NGF stimulation of erk phosphorylation is impaired by a point mutation in the transmembrane domain of trkA receptor

The nerve growth factor (NGF) trkA receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic tyrosine kinase region by a single transmembrane domain (TMD). To explore the role of TMD in the process of receptor activation, we substituted the hydrophobic amino-acid residue valine 432 with the charged amino-acid glutamic acid (designated V432E mutant) by utilizing in vitro site-directed mutagenesis. NIH 3T3 cells lacking endogenous NGF receptors were stably transfected with a pRc/CMV vector carrying either wild-type (trkA) or mutated (V432E) receptors. Stable transfectants were shown, using ¹²⁵I-NGF binding and Western-blot analysis, to express the trkA recombinant receptors. Scatchard analysis revealed similar affinity for NGF in wild-type and V432E receptors. Although the level of basal trkA receptor tyrosine phosphorylation was higher in the mutant than in the wild-type, NGF stimulation of WT 11 and V432E transfectants resulted in a rapid increase in receptor tyrosine phosphorylation and of its intracellular adaptor protein SHC. In contrast to WT 11, V432E mutants showed very low levels of NGF-, and moderate levels of FGF-induced erks phosphorylation, respectively. Collectively, these findings suggest that a single substitution (V432E) in the trkA TMD results in a selective impairment of trkA-mediated erks signaling pathway.     

骨关节炎关节软骨中软骨细胞线粒体DNA缺失突变的分析

目的：分析骨关节炎病人关节软骨组织中软骨细胞线粒体DNA序列变化，探讨骨关节炎发病的潜在病理机制。方法：应用gap—PCR扩增方法检测10例骨关节炎病人和3例正常关节软骨中软骨细胞线粒体DNA的4977bp缺失突变。采用2轮PCR扩增，第1轮PCR反应以骨关节炎病人的关节软骨中软骨细胞线粒体DNA为模板；第2轮PCR扩增反应以第1轮PCR扩增反应的产物为模板，再次进行扩增。结果：第1轮PCR反应结果显示，mtDNA524bp扩增产物呈阴性，而533bp扩增产物均呈阳性；第2轮PCR扩增反应结果显示，10例骨关节炎病人病例标本中有2例出现524bp区带，而533bp扩增产物均呈阳性。两次扩增，正常关节软骨中软骨细胞和外周血对照524bp扩增产物全部为阴性。结论：骨关节炎病人关节软骨组织中软骨细胞线粒体DNA有8470～13447nt之间4977bp大片段缺失，提示骨关节炎的发病与关节软骨中软骨细胞线粒体DNA的突变关系密切。     

豫医无毛小鼠无毛基因部分内含子突变研究

目的 研究豫医无毛小鼠突变的分子机制。方法 对豫医无毛小鼠和昆明小鼠的基因组DNA进行PCR扩增，对二者扩增片段克隆后测序，对结果进行比较，以确定豫医无毛小鼠无毛基因的特异性程度。结果 本实验共测出豫医无毛小鼠DNA序列1746bp和昆明小鼠DNA序列1733bp，两者不同之处有32处，均位于内含子，突变形式包括插入、缺失和点突变。结论 无毛性状的产生可能与内含子中的插入、缺失和点突变有关。     

Overview of HIV drug resistance and its implications for China

Since the initiation of China＇s nationwide free antiretroviral therapy （ART） in 2002, the availability of highly active antiretroviral therapy （HAART） in China has increased dramatically. HAART has been proven to prolong survival and control HIV disease progression. At the same time, experience in developed countries indicates that the high mutation rate of the virus in the presence of HAART is associated with drug resistance and diminished efficacy. The increase in HAART availability and use in China therefore also comes with the potential for increased resistance. Transmission of drug-resistant strains to individuals who have never been exposed to ART is on the rise. Moreover, studies have shown that new infections by drug-resistant virus result in suboptimal response to ART. With the rapid scale up of ART in China in recent years, the prevalence of HIV drug resistance will likely increase, posing a major public health concern in China. This review article provides an overview of ART resistance, the current worldwide trends in HIV drug resistance, the effect of HIV drug resistance in clinical management, and the implications for China＇s HIV treatment and care.     

