Autosomal dominant coralliform cataract related to a missense mutation of the γD-crystallin gene

Background Congenital cataract is a sight-threatening disease that affects about 1-6 cases per 10000 live births and causes 10%-30% of all blindness in children About 25% of all cases are due to genetic defects We identified autosomal dominant congenital coralliform cataracts-related genetic defect in a four-generation Chinese family Methods Complete ophthalmological examinations were performed prior to lens extraction Lens samples were then studied by electron microscopy Genomic DNA from family members were examined using whole-genomic linkage analysis, with two-point logarithm of odds (LOD) scores calculated using the Linkage program package (version 5 1) Mutation analysis of candidate genes was performed by direct sequencing Finally, a three-dimensional protein model was predicted using Swiss-Model (version 2 0) Results Eleven of the 23 examined individuals had congenital cataracts Ultrastructure studies revealed crystal deposits in the lens, and granules extensively dispersed in transformed lens fiber cells The maximum two-point LOD score, 3 5 at θ=0 1, was obtained for the marker D2S325 Mutation analysis of the γ-crystallin (CRYG) gene cluster identified a mutation (P23T) in exon 2 of γD-crystallin (CRYGD) Protein structure modeling demonstrated that the P23T mutation caused a subtle change on the surface of the γD protein Conclusions The results suggest that the coralliform cataract phenotype is due to a mutated CRYGD gene, and that this sequence change is identical to one reported by Santhiya to be related to another distinct clinical condition, lamellar cataract This study provides evidence that this same genetic defect may be associated with a different phenotype This is the first report identifying the genetic defect associated with an autosomal dominant congenital coralliform cataract

αB-crystallin基因在食管癌组织中的表达及其意义

目的探讨αB-crystallin基因在食管癌组织中的表达及其与食管癌临床病理的关系。方法采用RT-PCR方法检测食管癌组织及其正常组织黏膜中αB-crystallin基因的表达。结果αB-crystallin基因在食管癌组织中表达阳性率明显高于正常组织黏膜,深层浸润者的表达阳性率明显高于浅层浸润者,较大肿瘤者中的表达阳性率明显高于较小肿瘤者,有淋巴结转移者中的表达阳性率明显高于无淋巴结转移者(P均<0.05);αB-crystallin基因表达与患者年龄及肿瘤的分化程度无关(P>0.05)。结论αB-crystallin基因的高表达可能与食管癌的癌变及侵袭转移的发生有关。

Recombination and identification of human alpha B-crystallin

AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αBcrystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli(E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.RESULTS: Compared with the gene bank(GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin issuccessfully constructed, and the recombinant human αBcrystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.

Constitutively Expressed αB—Crystallin in Heat Schock Transcription Factor 1 Knockout Mice Myocardium

Objective:To invesligate the effects of heat shock transcription factor 1(HSF 1) gene on the constitutively expressed aB-Crystallin(αBC)in mice myocardium.Methods:The expression levels of constitutive αBC in HSF1 knockout(hsf1-/-) and HSF1 wild type (hsf1+/+) mice myocardium were evaluated by western blot and immunohistochemistry.Results:The αBC levels in hsf1-/- and hsf1+/_ were 68.42±4.16,100.00±7.58,respectively(P<0.05,cytosolic fraction),and 20.53±1.01,37.55±1.91,respectively(P<0.05,pellet fraction).The αBC signals decreased significantly in hsf1-/- myocardium when compared with those in hsf1+/+ myocardium stained with fluorescence immunohistochemistry.Conclusion.HSF1 is an important,but not the only factor,which mediates the constitutively expressed αBC.

Effects of Agrobacterial<i>rol</i>-Genes on the Thermodynamic and Structural Features of Starches Extracted from Potato Microtubers

Wild-type potato (Solanum tuberosum L.) plants and their transformants harboring agrobacterial rolB or rolC genes under control of the patatin class I promoter were cultured in vitro. These plants were used as a source of single-node stem cuttings. The structure of native starch in tubers formed on cuttings was determined using methods of X-ray scattering and differential scanning microcalorimetry (DSC). It was found that in starch from tubers of rolB plants the melting temperature of crystalline lamella was lower and their thickness was less than that in wild-type potato. In tubers of rolC plants starch differed from starch in wild-type plants by a higher melting temperature, reduced melting enthalpy, and a greater thickness of crystalline lamellae. The melting of starch from tubers of rolC plants proceeded as the melting of two independent crystalline structures with melting temperatures of 338.0°K and 342.8°K. Overall data show that starches of different structure can be obtained by using transgenic approach.

Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells

Objective To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared with that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases (P<0.01) and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in p16 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

先天性白内障晶状体蛋白致病基因及其功能研究进展

先天性白内障病因复杂,遗传性因素占22.3%。晶状体蛋白基因占突变基因的50.0%,包括α-晶状体蛋白、β-晶状体蛋白、γ-晶状体蛋白,其突变的致病机制主要是通过改变蛋白的二级或三级结构干扰蛋白质正确折叠方式从而影响蛋白的溶解度和稳定性,并形成细胞毒性的聚集小体;干扰蛋白与蛋白之间的相互作用并诱导细胞凋亡;消除三磷酸腺苷拮抗聚集效应等,目前针对致病机制的初步药物研究有羊毛甾醇等,可为治疗白内障提供新思路。因此,本文就晶状体蛋白基因的突变及其功能研究进行综述。

βB2晶体蛋白基因敲除小鼠模型的建立

目的:建立βB2晶体蛋白基因敲除小鼠模型。方法:采用美国iGTL实验室的技术构建打靶载体,建立基因敲除小鼠模型。采用PCR对小鼠基因型进行鉴定,Western印迹法对晶体蛋白的表达进行鉴定。结果:PCR成功检测出3种基因型;纯合子基因敲除的小鼠未检测到βB2晶体蛋白的表达。结论:成功构建了βB2晶体蛋白基因敲除的小鼠模型。

一个后极性白内障家系致病基因的筛查与鉴定

目的对中国一个具有常染色体显性遗传特点的后极性白内障家系进行致病基因的筛查。方法分别采集家系成员外周静脉血,提取基因组DNA,根据临床表型选取6个候选基因(CRYAA、CRYAB、PITX3、CHMP4B、MIP、CRYGD)设计引物,通过PCR扩增DNA片段,琼脂糖凝胶电泳分离DNA片段,直接测序法寻找致病基因及突变位点。结果该家系符合常染色体显性遗传家系特征,先证者表型为后极性白内障,通过候选基因外显子直接测序,发现家系内患者CRYGD基因第2个外显子第127位碱基有1个T→C的点突变,此突变导致蛋白第43位的色氨酸被精氨酸取代(W43R)。结论 CRYGD基因c.T127C(p.W43R)突变是该后极性白内障家系的致病原因。

年龄相关性白内障晶体上皮细胞的基因表达谱变化的初步分析

目的:探讨年龄相关性白内障与正常透明晶状体前囊上皮细胞基因表达差异,为年龄相关性白内障发病机制的研究及治疗提供理论依据。方法:提取年龄相关性白内障病人晶状体和正常透明晶状体前囊中央上皮细胞RNA,分别用cy3、cy5标记,再与含8064个基因点的芯片杂交,筛选两个样本间表达差异的基因,并对这些基因进行初步功能分析。按不同功能类型分别选择9个存在显著表达差异的基因,应用实时荧光定量PCR技术验证基因芯片可靠性。结果:年龄相关性白内障前囊中央上皮细胞中有724个差异表达基因,包括438个表达下调基因和286个表达上调基因。进行初步功能分类分析发现这些表达差异基因主要涉及晶状体结构蛋白基因、细胞骨架蛋白基因、细胞增殖及细胞凋亡以及细胞应激反应等方面。结论:年龄相关性白内障与透明晶状体前囊上皮细胞存在基因表达差异,其中多数基因呈表达下调。表达下调基因功能涉及的生物学过程导致晶状体维持内环境稳定的能力下降,形成白内障。

晶状体蛋白与先天性白内障

先天性白内障是全世界约1/10盲童失明的原因,绝大多数先天性白内障为单基因遗传病,其遗传方式包括常染色体显性遗传、常染色体隐性遗传和X连锁遗传3种。迄今为止在人类及其他动物定位的遗传性先天性白内障的致病基因主要包括编码晶状体结构蛋白、缝隙连接蛋白、膜蛋白、晶状体发育中的调节蛋白的基因。晶状体蛋白是晶状体中最主要的成分,其编码基因的突变与先天性白内障的发生密切相关。了解遗传性先天性白内障的发病机制有助于理解环境及营养因素在晶状体混浊中的作用。就晶状体蛋白的功能、晶状体蛋白基因突变及其导致的先天性白内障表型进行综述。

