Specificity of intracellular ribonucleases Pc1 and Pc2 of the fungus Penicillium claviforme]

The products of RNA and synthetic polynucleotides degradation by intracellular RNAses Pc1 and Pc2 of the fungus Penicillium claviforme were studied. It was shown that the enzymes possess the endonuclease activity and are not specific for the bases vicinal to the cleaved PDE bonds (EC 3.1.4.23). The increase of binding of the dinucleoside monophosphates by Pc1 and Pc2 dependent on the nucleoside at the 3'-end of the PDE bond is: A greater than C greater than G greater than U. This order is opposite for the rates of these substrates cleavage by the RNAses. A homologous specificity of the intracellular RNAse Pc1 and the extracellular RNAse II of Pen. claviforme has been revealed.     

Dramatic differences in the roles in lipid metabolism of two isoforms of diacylglycerol kinase

Lipid species changes for SV40-transformed fibroblasts from wild-type or from diacylglycerol kinase-epsilon (DGKepsilon) or diacylglycerol kinase-alpha (DGKalpha) knockout mice were determined for glycerophospholipids, polyphosphatidylinositides (GPInsP n ) and diacylglycerol (DAG) using direct infusion mass spectrometry. Dramatic differences in arachidonate (20:4 fatty acid)-containing lipids were observed for multiple classes of glycerophospholipids and polyphosphatidylinositides between wild-type and DGKepsilon knockout cells. However, no difference was observed in either the amount or the acyl chain composition of DAG between DGKepsilon knockout and wild-type cells, suggesting that DGKepsilon catalyzed the phosphorylation of a minor fraction of the DAG in these cells. The differences in arachidonate content between the two cell lines were greatest for the GPInsP n lipids and lowest for DAG. These findings indicate that DGKepsilon plays a significant role in determining the enrichment of GPInsP n with 20:4 and that there is a pathway for the selective translocation of arachidonoyl phosphatidic acid from the plasma membrane to the endoplasmic reticulum. In contrast, no substantial difference was observed in the acyl chain composition of any class of glycerophospholipid or diacylglycerol between lipid extracts from fibroblasts from wild-type mice or from DGKalpha knockout mice. However, the cells from the DGKalpha knockout mice had a higher concentration of DAG, consistent with the lack of downregulation of the major fraction of DAG by DGKalpha, in contrast with DGKepsilon that is primarily responsible for enrichment of GPInsP n with arachidonoyl acyl chains.     

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: Synthesis and in vitro studies

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the “window” between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH₂, the rate of oxidation of SkQH₂ being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH^·. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C _1/2 values for inhibition of the H₂O₂-induced apoptosis in fibroblasts appear to be as low as 1·10⁻¹¹ and 8·10⁻¹³ M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Δψ values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000: 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3·10⁸ times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 12, pp. 1589–1606. This and the following four articles were written by the request of the Editorial Board of Biochemistry (Moscow).     

Analgesic compound from sea anemone Heteractis crispa is the first polypeptide inhibitor of vanilloid receptor 1 (TRPV1)

Venomous animals from distinct phyla such as spiders, scorpions, snakes, cone snails, or sea anemones produce small toxic proteins interacting with a variety of cell targets. Their bites often cause pain. One of the ways of pain generation is the activation of TRPV1 channels. Screening of 30 different venoms from spiders and sea anemones for modulation of TRPV1 activity revealed inhibitors in tropical sea anemone Heteractis crispa venom. Several separation steps resulted in isolation of an inhibiting compound. This is a 56-residue-long polypeptide named APHC1 that has a Bos taurus trypsin inhibitor (BPTI)/Kunitz-type fold, mostly represented by serine protease inhibitors and ion channel blockers. APHC1 acted as a partial antagonist of capsaicin-induced currents (32 +/- 9% inhibition) with half-maximal effective concentration (EC(50)) 54 +/- 4 nm. In vivo, a 0.1 mg/kg dose of APHC1 significantly prolonged tail-flick latency and reduced capsaicin-induced acute pain. Therefore, our results can make an important contribution to the research into molecular mechanisms of TRPV1 modulation and help to solve the problem of overactivity of this receptor during a number of pathological processes in the organism.     

PCNA Up-Regulation in Cerebral Vessels with Angiotensin II-Hypertension: Abnormal Regulation of Ca2+ Channel and Nitrate Tolerance Associated with Alternative Splicing of cGKI