结直肠癌预防研究进展

38%的结直肠癌患者就诊时肿瘤局限于肠壁,大规模普查是降低结直肠癌发病率和死亡率有效方法。目前常用的普查方法为大便潜血试验、纤维乙状结肠镜、气钡双重造影、结肠镜等。虚拟内视镜和大便分子生物学检查正在研究中。随机对照研究发现4类化学药物对预防结直肠腺瘤和(或)癌有效,分别是硒、碳酸钙、激素替代疗法和非甾体类抗炎药。     

PCR-SSP法检测人胰腺癌细胞株PCNA-1的K-ras基因点突变的方式

目的检测人胰腺癌细胞株PANC-1的K-ras基因点突变方式,明确干扰Ras基因靶点的碱基序列。方法针对K-ras基因第十二位点密码子点突变最常见方式CAT,CGT,GTT设计顺序特异性引物(SSP),对人胰腺癌细胞株PANC1进行聚合酶链反应(PCR),扩增产物用聚丙烯酰胺凝胶电泳判定该细胞株有无点突变及突变方式。结果人胰腺癌细胞株PANC-1存在K-ras基因的点突变,其突变方式为CAT。结论 PCR-SSP法快速简便,特异性高,结果准确,为应用RNAi技术研究胰腺癌的基因治疗奠定了基础。     

应用DHPLC筛查先天性巨结肠RET基因突变研究

目的：应用DHPLC方法筛查先天性巨结肠RET基因突变。方法：提取32例先天性巨结肠患者的全血DNA，聚合酶链反应后应用DHPLC方法分析11、13、15号外显子的基因表达情况。结果：DHPLC技术分析有13例出现双峰，经测序后有1例突变为Gln849Lys，在15号外显子上；有3例患者有基因多态性：1例为Ser667Ser，在11号外显子上；2例为Leu746Leu，在13号外显子上。结论：DHPLC是一种快速有效的基因突变筛查方法，RET基因的突变和基因多态性与先天性巨结肠的发生密切相关。     

胰液细胞学及肿瘤相关标志物联合检测在胰腺癌诊断中的价值

目的通过内镜抽取纯胰液检测细胞学、K—rds基因点突变及端粒酶活性．探索早期胰腺癌的诊断方法。方法58例胰腺癌高危病人、单纯十二指肠镜收集胰液后．分别行巴氏染色（Papanicolaou）作细胞学检查、半巢式PCR检测K-ras基因12密码子点突变及PCR—TRAP检测端粒酶活性。结果5例胰液中发现可疑恶性细胞（8．62％），K-las基因点突变阳性22例（37．9％），端粒酶活性弱阳性8例（13，8％），三者均阳性4例（6．89％），K—Fas基因点突变及端粒酶活性两者阳性7例（12．06％），1例细胞学阳性但K—ras基因点突变及端粒酶活性均阴性f1．72％）。结论内镜抽取纯胰液联合检测细胞学、K—ras基因点突变及端粒酶活性对临床诊断早期胰腺癌具有重要价值。     

Sporadic case of X-chromosomal Alport syndrome in a consanguineous family

Alport syndrome (AS) is a genetic disorder of basement membranes caused by mutations in type IV collagen genes that is characterized by chronic hematuria and progressive nephropathy leading to renal failure. The main extrarenal features include sensorineural hearing loss and ocular lesions. The mode of inheritance is X-linked dominant in about 80%–85% of the affected families, whereas autosomal transmission is rarely encountered. We report a male patient originating from a healthy consanguineous Lebanese family who presented with an unusual association of obstructive uropathy and AS. Hematuria and proteinuria were initially attributed to a suspected poststreptococcal glomerulonephritis (GN) and high-grade subpelvic ureteral stenosis. Persistence of symptoms after medical treatment of poststreptococcal GN and surgical correction of obstructive uropathy finally led to renal biopsy. The observed ultrastructural changes of the glomerular basement membrane were typical for AS.Molecular genetic studies revealed a previously undescribed de novo mutation in the COL4A5 gene, excluding maternal heterozygotic carrier status. This case report emphasizes the importance of hereditary nephritis in the differential diagnosis of chronic hematuria, and demonstrates the value of molecular studies for genetic counselling in AS. Received: 9 October 1998 / Revised: 15 February 1999 / Accepted: 24 August 1999     

MTS1基因β启动子E2F1结合位点序列突变重组质粒的构建与表达

目的：深入研究MTS1基因β启动子的转录激活与E2F1转录因子的相互作用关系，阐明该转录水平的调控机制。方法：用PCR定点突变方法或酶切连接法，构建β启动子0.38kb SacⅡ-Sac I酶切片段中E2F1 A，B，C任意2个位点或3个位点均突变的pGL3重组质粒。用脂质体介导的基因瞬时转染法，将构建的重组质粒转染MTS1基因双等位缺失的急性T淋巴细胞白血病Jurkat细胞，检测pGL3重组质粒中荧光素酶报告基因的表达。结果：构建的E2F1 A，B，C结合位点突变的重组质粒经Sac I或Nae I酶切鉴定和DNA序列分析得到证实。与E2F1位点野生型重组质粒比较，突变型重组质粒在Jurkat细胞中荧光素酶报告基因的表达量减少，以3个位点均突变的重组质粒为明显。结论：构建的E2F1 A，B，C2个或3个结合位点均突变重组质粒成功，可通过基因转染用于研究MTS1基因的功能试验中；MTS1基因β启动子的转录活性可能与E2F1转录因子的反式激活有关。