溶葡球菌酶高效表达与应用

溶葡球菌酶(lysostaphin,Lys)是采用基因克隆技术使溶葡球菌酶基因实现外源表达所产生的蛋白质。它是Zn2+依赖的金属蛋白酶,具有肽链内切酶活性,能专一性地水解葡萄球菌细胞壁Gly五肽桥联,使金黄色葡萄球菌(特别是MRSA)细胞壁破裂,达到溶菌杀菌作用,而不产生耐药性。作为一种抗菌剂,在兽药与临床等领域具有巨大的应用潜力。综述对溶葡球菌酶的来源、作用机制、不同表达系统及前景与展望进行综述。     

Preoperative Monocyte-to-Lymphocyte Ratio in Peripheral Blood Predicts Stages,Metastasis, and Histological Grades in Patients with Ovarian Cancer

PURPOSE: The monocyte-to-lymphocyte ratio (MLR) has been shown to be associated with the prognosis of various solid tumors. This study sought to evaluate the important value of the MLR in ovarian cancer patients. METHODS: A total of 133 ovarian cancer patients and 43 normal controls were retrospectively reviewed. The patients'' demographics were analyzed along with clinical and pathologic data. The counts of peripheral neutrophils, lymphocytes, monocytes, and platelets were collected and used to calculate the MLR, neutrophil-to-lymphocyte ratio (NLR). and platelet-to-lymphocyte ratio (PLR). The optimal cutoff value of the MLR was determined by using receiver operating characteristic curve analysis. We compared the MLR, NLR, and PLR between ovarian cancer and normal control patients and among patients with different stages and different grades, as well as between patients with lymph node metastasis and non–lymph node metastasis. We then investigated the value of the MLR in predicting the stage, grade, and lymph node positivity by using logistic regression. The impact of the MLR on overall survival (OS) was calculated by Kaplan-Meier method and compared by log-rank test. RESULTS: Statistically significant differences in the MLR were observed between ovarian cancer patients and normal controls. However, no difference was found for the NLR and PLR. Highly significant differences in the MLR were found among patients with different stages (stage I-II and stage III-IV), grades (G1 and >G1), and lymph node metastasis status. The MLR was a significant and independent risk factor for lymph node metastasis, as determined by logistic regression. The optimal cutoff value of the MLR was 0.23. We also classified the data according to tumor markers (CA125, CA199, HE4, AFP, and CEA) and conventional coagulation parameters (International Normalized Ratio [INR] and fibrinogen). Highly significant differences in CA125, CA199, HE4, INR, fibrinogen levels, and lactate dehydrogenase were found between the low-MLR group (MLR ≤ 0.23) and the high-MLR group (MLR > 0.23). Correspondingly, dramatic differences were observed between the two groups in OS. CONCLUSION: Our results show that the peripheral blood MLR before surgery could be a significant predictor of advanced stages, advanced pathologic grades, and positive lymphatic metastasis in ovarian cancer patients.     

Sequence CLCN1 and SCN4A in patients with Nondystrophic myotonias in Chinese populations: Genetic and pedigree analysis of 10 families and review of the literature

Myotonia congenita (MC), paramyotonia congenita (PC) and sodium channel myotonias(SCM) were belonged to Non-dystrophic myotonias, in which muscle relaxation is delayed after voluntary or evoked contraction. These diseases can not be simply distinguished only based on symptoms and signs but also on genetics: more than 100 mutations in the CLCN1 gene have been associated with MC, while at least 20 mutations in the SCN4A gene have been associated with PC and SCM. Most of these genetics studies have been conducted outside China, only several MC, PC, and SCM families accepted gene scan were reported in China. Therefore we analyzed genetic mutations in CLCN1 and SCN4A in 10 Chinese families clinically diagnosed with Non-dystrophic myotonias. Our result revealed 12 potential disease-causing mutations(3 mutations were novel) that were present in the probands and affected family members. We also reviewed all available literature on mutations linked to these 3 disease in Chinese populations. Our results may help identify genetic determinants as well as clarify genotype-phenotype relationships.     

