Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.     

Induction of defence gene expression by oligogalacturonic acid requires increases in both cytosolic calcium and hydrogen peroxide in Arabidopsis thaliana

Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings express-ing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca^2 ]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H202. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca^2 ]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.     

SNPs基因分型技术

介绍了SNP的概念、特点,集中讨论了各种技术的原理及优缺点,并对目前SNP在遗传图的绘制、疾病防治、药物设计及法医学等方面的应用及研究进展进行了综述。     

DNA chip-based expression profile analysis indicates involvement of the phosphatidylinositol signaling pathway in multiple plant responses to hormone and abiotic treatments

The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses.Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK),phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms.In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid).Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions.In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.     

多巴胺D1和D2受体激动剂和拮抗剂对脑缺血/再灌注损伤的影响

实验应用开阔法、组织病理学方法、原位末端标记(in situ terminal deoxynucleotidyl transferase-metliated de-oxy-UTP mick end labeling,TUNEL)法及免疫组织化学等方法,探讨多巴胺D1、D2受体激动剂和拮抗剂对沙土鼠前脑缺血／再灌注损伤海马CA1区神经元凋亡及凋亡相关基因bcl-2、bax表达的影响。结果显示：前脑缺血5min可引起沙土鼠探索活动增加;再灌注3d,海马CA1区约95％的锥体细胞凋亡;再灌注7d,海马CA1区仅残存约2％—7％的存活锥体细胞;前脑缺血5min可抑制bcl-2的表达并诱导bax表达增高;预先应用D2受体激动剂培高利特可减轻缺血后沙土鼠行为学异常、抑制海马CA1区锥体细胞凋亡、提高锥体细胞存活数、显著诱导bcl-2的表达并抑制bax的表达。预先应用SKF38393、SCH23390及螺哌隆对以上结果无明显影响。实验结果提示,培高利特具有确切的脑保护作用,诱导bcl-2并抑制bax的表达可能是其脑保护作用机制之一。     

肿瘤相关基因表达检测寡核苷酸芯片的制备及其初步应用

为研制肿瘤相关寡核苷酸芯片,并实现其在抗肿瘤反义核酸“癌泰得”作用机理研究方面的初步应用,制备了包含近450种肿瘤相关基因特异寡核苷酸探针的寡核苷酸芯片,建立了相应的质控标准.“癌泰得”用脂质体转染HepG2肿瘤细胞,提取细胞总RNA反转录并荧光标记cDNA,用制备的寡核苷酸芯片检测肝癌细胞HepG2的肿瘤相关基因表达水平,用软件分析获得其差异基因表达谱.0.4 μmol/L的反义核酸“癌泰得”作用于HepG2细胞15 h后,MDNCF、DHS等基因mRNA表达下调,MUC2、MPP11、LAT、HRIF-B、JNK3A1等mRNA基因表达上调,初步检测到了“癌泰得”的抗肿瘤作用可能的相关基因,为进一步的分子作用机理的探讨奠定基础.结果表明,制备的肿瘤相关芯片敏感度高、特异性高、重复性均较好,可用于检测肿瘤相关基因的表达谱,为临床诊断和基础研究提供了技术平台.     

db/db小鼠糖尿病肾病相关基因的分析和克隆

用GM-U74A基因芯片分别检测了正常对照组（db/m小鼠）、糖尿病肾病组（db/db小鼠）、大黄酸治疗组（大黄酸150 mg/kg治疗12周）肾脏基因表达谱.发现在12 437个基因（包括表达序列标签）中,与正常对照组相比,糖尿病肾病组有1 085个基因表达下调,37个基因表达上调,其中变化幅度大于2倍,表达下调的有166个和表达上调的有29个.与糖尿病肾病组相比,大黄酸治疗组有384个基因表达下调,155个表达上调,其中变化幅度大于2倍,表达下调的有47个和表达上调的有30个.在此基础上,对其中的一个差异表达的表达序列标签(EST)进行了详细的生物信息学分析,发现它是一个未知功能基因——“REKEN cDNA 0610006H10”基因的一部分.在用RT-PCR进一步验证了其与糖尿病肾病的相关性后,对“REKEN cDNA 0610006H10”基因进行了克隆.     

Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis

To determine the possible role of the epigenetic mechanisms in carcinogenesis of the hepatocellular carcinoma, we methylation-profiled the promoter CpG islands of twenty four genes both in HCC tumors and the neighboring non-cancerous tissues of twenty eight patients using the methylation-specific PCR (MSP) method in conjunction with the DNA sequencing. In comparison with the normal liver tissues from the healthy donors, it was found thatwhile remained unmethylated the ABL, CAV, EPO, GATA3, LKB 1, NEP, NFL, NIS and p27KIPI genes, varying extents of the HCC specific loss of the epigenetisoermethvlation were found associated with the ABO, AR. CSPG2. cvclin al. DBCCR1,GALR2, IRF7, MGMT, MT 1 A, MYOD 1, OCT6, p57KP2, p73, WT 1 genes, and demethylation with the MAGEA 1 gene, respectively. Judged by whether the hypermethylated occurred in HCC more rrequenuy man in tneir neignboring normal tissues, the hypermethylation status of the AR, DBCCR1, IRF7, OCT6, and p73 genes was considered as the event specific to the late stage, while that the rest that lacked such a distinguished contrast, as the event specific to the early stage of HCC carcinogenesis. Among all the clinical pathological parameters tested for theassociation with, the hypermethylation of the cyclin al gene was more prevalent in the non-cirrhosis group (P=0.02 1) while the hvoermethvlated p16^INK4a gene was more common in the cirrhosis group (P=0.017). The concordant dant methylation behaviors of nineteen genes, including the four previously studied and their association With clrrllosis has been evaluated by the best subgroup selection method. The data presented in this report would enable us toshape our understanding of the mechanisms for the HCC specific loss of the epigenetic stability of the genome, aswell as the strategy of developing the novel robust methylation based diagnostic and prognostic tools.     

两种过滤特征基因选择算法的有效性研究

对基因表达谱进行特征基因选择不仅能改善疾病分类方法的效能,而且为寻找与疾病相关的特征基因提供新的途径．通过比较用调整p值的t检验、非参数评分两种特征基因选择算法后和未进行选择时支持向量机(SVM)分类器的分类性能、支持向量(SV)的吻合度、错分样本ID的吻合度和对样本均匀翻倍后的稳定性．结果发现：特征选择后线性、核函数为二阶多项式和径向基的SVM分类性能明显提高;特征选择前后的SV及错分样本ID的吻合度均较高;SVM的稳定性较好．由此得出结论：这两种特征选择算法具有一定的有效性．     

加工产品中转基因玉米Bt11成分实时荧光PCR定量(性)检测

实验在玉米自身基因和外源基因的边界序列之间设计了具有品种和品系特异性的引物和探针 ,并以实时荧光PCR技术 ,建立了加工产品中转基因玉米Bt1 1成分品系鉴定检测和定量检测的方法。实验对加热条件和时间对检测转基因成分的影响作了探讨 ,并检测了部分市售食品和饲料。检测结果发现 ,加热时间温度越高、时间越长 ,对转基因成分定量检测的影响越大 ;在所检测的样品中可以检测出转基因玉米Bt1 1成分 ,有些样品还同时检出其他转基因成分。本研究实验建立的方法 ,可以用于加工产品中转基因成分的定量检测 ,也可以用于定性检测 ,或作为常规PCR定性检测后的确证实验方法。