耐甲氧西林葡萄球菌3种检测方法的实验比较及临床应用

目的 通过对200株临床分离的葡萄球菌耐苯唑西林的检测,比较3种表型检测法的阳性率并对其临床实用性进行评价。方法 采用美国NCCLS 2004年制订的头孢西丁-纸片扩散法、VITEK32微生物鉴定仪检测及苯唑西林盐琼脂法测试mecA介导的葡萄球菌耐药。结果 200株被检测的葡萄球菌中,头孢西丁-纸片扩散法检出耐苯唑西林的阳性率为68％（136／200）;苯唑西林盐琼脂法的阳性率为67．5％（135／200）;VITEK32仪的阳性率为68．5％（137／200）;且耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）的耐药率（86．2％）高于耐甲氧西林金黄色葡萄球菌（26．23％）。3种检测方法结果经χ^2检测差异无显著性。结论 3种方法操作都简便,但各有优缺点,VITEK32仪器检测葡萄球菌耐笨唑西林费用高,一般小型医院不具备条件;盐琼脂筛选法判读直观,但要掌握好孵育时间;NCCLS2004版的纸片扩散法较经济实用,不仅可检测被检菌是否耐笨唑西林,同时还可获得大环内酯-林可霉素-链阳霉素（MLSB）结果,但是当头孢西丁的判断折点在临界时应当复检,建议与盐琼脂筛选法同步检测。     

草鱼TRIM32基因克隆表达及功能研究

TRIM (Tripartite-motif protein) 家族是机体先天性免疫的重要成员, 参与细胞增殖分化、细胞凋亡、肿瘤抑制、抗病毒等过程。为了进一步探究TRIM蛋白在鱼类免疫中的作用, 实验利用PCR方法克隆了草鱼TRIM32基因的编码区全长, 共1980 bp, 编码660个氨基酸。草鱼TRIM32具有TRIM家族典型的3个结构域, 与斑马鱼TRIM32的核苷酸序列同源性较高, 遗传进化分析也聚为一支。间接免疫荧光、Western-blotting检测结果显示草鱼TRIM32在EPC细胞中成功表达, 主要以点状分布在细胞质中, 细胞核中表达量较少; 组织分布分析表明TRIM32在被检测的11个组织中均有表达, 其中在头肾组织中的表达量明显高于其他组织; 不同胚胎发育时期的表达分析进一步表明, TRIM32在受精卵时期、卵裂期和囊胚期的表达量显著高于其他时期(P<0.05), 随后表达量下降, 直到出膜后表达量再次显著性升高。此外, 双荧光素酶检测系统显示TRIM32能够激活NF-κB信号通路, 诱导下游的炎症反应。结果显示, 草鱼TRIM32广泛分布于鱼体内, 并参与机体的天然免疫反应, 对于抵抗病原体的入侵具有重要作用。     

Potential‐resolved electrochemiluminescence of Ru(bpy)32+/C2O42− system on gold electrode

The electrogenerated chemiluminescence of Ru(bpy)₃²⁺/C₂O₄^2? system on a pre‐polarized Au electrode was studied using a potential‐resolved electrochemiluminescence (PRECL) method. Two anodic ECL peaks were observed at 1.22 V (vs. SCE) (EP1), 1.41 V (vs. SCE) (EP2), respectively. The effects of the concentration of oxalate and Ru(bpy)₃²⁺, adsorbed sulphur, CO₂, O₂, pH of the solution and pretreatment of the Au electrode on the two PRECL peaks were examined. The surface state of the pre‐oxidized gold electrode was also studied using the X‐ray photoelectron spectroscopy (XPS) technique. Moreover, comparative studies on i–E and I–E curves were carried out and a possible mechanism involving both the catalytic and the direct electro‐oxidation pathways was proposed for the ECL of Ru(bpy)₃²⁺/C₂O₄^2? system. EP1 is attributed to the Ru(bpy)₃^2/3+ reaction catalysed by C₂O₄^2? to generate Ru(bpy)₃^2+*. EP2 is likely because C₂O₄^2? was oxidized at the electrode to form CO₂^?·, followed by reaction with Ru(bpy)₃³⁺ to generate Ru(bpy)₃^2+*. Copyright © 2002 John Wiley & Sons, Ltd.     

