成人腹泻轮状病毒的提纯及其高价免疫血清的制备

本文采用交叉免疫电泳技术,首先提纯了成人腹泻轮状病毒(Adult DiarrhoeaRotavirus简称:ADRV),并经电镜确认,用作免疫抗原,首次制备了高价兔与豚鼠抗ADRV血清和小鼠抗ADRV腹水,其特异性经交叉免疫电泳和对流免疫电泳鉴定,其效价经酶联免疫吸附试验(ELISA)检测均在1:2,000以上。     

Thyroid-hormone effects on putative biochemical pathways involved in UCP3 activation in rat skeletal muscle mitochondria

In vitro, uncoupling protein 3 (UCP3)-mediated uncoupling requires cofactors [e.g., superoxides, coenzyme Q (CoQ) and fatty acids (FA)] or their derivatives, but it is not yet clear whether or how such activators interact with each other under given physiological or pathophysiological conditions. Since triiodothyronine (T3) stimulates lipid metabolism, UCP3 expression and mitochondrial uncoupling, we examined its effects on some biochemical pathways that may underlie UCP3-mediated uncoupling. T3-treated rats (Hyper) showed increased mitochondrial lipid-oxidation rates, increased expression and activity of enzymes involved in lipid handling and increased mitochondrial superoxide production and CoQ levels. Despite the higher mitochondrial superoxide production in Hyper, euthyroid and hyperthyroid mitochondria showed no differences in proton-conductance when FA were chelated by bovine serum albumin. However, mitochondria from Hyper showed a palmitoyl-carnitine-induced and GDP-inhibited increased proton-conductance in the presence of carboxyatractylate. We suggest that T3 stimulates the UCP3 activity in vivo by affecting the complex network of biochemical pathways underlying the UCP3 activation.     

A variational constitutive model for soft biological tissues

In this paper, a fully variational constitutive model of soft biological tissues is formulated in the finite strain regime. The model includes Ogden-type hyperelasticity, finite viscosity, deviatoric and volumetric plasticity, rate and microinertia effects. Variational updates are obtained via time discretization and pre-minimization of a suitable objective function with respect to internal variables. Genetic algorithms are used for model parameter identification due to their suitability for non-convex, high dimensional optimization problems. The material behavior predicted by the model is compared to available tests on swine and human brain tissue. The ability of the model to predict a wide range of experimentally observed behavior, including hysteresis, cyclic softening, rate effects, and plastic deformation is demonstrated.     

Discovery of a novel site regulating glucokinase activity following characterization of a new mutation causing hyperinsulinemic hypoglycemia in humans

Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S(0.5), 3.3 ± 0.1 and 3.5 ± 0.1 mm, respectively) versus wild type (7.5 ± 0.1 mm). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (<0.1). T103I had an altered affinity for glucose (S(0.5), 24.9 ± 0.6 mm), whereas V389D also exhibited a reduced affinity for ATP and decreased catalysis rate (S(0.5), 78.6 ± 4.5 mm; ATP(K(m)), 1.5 ± 0.1 mm; K(cat), 10.3 ± 1.1s(-1)) compared with wild type (ATP(K(m)), 0.4 ± <0.1; K(cat), 62.9 ± 1.2). Both Thr-103 mutants showed reduced inhibition by the endogenous hepatic inhibitor glucokinase regulatory protein. Molecular modeling demonstrated that Thr-103 maps to the allosteric activator site, whereas Val-389 is located remotely to this position and all other previously reported activating mutations, highlighting α-helix 11 as a novel region regulating GCK activity. Our data suggest that pharmacological manipulation of GCK activity at locations distal from the allosteric activator site is possible.     

IL‐6 Trans‐Signaling Plays Important Protective Roles in Acute Liver Injury Induced by Acetaminophen in Mice

Our study was undertaken to evaluate the important role of interleukin‐6 (IL‐6) trans‐signaling in acetaminophen (AAP)‐induced liver injury. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL‐6 trans‐signaling, whereas an IL‐6/soluble IL‐6 receptor (sIL‐6R) fusion protein (hyper‐IL‐6) mimics IL‐6 trans‐signaling. Using these tools, we investigated the role of IL‐6 trans‐signaling in AAP‐induced liver injury. Blockade of IL‐6 trans‐signaling during AAP‐induced liver injury remarkably increased the levels of serum aspartate aminotransferase and alanine aminotransferase; lowered the level of serum sIL‐6R; aggravated liver injury; inhibited the expression of phosphorylation of STAT3 (pSTAT3), proliferating cell nuclear antigen, vascular endothelial growth factor, and glycogen synthesis; and induced the expression of Caspase3, cytochrome P450 2E1 (CYP2E1), and hepatocyte apoptosis in the liver of mice. In summary, our study suggested that IL‐6 trans‐signaling plays important protective roles by regulating the hepatocyte proliferation and apoptosis, angiogenesis, CYP2E1 expression, and glycogen metabolism during AAP‐induced liver injury in mice.     