Analysis of spatial and temporal patterns of aboveground net primary productivity in the Eurasian steppe region from 1982 to 2013

To explore the importance of the Eurasian steppe region (EASR) in global carbon cycling, we analyzed the spatiotemporal dynamics of the aboveground net primary productivity (ANPP) of the entire EASR from 1982 to 2013. The ANPP in the EASR was estimated from the Integrated ANPP_NDVI model, which is an empirical model developed based on field‐observed ANPP and long‐term normalized difference vegetation index (NDVI) data. The optimal composite period of NDVI data was identified by considering spatial heterogeneities across the study area in the Integrated ANPP_NDVI model. EASR's ANPP had apparent zonal patterns along hydrothermal gradients, and the mean annual value was 43.78 g C m^?2 yr^?1, which was lower than the global grasslands average. Compared to other important natural grasslands, EASR's ANPP was lower than the North American, South American, and African grasslands. The total aboveground net primary productivity (TANPP) was found to be 378.97 Tg C yr^?1, which accounted for 8.18%–36.03% of the TANPP for all grasslands. In addition, EASR's TANPP was higher than that of the grasslands in North America, South America, and Africa. The EASR's TANPP increased in a fluctuating manner throughout the entire period of 1982–2013. The increasing trend was greater than that for North American and South American and was lower than that for African grasslands over the same period. The years 1995 and 2007 were two turning points at which trends in EASR's TANPP significantly changed. Our analysis demonstrated that the EASR has been playing a substantial and progressively more important role in global carbon sequestration. In addition, in the development of empirical NDVI‐based ANPP models, the early–middle growing season averaged NDVI, the middle–late growing season averaged NDVI and the annual maximum NDVI are recommended for use for semi‐humid regions, semi‐arid regions, and desert vegetation in semi‐arid regions, respectively.     

Quantifying ecosystem service trade‐offs for plantation forest management to benefit provisioning and regulating services

There is increasing interest worldwide regarding managing plantation forests in a manner that maintains or improves timber production, enhances ecosystem services, and promotes long‐term sustainability of forest resources. We selected the Gan River Basin, the largest catchment of Poyang Lake and a region with a typical plantation distribution in South China, as the study region. We evaluated and mapped four important forest ecosystem services, including wood volume, carbon storage, water yield, and soil retention at a 30 × 30 m resolution, then quantified their trade‐offs and synergies at the county and subwatershed scales. We found that the wood volume and carbon storage services, as well as the soil retention and water yield, exhibited synergistic relationships. However, the carbon storage displayed a trade‐off relationship with the water yield. Additionally, we compared the beneficial spatial characteristics among dominant species in the study region. The results showed that the Chinese fir forest and the pine forest exhibited lower overall benefits than natural forests including the broad‐leaved forest and the bamboo forest. To propose a suitable management strategy for the study region, method of spatial cluster analysis was used based on the four eco‐services at the subwatershed scale. The basin was divided into four management groups instead of treating the region as a homogenous management region. Finally, we proposed more specific and diverse management strategies to optimize forest benefits throughout the entire region.     

中亚热带4种森林类型土壤活性有机碳的季节动态特征

2011年12月至2012年9月, 在湘中丘陵区杉木(Cunninghamia lanceolata)人工林、马尾松(Pinus massoniana)-石栎(Lithocarpus glaber)针阔混交林、南酸枣(Choerospondias axillaries)落叶阔叶林、石栎(Lithocarpus glaber)-青冈(Cyclobalanopsis glauca)常绿阔叶林1 hm²的长期定位观测样地, 采集0-15 cm、15-30 cm土层土壤样品, 测定土壤微生物生物量碳(MBC)、可矿化有机碳(MOC)、易氧化有机碳(ROC)、水溶性有机碳(DOC)含量, 分析4种森林土壤MBC、MOC、ROC、DOC含量的季节变化特征, 为揭示天然林保护与恢复对土壤有机碳(SOC)库的影响机理过程提供基础数据。结果表明: 森林土壤MBC、MOC、ROC、DOC含量具有明显的季节动态, 且不同森林同一土壤活性有机碳组分的季节变化节律基本一致, MBC、MOC、ROC含量表现为夏、秋季较高, 春、冬季较低; DOC含量表现为春、夏、冬季较高, 秋季最低; 同一森林不同土壤活性有机碳组分含量的季节变化节律不同; 土壤MBC、MOC、ROC、DOC含量与土壤自然含水率、SOC、全N、水解N、全P (除杉木人工林土壤MBC、MOC、ROC外)、速效P含量显著或极显著正相关, 与土壤pH值、全K、速效K含量相关性不显著, 表明不同森林类型外源碳库投入和土壤理化性质的差异是导致不同森林类型土壤活性有机碳含量差异显著的主要原因, 该区域森林土壤活性有机碳各组分含量的季节变化与各森林类型组成树种生长节律及其土壤水分含量和SOC、N、P的可利用性, 以及土壤活性有机碳各组分的来源有关, 森林土壤MBC、MOC、ROC、DOC含量可作为衡量森林土壤C、N、P动态变化的敏感性指标。     