Correlation between heat shock protein 32 and chronic heat-induced liver injury in developing mice

Heat stress is one of a wide variety of factors causing liver injury, a small heat shock protein (HSP), HSP32, is induced by heat stress in the liver. But the biological function of HSP32 in this injury is unclear. To investigate the underlying role of HSP32, RT-PCR, immunocytochemical staining and ELISA were applied to confirm the expression of HSP32. And the underlying mechanism in the pathogenesis of hepatic dysfunction following hyperthermic challenge and the possible involvement of oxidative stress to induce oxidative deterioration of liver functions in developing mice were investigated in this study. Caspase-3mRNA expression and caspase-3 activity of heated liver were also analysed. The results showed that liver injury caused by chronic heat stress(39 °C, 1.5 h/day for 6 weeks) was reversible, caspase-3mRNA expression and caspase-3 activity of heat treated mice were increased after the first three weeks of heat exposure (P<0.05) and high expression levels of HSP32 were observed throughout the duration of experiment (P<0.01). A strong correlation exists between heat-induced liver injury and the induction of HSP32, which suggested that the reversibility of liver injury is involved in the induction of HSP32 in the hepatic cells under continuing heat stress.     

白浆土－植物系统营养物质转化机制及其有效性研究——Ⅰ．32P同位素示踪法对白浆土中P肥利用率

利用³²P同位素示踪法对白浆土中P肥利用率进行了研究.结果表明,在岗地白浆土上,表层土壤表现出一定程度的供P不足现象,白浆层土壤有效P含量严重缺乏.表层土壤中当季P肥利用率的变幅范围为6.09～12.35%,白浆层土壤P肥利用率为13%左右.施用有机肥能明显提高白浆土Olsen P的含量,加速土壤本身P的活化,各种有机物中,以猪粪对土壤潜在P的活化效果最好.将Olsen P与X值、A值比较,认为Olsen P是评价白浆上P肥力简便易行的可靠指标.     

地中海拟无枝酸杆菌甲基丙二酰CoA变位酶和消旋酶的纯化及性质

采用原生质体裂解方法确定甲基丙二酰CoA变位酶(MCM)和消旋酶(MCR)均是胞浆内酶。各经过四步纯化得到电泳纯酶。纯化MCM酶的比活力为12.84u/mg,纯化倍数为528,酶活回收为60％,纯化的MCM酶服从典型的米氏底物饱和曲线,对琥珀酰CoA和辅酶B_(12)的K_m值分别为9.723#mol/L和0.1277#mol/L。经SephadexG-150测定MCM分子量约为134.000±2000,SDS-聚丙烯酰胺凝胶电泳显示两条分子量分别为67000和65000的蛋白带,说明该酶由两个大小不等亚基组成。吸收光谱测定每摩尔纯化全酶含两摩尔辅酶B_(12)。纯化MCR酶比活力为2.305u/mg,纯化倍数96,酶活回收46.7％。MCR酶由两个分子量均为17500的亚基组成。MCR酶活性能被二价金属离子Cu²⁺、Co²⁺、Mg²⁺、Mn²⁺和Fe²⁺所促进。两酶的酶学性质和其他生物来源的MCM、MCR酶明显相似。     