Preliminary results on the reproduction of a deep-sea snailfish Careproctus rhodomelas around the active hydrothermal vent on the Hatoma Knoll, Okinawa, Japan

Deep-sea snailfish Careproctus rhodomelas were collected from an active hydrothermal vent using a remotely operated vehicle (R.O.V. Hyper-dolphin) and a pressurized device (Deep-Aquarium). Careproctus rhodomelas exhibited a cystovarian-type ovary containing a small number of developing oocytes at different stages, suggesting that the fish is a batch-spawner that spawns large eggs (c. 6·0 mm) several times within its life span. In vitro culture of the oocytes in the presence of human chorionic gonadotropin showed that oestradiol-17β production fluctuated with oocyte development, suggesting that the oocytes were at the vitellogenic stage.     

渗透压冲击下中度嗜盐菌Halomonas sp. Y四氢嘧啶类相容性溶质的合成与释放

【背景】四氢嘧啶类物质在高温、冷冻和干燥等逆境条件下,对酶、蛋白质、核酸及整个细胞具有良好的保护作用,已经应用于酶制剂、生物医药及护肤品等相关领域。目前此类物质只能依赖中度嗜盐菌采用细菌泌乳工艺进行商业化生产,因此四氢嘧啶类高产菌株及其发酵技术的研究日益受到国内外研究者关注。【目的】分离获得高产合成四氢嘧啶类相容性溶质的中度嗜盐细菌,研究渗透压冲击对其胞内四氢嘧啶合成与释放的影响,探索细菌泌乳法制备四氢嘧啶的可行性。【方法】采用涂布平板法分离中度嗜盐菌,对分离菌株进行形态、生理生化和16S rRNA基因序列分析,鉴定其种属;采用高效液相色谱法(HPLC)和质谱法(MS)分析四氢嘧啶类物质,细菌泌乳法制备四氢嘧啶类物质。【结果】从盐池土样中分离到一株以四氢嘧啶类物质为主要相容性溶质的中度嗜盐菌Y,鉴定为盐单胞菌(Halomonas sp.)Y。盐单胞菌Y能在NaCl质量浓度为10-250 g/L的培养基中生长,最适生长的NaCl浓度为100 g/L;HPLC-MS测试结果证明盐单胞菌Y可同时合成四氢嘧啶和羟基四氢嘧啶2种相容性溶质,在最适生长的盐浓度下其合成量分别达175.5 mg/g和47.9 mg/g;在NaCl质量浓度为0-30 g/L的低渗溶液中胞内四氢嘧啶类物质经5 min即可达到最大释放率,而细菌泌乳工艺中最适合诱导四氢嘧啶释放的低渗溶液为质量浓度为10 g/L的NaCl溶液;采用细菌泌乳工艺制备四氢嘧啶,经连续11轮的高渗/低渗冲击,四氢嘧啶总合成量为6.0 g/L,总释放量为5.7 g/L,平均释放率为64.5%,底物转化率为128.9 mg/g。【结论】盐单胞菌Y是一株较高产合成四氢嘧啶类的中度嗜盐菌,能够耐受反复的渗透压冲击,采用细菌泌乳工艺显著提高了四氢嘧啶的制备效率。     

猪流行性腹泻病毒流行株S蛋白高变区B细胞线性表位肽的鉴定

【背景】对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)经典毒株与流行毒株S蛋白氨基酸序列比对显示,变异位点主要集中在S蛋白的N端。【目的】鉴定PEDV流行毒株S蛋白高变区(S10A,1-496 aa)的B细胞线性表位肽,特别是流行毒株特异性B细胞表位肽。【方法】以SDS-PAGE割胶纯化大肠杆菌表达的PEDV流行株SD2014 S蛋白高变区片段(S10ASD2014),免疫新西兰大白兔制备多克隆抗体;在E. coli中谷胱甘肽巯基转移酶(Glutathione S-transferase,GST)融合表达覆盖S10ASD2014序列全长且彼此重叠8个氨基酸残基的系列16肽。以制备的抗S10ASD2014多抗为一抗,通过Western blot筛选系列16肽中的阳性反应性16肽,鉴定S10ASD2014上的B细胞线性表位肽;利用抗PEDV经典毒株(CV777)S蛋白高变区片段(S10ACV777)多抗血清鉴定S10ASD2014上的保守性B细胞线性表位肽。【结果】重组表达的S10ASD2014相对分子质量约为72 kD;纯化的S10ASD2014表位肽能被PEDV阳性血清识别。制备的抗S10ASD2014多抗可以识别PEDV DR13弱毒株。利用抗S10ASD2014血清和抗S10ACV777血清从61个GST融合表达的16肽中鉴定到28个阳性反应16肽,其中13个为2种血清共同识别16肽。对24株PEDV不同亚群代表毒株的对应区域进行同源性分析表明2个阳性16肽为保守性表位肽。【结论】确定了PEDV流行株SD2014 S蛋白高变区的B细胞线性表位肽,B细胞线性表位肽,特别是流行毒株特异性表位肽的确定有助于了解S蛋白的结构与功能,为建立以表位为基础的PEDV感染检测方法打下良好基础。