贵州矮马IGF-I启动子-529 CpG甲基化及个别核苷酸变异与贵州矮马矮小表型有关

胰岛素样生长因子（insulin-like growth factors,IGFs）和生长（激）素（growth hormone,GH）是动物机体主要的促生长因子,它们的表达水平直接决定成熟个体的高度。贵州矮马的体高明显低于伊犁马等大型马,但其原因尚不清楚。本研究从贵州矮马基因组DNA中克隆了GH和IGF-I基因5′-侧翼序列,分别为239 bp和817 bp,包括部分启动子结构;进而采用生物信息学、比较基因组学方法对比分析了贵州矮马与伊犁马两个基因5′-侧翼序列/启动子的转录因子结合位点及潜在甲基化位点（CpG岛）分布。亚硫酸氢盐PCR测序法（bisulfite sequencing PCR,BSP）显示,两个马群的GH基因5′-侧翼区域（239 bp）内的6个CpG位点均发生了甲基化,甲基化频率无明显差异。然而,在IGF-I基因5′-侧翼区的148 bp片段内含有4个CpG位点中,贵州矮马的-529 bp处CpG位点的甲基化程度明显高于伊犁马（P < 0.01）,且该甲基化位点处于基本启动子邻近3′端;此外,两个马群IGF-I基因5′-侧翼区的-561 bp处检测到T、C碱基改变,导致贵州矮马的顺式调控元件/转录因子结合位点较伊犁马少1个,有可能影响IGF-I基因的转录效率。血清IGF-I浓度测定揭示,贵州矮马血清IGF-I含量极显著低于伊犁马（P< 0.01）。Spearman相关性结果显示,贵州矮马及伊犁马的IGF-Ⅰ基因甲基化频率与血清IGF-I浓度呈中度负相关（r=-0.468）,提示IGF-Ⅰ基因甲基化抑制其编码蛋白质的表达。结果证明,IGF-I启动子高甲基化及某些核苷酸（碱基）序列变异可能是贵州矮马个体矮小的部分原因。     

重组人组蛋白H3和H4的表达纯化及其细胞毒性分析

细胞外组蛋白在脓毒症、类风湿性关节炎、急性肺损伤等多种疾病的发生发展中起关键作用,但由于缺乏合适的标准品,至今无法对患者体内的胞外组蛋白进行精确定量,导致在多种感染性疾病中无法根据血清组蛋白含量对疾病进行精确分级,也无法据此合理用药。同时,对患者体内胞外组蛋白精确定量也有助于确定细胞毒性机制研究的使用剂量。本研究用大肠杆菌表达单体变性组蛋白H3和H4,亲和纯化后用梯度稀释和透析方法,可以得到复性的组蛋白单体H3、H4以及H3/H4复合物。通过对蛋白质在纯化过程中稳定性的比较,发现H3/H4复合物较单体更为稳定。 以该复合物（50 μg/mL）处理HUVEC细胞,细胞存活率约为20%,与小牛胸腺组蛋白（200 μg/mL）的毒性类似。 该复合物引起的细胞毒性可被人血清白蛋白以浓度依赖的形式（0.625~10 mg/mL）缓解,提示其构象基本正确。 因此,重组组蛋白H3/H4复合物可以作为精确定量组蛋白的标准品,对基于组蛋白含量的疾病分级和组蛋白毒性机制的研究均有应用价值。     

抗人AEG-1单克隆抗体可变区基因克隆及序列分析

AEG-1基因位于人染色体8q22,编码582个氨基酸,参与多种信号转导途径并与多种恶性肿瘤的发生、发展及生物学表型密切相关。为更好地探讨AEG-1生物学功能,以纯化的pGSTag-AEG-1蛋白免疫BALB/c小鼠,应用细胞融合技术并经筛选及鉴定,获得了分泌抗人AEG-1单克隆抗体的杂交瘤细胞株1E3;Western blot及免疫组化证实该细胞株分泌的单克隆抗体能与肿瘤细胞中AEG-1蛋白特异性结合;RT-PCR方法从1E3细胞中克隆出抗AEG-1抗体的VH和VL基因片段,通过测序分析、碱基和蛋白序列的比对确认该株抗体为鼠源性IgG的轻、重链可变区基因。进一步运用Kabat System在线分析系统对VH和VL基因进行结构分析,确证FWRs和CDRs的结构完整,VH编码117个氨基酸;VL编码119个氨基酸,属于轻链κV家族。实验结果为进一步研究AEG-1与恶性肿瘤发生、发展的关系及在其临床诊断中的应用奠定了基础。