大肠杆菌rpoH基因的克隆,表达及其产物的纯化

本文用ＰＣＲ方法获得大肠杆菌热休克蛋白转录因子σ３２的编码基因ｒｐｏＨ,并克隆在含有ｔａｃ启动子的表达载体ｐＵＨＥ中,经ＩＰＴＧ诱导,在大肠杆菌中表达了Ｃ端融合有６个寡聚组氨酸的σ３２。表达产物经金属螯合层析一步纯化,达到ＳＤＳ－ＰＡＧＥ银染一条带纯度,氨基酸组成分析及Ｎ端序列分析结果与文献报道一致。３５Ｓ细胞内参入实验表明：即使在较低的温度下,表达产物σ３２（Ｈｉｓ）６也能导致热休克蛋白如ＧｒｏＥｌ、ＤｎａＫ、Ｈｔｐ的大量合成．     

Important roles of CD32 in promoting suppression of IL-4 induced immune responses by a novel anti-IL-4Rα therapeutic antibody

ABSTRACT

Asthma is characterized by airway hyperresponsiveness and inflammation, as well as underlying structural changes to the airways. Interleukin-4 (IL-4) is a key T-helper type 2 (Th2) cytokine that plays important roles in the pathogenesis of atopic and eosinophilic asthma. We developed a novel humanized anti-IL-4Rα antibody that can potently inhibit IL-4/IL-13-mediated TF-1 cell proliferation. Using monocytes isolated from human peripheral blood mononuclear cells (PBMCs), we revealed a critical role of CD32 in modulating the immune responses of monocytes in response to blockade of IL-4Rα signaling pathway. We, therefore, devised a new strategy to increase the efficacy of the anti-IL-4Rα monoclonal antibody for the treatment of asthma and other atopic diseases by co-engaging CD32 and IL-4Rα on monocytic cells by choosing IgG classes or Fc mutations with higher affinities for CD32. The antibody with selectively enhanced affinity for CD32A displayed superior suppression of IL-4-induced monocytes’ activities, including the down-regulation of CD23 expression. Intriguingly, further analysis demonstrated that both CD32A and CD32B contributed to the enhancement of antibody-mediated suppression of CD23 expression from monocytes in response to blockade of IL-4Rα signaling. Furthermore, inhibition of IgE secretion from human PBMC by the antibody variants further suggests that the complex allergic inflammation mediated by IL-4/IL-4Rα signaling might result from a global network where multiple cell types that express multiple FcγRs are all involved, of which CD32, especially CD32A, is a key mediator. In this respect, our study provides new insights into designing therapeutic antibodies for targeting Th2 cytokine-mediated allergic pathogenesis.     

RGC-32 Deficiency Protects against Hepatic Steatosis by Reducing Lipogenesis

Hepatic steatosis is associated with insulin resistance and metabolic syndrome because of increased hepatic triglyceride content. We have reported previously that deficiency of response gene to complement 32 (RGC-32) prevents high-fat diet (HFD)-induced obesity and insulin resistance in mice. This study was conducted to determine the role of RGC-32 in the regulation of hepatic steatosis. We observed that hepatic RGC-32 was induced dramatically by both HFD challenge and ethanol administration. RGC-32 knockout (RGC32^−/−) mice were resistant to HFD- and ethanol-induced hepatic steatosis. The hepatic triglyceride content of RGC32^−/− mice was decreased significantly compared with WT controls even under normal chow conditions. Moreover, RGC-32 deficiency decreased the expression of lipogenesis-related genes, sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase, and stearoyl-CoA desaturase 1 (SCD1). RGC-32 deficiency also decreased SCD1 activity, as indicated by decreased desaturase indices of the liver and serum. Mechanistically, insulin and ethanol induced RGC-32 expression through the NF-κB signaling pathway, which, in turn, increased SCD1 expression in a SREBP-1c-dependent manner. RGC-32 also promoted SREBP-1c expression through activating liver X receptor. These results demonstrate that RGC-32 contributes to the development of hepatic steatosis by facilitating de novo lipogenesis through activating liver X receptor, leading to the induction of SREBP-1c and its target genes. Therefore, RGC-32 may be a potential novel drug target for the treatment of hepatic steatosis and its related diseases.