ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III₂ + IV (S₁) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.     

Ascorbic acid prevents high glucose-induced apoptosis in human brain pericytes

High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5 mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24 h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.     

High rate algal pond operating strategies for urban wastewater nitrogen removal

Two experimental high rate algal ponds (HRAPs) (1.5m², 570 L per unit), each with a secondaryclarifier for algal biomass separation (0.025 m²,without recirculation), were fed with urban wastewaterfor a one-year period (June 1993 to July 1994). TheHRAPs were installed on the roof of the Department ofHydraulic, Coastal and Environmental Engineering ofthe Technical University of Catalonia, Barcelona,Spain (lat. 41^° 24 42 N; long. 2^° 742 E). Nitrogen removal efficiency and changes intotal nitrogen, total organic nitrogen,NH₄ ⁺-N, and oxidized nitrogen underdifferent hydraulic retention times (HRTs) werecompared. HRAP A was always operated at a higherHRT than HRAP B. Both HRAPs were subjected to thesame environmental conditions of solar radiation, airtemperature and influent water quality. Grab samplesof influent, effluent of the HRAP (mixed liquor) andfinal effluent from the clarifiers were taken once aweek. The annual average nitrogen removal was 73% forHRAP A, and 57% for HRAP B. Higher removal in HRAP Awas due to a lower inorganic nitrogen concentration inits effluent. Significant differences (p> 0.05) inthe relative proportions of nitrogen forms between thetwo HRAPs were observed only in autumn and winter.This was mainly because HRAP B did not achieve a highlevel of NH₄ ⁺-N removal by stripping andalgal uptake, as observed in HRAP A. NH₄ ⁺-Nstripping was the most important mechanism fornitrogen removal (mean efficiency of 47% and 32% inHRAP A and B, respectively) followed by algal uptake,and subsequent algal separation in the clarifiers(mean efficiency of 26% and 25% in HRAP A and Brespectively). The conclusion of this study is thatHRT determines both the nitrogen removal efficiencyand the distribution of nitrogen forms in the effluentof a HRAP. The nitrogen removal level can becontrolled through suitable HRT operating strategies.By operating at a HRT of 4 days in spring and summer,and 10 days in autumn and winter, nitrogenconcentration in the effluent of a HRAP system can bereduced to less than 15 mg L^-1 N.     

山梨酸钾和D-异抗坏血酸钠联合作用HepG2细胞的生物学效应分析

该文研究食品添加剂联合作用人肝癌HepG2细胞后的多参数生物学指标,并探索可能存在的损伤机制。CCK-8法检测受试物山梨酸钾及D 异抗坏血酸钠0.13～ 2.00 g/L分别作用HepG2细胞24、48及72 h后的细胞增殖活力,显示山梨酸钾及D-异抗坏血酸钠均呈显著的时间、剂量依赖性的抑制HepG2细胞增殖,其24 h的IC50分别为1.35±0.11 g/L和1.58±0.17 g/L。高内涵分析结果表明,与单独组相比,联合组显著降低细胞数量和线粒体膜电位,增大细胞膜通透性及活性氧水平(P＜0.05);高剂量联合组(0.30+0.41 g/L)显著增加DNA损伤(P＜0.01),表现为协同作用。qRT-PCR和Western印迹法显示,山梨酸钾及D 异抗坏血酸钠可显著提高P53、Caspase 3、Bax、γ-H2AX表达,同时显著降低Bcl-2表达,从而抑制HepG2细胞增殖。     

Fasciola hepatica: humoral and cytokine responses to a member of the saposin-like protein family following delivery as a DNA vaccine in mice

The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG(1), IgG(2), and IgE as well as high levels of IL-10 and IFNgamma indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG(2a) isotype antibody as well as a dominant IFNgamma cytokine, with significant IgE, IgG(1), and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica.     

转化生长因子α－绿脓杆菌外毒素融合蛋白TGFα－PE40高效表达质粒的构建和表达

利用基因工程技术,将质粒pYX382用xbaI和EcoRl切下插入的TGFa—PE40融合 基因片段-连接到可表达载体pCB604的XbaⅠ／EcoR Ⅰ位点中,构建成新的重组质粒p2x—TP1。P2x—TP1转化E.coli BL2l感受志菌后,在ⅠPTG诱导下Ⅰpp启动子转录表达TGFα—PE40融合蛋白。表达产物主要以包涵体形式沉积在细胞内。融合蛋白表达量的高低与诱导时的细胞密度,诱导的温度以及培养基有关。在一定范围内与诱导剂的剂量以及诱导时间的长短无关。P2x—TPl重组质粒在工程菌中的表达TGFα－PE40融合蛋白量约为50mg／L。     

Determination of carbamazepine in human urine and serum samples by high‐performance liquid chromatography with post‐column Ru(bipy)‐Ce(SO4)2 chemiluminescence detection

A novel, sensitive and rapid CL method coupled with high‐performance liquid chromatography separation for the determination of carbamazepine is described. The method was based on the fact that carbamazepine could significantly enhance the chemiluminescence of the reaction of cerium sulfate and tris(2,2‐bipyridyl) ruthenium(II) in the presence of acid. The chromatographic separation was performed on a Kromasil® (Sigma‐Aldrich) TM RP‐C18 column (id: 150 mm × 4.6 mm, particle size: 5 µm, pore size: 100 Å) with a mobile phase consisting of methanol–water‐glacial acetic acid (70:29:1, v/v/v) at a flowrate of 1.0 mL/min, the total analysis time was within 650 s. Under optimal conditions, CL intensity was linear for carbamazepine in the range 2.0 × 10^?8 ~ 4.0 × 10^?5 g/mL, with a detection limit of 6.0 × 10^?9 g/mL (S/N = 3) and the relative standard detection was 2.5% for 2.0 × 10^?6 g/mL (n = 11). This method was successfully applied to the analysis of carbamazepine in human urine and serum samples. The possible mechanism of the CL reaction is also discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd.     

神农架川金丝猴栖息地优势乔木树种遥感识别及其分布特征

针对神农架川金丝猴生境基础研究中乔木树种大范围分布数据难以获取问题,尝试利用多源多时相遥感数据结合专家知识分层次实现树种识别。首先采用冬季Landsat8/OLI数据根据物侯特性分层提取常绿、落叶林的地域范围;进而依据夏季Worldview-2高分遥感影像的实地乔木样本的光谱特征分层次完成常绿树种(巴山冷杉、华山松、青$、刺叶栎)和落叶树种(红桦、日本落叶松、米心水青冈、漆树、锐齿槲栎、椅杨)的识别;并通过实地植被样方及专家知识通过高程数据完成分类结果的修正;最后结合GIS对主要优势树种的地形及地域分布特征进行了空间分析。实验精度表明常绿林中巴山冷杉、华山松、刺叶栎、虫害华山松整体精度较高,落叶林中红桦、漆树等识别精度相对较高,部分树种如椅杨、锐齿槲栎识别精度较低;总体上常绿树种的精度要优于落叶树种。从植物地理学、遥感、GIS三者相结合的角度,将多源、多时相遥感数据与物种物候特性、专家知识进行有效整合,提出了一种乔木树种识别的方法(1)提供了复杂山地环境的主要乔木优势种识别途径,且具有通用性;(2)完成了物种物候特性与遥感数据特性的整合利用,有效降低数据成本费用;(3)配合地面样方及专家知识修正结果,避免了过分依赖光谱特征引起的误判。这将为神农架川金丝猴栖息地保护与恢复提供更精确的数据依据。