We studied the responses of the basilar arteries from control rats and from rats infused with angiotensin II (Ang; 240 μg/kg/h × 4 weeks), which were hypertensive (137 ± 13 vs 205 ± 10 mm Hg). Ang-hypertensive rats (AHR) showed significant up-regulation of the expression of proliferative cell nuclear antigen (PCNA) (PCNA index, 0.65) in vascular smooth muscle cell (VSMC) layers. Both hypertension and PCNA up-regulation were absent in animals co-treated with the hydrophilic dihydropyridine Ca²⁺ channel blocker amlodipine (100 mg/liter in the drinking water). Quantitative patch-clamp analysis of freshly isolated VSMC showed a significant increase in L-type Ca²⁺ channel currents in AHR that was attributed to an increase in the open channel probability, with no change in other biophysical properties, pharmacological characteristics, or in the channel expression. Compared with controls, regulation of Ca²⁺ channels in AHR was abnormal, with nitrate tolerance manifested as a reduction in down-regulation of the Ca²⁺ channel activity in response to the NO donor Na nitroprusside. A diminished sensitivity to 8-Br-cGMP was also observed, consistent with a mechanism downstream of soluble guanylyl cyclase. The nitrate tolerance was found to be attributable to alternative splicing of cGKI, with a decrease in the cGKIalpha expression and an increase in the cGKIbeta expression, with the latter known to be less sensitive to activation by cGMP and related analogs. We conclude that the increase in the proliferative response of VSMC in AHR is associated with an abnormal response to NO by VSMC due to alternative splicing of cGKI, resulting in an increase in the Ca²⁺ channel activation and up-regulation of the pro-proliferative Ca²⁺-sensitive gene for PCNA.     

榆科榉属的植物地理学

榉属是东亚—地中海间断分布局,分属于东亚植物区、地中海植物区及伊朗—土兰植物区。根据现存榉属植物的形态演化趋势、细胞学、孢粉学证据及其化石资料和古地质历史事件相结合分析,该属植物是第三纪温带森林的孑遗成分而且第三纪末期的喜马拉雅造山运动及第四纪的冰川作用是样属植物演化的主要动力。在欧洲,由于喜马拉雅造山运动及第四纪冰川作用的较强烈,使分布于此的榉属植物受到严厉的生存竞争,未能适应环境的物种遭受灭绝,少数进行适应性变异的物种在极端环境条件下生存了下来。而东亚地区由于自然环境相对较稳定,便成了榉属植物的避难所,因此也保留了榉属植物种较为古老的种类。     

天麻抗真菌蛋白基因的克隆及其启动子活性

通过构建和筛选天麻(Gastrodia elata Bl.)基因组文库,克隆了一个天麻抗真菌蛋白基因组DNA.该基因组DNA含有一个516碱基组成的编码区,没有内含子结构.其启动子区含有保守的TATA盒及CAAT盒.为研究启动子活性,构建了-1 157 bp启动子区与GUS基因的融合表达载体.并将其用农杆菌(Agrobacterium tumefaciens)介导的遗传转化方法导入烟草(Nicotiana tabacum)中,获得了稳定转化的烟草.利用荧光检测及组织化学染色法对GUS表达进行了分析.结果表明,该启动子能够启动GUS基因在转基因烟草中组织特异性地表达.GUS基因在根中的表达水平最高,茎中次之,叶中只有低水平表达.而且该启动子具有诱导表达活性,可被真菌及水杨酸、茉莉酸强烈诱导表达.     

Increased content of very-long-chain fatty acids in the lipids of halophyte vegetative organs

Qualitative and quantitative compositions of esterified fatty acids (FAs) in the total lipids from the leaves, shoots, and roots of halophile plants, such as suaeda (Suaeda altissima), samphire (Salicornia europaea), and wormwood (Artemisia lerchiana), collected in their natural environments were estimated by GLC techniques. It was shown that the vegetative organs of these halophytes contained 24 FA species, and 16 of them were tentatively identified as the very-long-chain FAs (VLCFAs). There were four VLCFA groups, viz. C₂₀, C₂₁, C₂₂, and C₂₃, each including saturated, mono-, and diunsaturated components; C₂₄ and C₂₅ FAs were also present. The concentration of VLCFAs in the total FAs comprised 4–64%. In vegetative organs of higher plants not subjected to genetic transformation, such a high VLCFA content was found for the first time. Saturated and even-numbered components predominated among the VLCFAs, and the roots exceeded severalfold the above-ground organs in the total VLCFA content. Possible pathways of VLCFA biosynthesis in plants, VLCFA content in the vegetative tissues, and the physiological role of membrane lipid FA composition in the plant salt metabolism are discussed.     

Complete genome sequence of Kytococcus sedentarius type strain (541)

Kytococcus sedentarius (ZoBell and Upham 1944) Stackebrandt et al. 1995 is the type strain of the species, and is of phylogenetic interest because of its location in the Dermacoccaceae, a poorly studied family within the actinobacterial suborder Micrococcineae. Kytococcus sedentarius is known for the production of oligoketide antibiotics as well as for its role as an opportunistic pathogen causing valve endocarditis, hemorrhagic pneumonia, and pitted keratolysis. It is strictly aerobic and can only grow when several amino acids are provided in the medium. The strain described in this report is a free-living, nonmotile, Gram-positive bacterium, originally isolated from a marine environment. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Dermacoccaceae and the 2,785,024 bp long single replicon genome with its 2639 protein-coding and 64 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.