerve crush while transiently delaying regeneration after sciatic nerve transection and repair. On the basis of the finding that both S100A4 and H3 increased neurite branching in vitro, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P₀ null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1 disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss, such as Charcot-Marie-Tooth type 1 disease. Moreover, our data suggest that S100A4 is a neuroprotectant in PNS and that other S100 proteins, sharing high homology in the H3 motif, may have important functions in PNS pathologies.     

Conformational diversity of the quercetin molecule: a quantum-chemical view

Abstract

This paper focuses on the comprehensive conformational analysis of the quercetin molecule with a broad range of the therapeutic and biological actions. All possible conformers of these molecule, corresponding to the local minima on the potential energy hypersurface, have been obtained by the sequential rotation of its five hydroxyl groups and also by the rotation of its (A?+?C) and B rings relatively each other. Altogether, it was established 48 stable conformers, among which 24 conformers possess planar structure and 24 conformers – nonplanar structure. Their structural, symmetrical, energetical and polar characteristics have been investigated in details. Quantum-mechanical calculations indicate that conformers of the quercetin molecule are polar structures with a dipole moment, which varies within the range from 0.35 to 9.87 Debay for different conformers. Relative Gibbs free energies of these conformers are located within the range from 0.0 to 25.3?kcal·mol^?1 in vacuum under normal conditions. Impact of the continuum with ε?=?4 leads to the decreasing of the Gibbs free energies (–0.19–18.15?kcal·mol^?1) and increasing of the dipole moment (0.57–12.48?D). It was shown that conformers of the quercetin molecule differ from each other by the intramolecular specific contacts (two or three), stabilizing all possible conformers of the molecule – H-bonds (both classical ОН…О and so-called unusual С′Н…О and ОН…С′) and attractive van-der-Waals contacts О…О. Obtained conformational analysis for the quercetin molecule enables to provide deeper understanding of the ‘structure-function’ relationship and also to suggest its mechanisms of the therapeutic and biological actions.

Communicated by Ramaswamy H. Sarma     

The Sulfonylurea Receptor 1 (Sur1)-Transient Receptor Potential Melastatin 4 (Trpm4) Channel

The sulfonylurea receptor 1 (Sur1)-NC_Ca-ATP channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NC_Ca-ATP channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and K_ATP channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.     

绝经对血液单核细胞基因表达谱的影响:基因芯片初步研究

绝经是女性一生中很重要的生理现象之一,它能增加一系列复杂免疫、神经退化、新陈代谢和心血管方面的疾病。血液单核细胞能分化成各种各样的细胞,这些细胞在组织形态发生和免疫应答方面起着很重要的作用。本研究中采用了包含大约14,500个基因探针的Affymetrix Human U133A基因芯片来研究健康的绝经前和绝经后女性外周血液单核细胞中的基因表达谱。样本之间的对比分析表明有20个基因上调,20个基因下调。其中的28个基因根据它们的生物过程如细胞繁殖、免疫应答、细胞代谢等等被分成了6个主要的GO类别;剩下的12个基因其生物学功能还没有被鉴定。研究结果支持了我们的假设:血液单核细胞的功能状态确实受到绝经的影响,而且由此带来的改变可能是由全基因组范围的基因表达谱而决定的。本研究中鉴定的一些差异表达基因有可能作为以后研究与绝经相关的系统免疫、神经退化和心血管疾病的候选基因研究。此工作是这个研究方向的第一次尝试,为将来的进一步研究奠定了基础。     

Jekyll encodes a novel protein involved in the sexual reproduction of barley

Cereal seed development depends on the intimate interaction of filial and maternal tissues, ensuring nourishment of the new generation. The gene jekyll, which was identified in barley (Hordeum vulgare), is preferentially expressed in the nurse tissues. JEKYLL shares partial similarity with the scorpion Cn4 toxin and is toxic when ectopically expressed in Escherichia coli and tobacco (Nicotiana tabacum). In barley, jekyll is upregulated in cells destined for autolysis. The gene generates a gradient of expression in the nucellar projection, which mediates the maternal-filial interaction during seed filling. Downregulation of jekyll by the RNA interference technique in barley decelerates autolysis and cell differentiation within the nurse tissues. Flower development and seed filling are thereby extended, and the nucellar projection no longer functions as the main transport route for assimilates. A slowing down in the proliferation of endosperm nuclei and a severely impaired ability to accumulate starch in the endosperm leads to the formation of irregular and small-sized seeds at maturity. Overall, JEKYLL plays a decisive role in the differentiation of the nucellar projection and drives the programmed cell death necessary for its proper function. We further suggest that cell autolysis during the differentiation of the nucellar projection allows the optimal provision of basic nutrients for biosynthesis in endosperm and embryo.     

Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.