Ser203在大鼠脑驱动蛋白水解ATP中重要作用的晶体学分析

鼠脑驱动蛋白（rat brain kinesin）是一种利用水解ATP所释放的能量在微管束上高速并且连续性运动的常规驱动蛋白. 它在神经突触的物质运输中起着重要作用. 研究驱动蛋白是如何将ATP中储藏的化学能转化为机械动能是理解其运动机能的重要课题. 本课题获得了鼠脑驱动蛋白单体与ATP结构类似物AMPPCP形成的复合物晶体结构. 将这个晶体结构与鼠脑驱动蛋白单体-另一种ATP结构类似物AMPPNP形成的复合物晶体结构以及鼠脑驱动蛋白单体-ATP水解产物ADP形成的复合物晶体结构进行相互比较,揭示了活性中心的开关区域I中丝氨酸203可能作为质子的供体,加速了ATP中gamma-磷酸和beta-磷酸的断裂,从而导致ATP的水解.     

醛糖还原酶基因5′调控区新的点突变对基因表达调控的影响

为了进一步研究醛糖还原酶 (AR)基因 5′调控区存在的可引起蛋白质表达发生改变的遗传变异及其对糖尿病并发症的影响 ,应用PCR SSCP对中国人 2型糖尿病患者的AR基因 5′调控区进行筛选 ,在两名糖尿病患者中发现一新点突变C- 1 6 7→A ,使AR基因 5′调控区产生一个新的CCAAT盒。含点突变的两名患者尽管患病多年 ,长期处于高血糖状态 ,但无糖尿病并发症发生。而且他们的红细胞中AR活性都很低 ,处于无视网膜病变患者组的下限范围。将含野生型和点突变DNA片段分别克隆至氯霉素乙酰转移酶报告基因载体 (pCAT) ,检测CAT的活性确定野生型和突变型序列的转录活性。同时进行凝胶滞留试验以观测DNA与蛋白质的相互作用。结果显示 :含C- 1 6 7→A的启动子相对转录活性 (5 .7% )明显低于野生型(15 .7% )。凝胶滞留试验中 ,突变序列迁移速率较野生型慢。以上结果说明 ,AR基因 5′调控区C- 1 6 7→A点突变干扰了启动子区顺式作用元件与反式作用因子的结合 ,导致AR基因转录活性降低 ,使患者组织中AR活性下降 ,从而阻止或减缓 2型糖尿病视网膜病变发生发展。     

口服基因疫苗质粒稳定性测定

在研究口服基因疫苗的过程中 ,为确保口服疫苗剂量 ,对转化细菌中质粒稳定性的动态变化 ,细菌浓度OD60 0 值与有效细菌 (携带质粒的细菌 )的关系等进行了测定 ,结果显示PAR3132在培养 10h前 ,pcDNA3 VP1在培养 6h前质粒在细菌中的稳定性可维持在 95 %以上 ,但当培养时间超过 16h ,2种质粒的稳定性在Amp为 5 0g/L时均不足 2 0 %。高浓度Amp可提高质粒稳定性。在质粒稳定性维持于 95 %的时段内 ,细菌浓度的OD60 0 值与有效细菌的量呈线性关系。因此 ,在确定口服基因疫苗剂量时 ,应根据细菌各自的情况决定振摇的时间和采用的OD值。     

A ginger derivative,zingerone—a phenolic compound—induces ROS‐mediated apoptosis in colon cancer cells (HCT‐116)

Zingerone (ZO), an active phenolic agent derived from Zingiber officinale (Ginger), has many pharmacological properties such as antioxidant, antiangiogenic, and antitumor. However, its potential value in cancer and the mechanism by which ZO wields its therapeutic effects remain obscure. Therefore, in this current study, we explored the effects of ZO on suppressing cell proliferation and enhancing apoptosis in colon cancer cells (HCT116). Our results indicated that ZO significantly enhances the production of reactive oxygen species, lipid peroxidation (thiobarbituric acid reactive substance [TBARS]), and loss of cell viability; and reduces mitochondrial membrane potential and antioxidant levels (SOD, CAT, and GSH) in ZO‐treated HCT116 cells in a dose‐dependent (2.5, 5, and 10 µM) manner. Furthermore, ZO induces oxidative stress‐mediated apoptosis as evidenced by apoptotic morphological changes predicted by AO/EtBr, Hoechst staining and further confirmed by comet assay. Moreover, immunoblotting techniques showed that ZO treatment effectively enhances Bax, caspase‐9, and caspase‐3 expressions and decreases the expression of Bcl‐2 in colon cancer cells. Together, our results evidenced that the antitumor effects of ZO reduce cell proliferation and stimulate apoptosis through modulating pro‐ and antiapoptotic molecular events in HCT116 colon cancer cells. Therefore, based on our findings, ZO may be used as a therapeutic agent for the treatment of colon cancer.     

Trans‐3,5,4´‐trimethoxystilbene reduced gefitinib resistance in NSCLCs via suppressing MAPK/Akt/Bcl‐2 pathway by upregulation of miR‐345 and miR‐498

Despite initial dramatic efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR‐TKIs) in EGFR‐mutant lung cancer patients, subsequent emergence of acquired resistance is almost inevitable. Resveratrol and its derivatives have been found to exert some effects on EGFR‐TKI resistance in non‐small cell lung cancer (NSCLC), but the underlying mechanisms remain unclear. We screened several NSCLC cell lines with gefitinib resistance by MTT assay and analysed the miR‐345/miR‐498 expression levels. NSCLC cells were pre‐treated with a resveratrol derivative, trans‐3,5,4‐trimethoxystilbene (TMS) and subsequently challenged with gefitinib treatment. The changes in apoptosis and miR‐345/miR‐498 expression were analysed by flow cytometry and q‐PCR respectively. The functions of miR‐345/miR‐498 were verified by CCK‐8 assay, cell cycle analysis, dual‐luciferase reporter gene assay and immunoblotting analysis. Our results showed that the expression of miR‐345 and miR‐498 significantly decreased in gefitinib resistant NSCLC cells. TMS pre‐treatment significantly upregulated the expression of miR‐345 and miR‐498 increasing the sensitivity of NSCLC cells to gefitinib and inducing apoptosis. MiR‐345 and miR‐498 were verified to inhibit proliferation by cell cycle arrest and regulate the MAPK/c‐Fos and AKT/Bcl‐2 signalling pathways by directly targeting MAPK1 and PIK3R1 respectively. The combination of TMS and gefitinib promoted apoptosis also by miR‐345 and miR‐498 targeting the MAPK/c‐Fos and AKT/Bcl‐2 signalling pathways. Our study demonstrated that TMS reduced gefitinib resistance in NSCLCs via suppression of the MAPK/Akt/Bcl‐2 pathway by upregulation of miR‐345/498. These findings would lay the theoretical basis for the future study of TMS for the treatment of EGFR‐TKI resistance in NSCLCs.     

Microbial Community Structure and Arsenic Biogeochemistry in an Acid Vapor-Formed Spring in Tengchong Geothermal Area,China

Arsenic biogeochemistry has been studied extensively in acid sulfate-chloride hot springs, but not in acid sulfate hot springs with low chloride. In this study, Zhenzhuquan in Tengchong geothermal area, a representative acid sulfate hot spring with low chloride, was chosen to study arsenic geochemistry and microbial community structure using Illumina MiSeq sequencing. Over 0.3 million 16S rRNA sequence reads were obtained from 6-paired parallel water and sediment samples along its outflow channel. Arsenic oxidation occurred in the Zhenxhuquan pool, with distinctly high ratios of arsenate to total dissolved arsenic (0.73–0.86). Coupled with iron and sulfur oxidation along the outflow channel, arsenic accumulated in downstream sediments with concentrations up to 16.44 g/kg and appeared to significantly constrain their microbial community diversity. These oxidations might be correlated with the appearance of some putative functional microbial populations, such as Aquificae and Pseudomonas (arsenic oxidation), Sulfolobus (sulfur and iron oxidation), Metallosphaera and Acidicaldus (iron oxidation). Temperature, total organic carbon and dissolved oxygen significantly shaped the microbial community structure of upstream and downstream samples. In the upstream outflow channel region, most microbial populations were microaerophilic/anaerobic thermophiles and hyperthermophiles, such as Sulfolobus, Nocardia, Fervidicoccus, Delftia, and Ralstonia. In the downstream region, aerobic heterotrophic mesophiles and thermophiles were identified, including Ktedonobacteria, Acidicaldus, Chthonomonas and Sphingobacteria. A total of 72.41–95.91% unassigned-genus sequences were derived from the downstream high arsenic sediments 16S rRNA clone libraries. This study could enable us to achieve an integrated understanding on arsenic biogeochemistry in acid hot springs.     

Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL⁺ CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL⁺ CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL⁺ cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL⁺ cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.     

地方稻资源D43的开花期耐热特性研究

水稻在抽穗开花期对高温胁迫非常敏感,通过挖掘耐热资源,培育耐热水稻品种是应对高温热害最有效的方式。前期研究发现,地方稻资源D43在花期连续高温条件下能保持较高的结实率。本研究在大田和人工气候室不同高温处理下,分析了D43的开花时间与耐热性之间的相互关系。结果表明,高温能够使水稻的开花时间提前,D43表现出稳定的早花时特性,高温胁迫下的开花时间集中在8∶30~10∶00;在开花时间段恒定高温胁迫下,D43的结实率较低;但在大田高温和人工气候室模拟高温胁迫下,D43的开花时间避开了日高温段,从而表现出较高的结实率;花器官形态性状包括花药开裂率、柱头上的花粉附着数、花粉萌发数与结实率之间呈显著正相关,因此可用于评价水稻的花期耐热性。     

QTL复合区间作图中标记筛选的效率及其影响因素研究

高效地筛选标记 ,是复合区间作图方法定位QTLs的基础。筛选出的主效标记和互作标记 ,除了用作控制背景遗传效应外 ,在定位具有上位性效应的QTLs时 ,还将用于构筑两维搜索区间。因而 ,标记筛选的效率将直接影响QTL定位的功效和精度。通过对不同方法筛选标记的效率进行模拟研究 ,发现回归方法明显优于随机效应预测方法 ,同归方法中又以前向选择法简单有效。普通遗传力和基因型×环境互作遗传力的增加都能提高标记筛选效率 ,前者对主效应较大的QTLs影响明显 ,后者对主效应较小的QTLs作用较大。过多过密的标记会降低标记筛选效率 ,其中密度增加对标记筛选的负作用更为突出。为了缓解标记筛选效率制约QTL定位功效的缺陷 ,可以用多环境下筛选出的标记共同构建两维搜索区间     

南瓜果肉色素的提取及稳定性的研究

本文研究了从南瓜果肉中提取色素的方法,并对它的光、热、酸、碱稳定性进行了研究,发现其性质较稳定,且原料来源广泛,提取工艺简单,着色效果好,是食品、医药、化妆品等领域的理想添加剂。