慢病毒介导的αB-晶状体蛋白基因沉默对胃癌细胞SGC-7901增殖和迁移能力的影响

目的应用慢病毒转染技术研究αB-晶状体蛋白(CRYAB)基因沉默对人胃癌SGC-7901细胞增殖和迁移能力的影响。方法构建CRYAB慢病毒载体转染人胃癌细胞株SGC-7901,获得稳定转染的细胞株,制备空载体作为阴性对照组(sh-ctrl组),实验组为CRYAB病毒转染组(sh-CRYAB组)。提取总RNA和蛋白,应用荧光定量PCR和蛋白免疫印迹法检测CRYAB的表达,应用CCK-8、Transwell和细胞划痕实验分别检测CRYAB基因沉默后细胞增殖和迁移能力的变化。结果慢病毒转染SGC-7901细胞后,CRYAB mRNA及蛋白水平显著降低(P<0.01,P<0.001),表明成功构建了稳定转染的CRYAB-shRNA胃癌细胞株。与sh-ctrl组比较,sh-CRYAB组细胞的增殖能力在第4天、第5天显著降低(P<0.05,P<0.01),sh-CRYAB组细胞迁移率显著降低(P<0.01)。结论 CRYAB表达沉默可降低胃癌细胞的增殖和迁移能力,CRYAB有望成为胃癌基因治疗的新靶点。

晶状体蛋白基因在一先天性白内障家系中的突变筛查

目的研究晶状体蛋白基因与先天性白内障的关系。方法收集1个先天性白内障家系,制备外周血白细胞基因组DNA。除对CRYGD基因直接测序外,在距离已知晶状体蛋白基因5个厘摩范围内选取微卫星标记进行连锁分析,计算微卫星位点与致病基因之间的最大优势对数值(LOD SCORE),来确定晶状体蛋白基因与此家系致病基因的关系。结果当重组分数分别为0.1、0.2、0.3、0.4时,所选取的位于晶状体蛋白基因附近的14个微卫星位点与该家系致病基因之间连锁的LOD值均为负值,故排除连锁。CRYGD基因测序后在基因编码区及启动子、内含子与外显子连接处的剪切位点均未见任何碱基改变。结论晶状体蛋白基因非此家系的致病基因,为进一步定位与克隆该家系的致病基因,需进行全基因组扫描,以探求先天性白内障的分子发病机制。

珊瑚状先天性白内障一家系致病基因筛查与鉴定

目的:对一个珊瑚状先天性白内障家系进行致病基因的筛查。方法:采集家系中2例患者和1例正常对照者的外周静脉血,提取基因组DNA。选择与珊瑚状白内障相关的候选基因GJA3、GJA8、CRYGC及CRYGD设计引物,进行聚合酶链反应(PCR)扩增候选基因,并对扩增片段进行Sanger测序。结果:该家系疾病表型为珊瑚状白内障,呈常染色体显性遗传。通过对扩增产物测序,发现家系内患者CRYGD第2个外显子第70位有1个C>A碱基的杂合突变(c.70C>A),正常对照未见该点突变。结论:CRYGD基因的错义突变c.70C>A是该珊瑚状白内障家系的致病原因。

亚硒酸钠对大鼠晶体上皮细胞αA晶体蛋白基因转录的影响

在体外观察了亚硒酸钠（Ｎａ＿２ＳｅＯ＿３）作用于大鼠晶体上皮细胞（ＲＬＥｃｅｌｌｓ）而引起其晶体蛋白基因转录的改变，对不同浓度的硒在体外对αＡ晶体蛋白基因转录的影响作了初步的研究。结果发现，随着亚硒酸钠浓度的升高，αＡ基因的转录下降；而当亚硒酸钠浓度升至５×１０（－５）ｍｏ１／Ｌ时，αＡ基因的转录又呈反跳性回升。提示硒在致障过程中对晶体蛋白基因转录的影响作用不可忽视；同时αＡ晶体蛋白在晶体细胞内，至少应答于高浓度的硒，可能作为一种应激蛋白表达。

遗传性全白甲一家系的CRYGA、CRYGB、CRYGC和CRYGD基因突变分析

目的：分析常染色体显性遗传全白甲病家系的候选基因γ晶状体球蛋白基因（CRYGA、CRYGB、CRYGC和CRYGD）的突变与本病的相关性。方法：对以上4个基因的全部外显子区域及邻近内含子区域进行PCR扩增，其产物进行直接测序，根据测序结果分析此四个基因突变。结果：在CRYGA、CRYGB、CRYGC和CRYGD基因外显子区域及邻近内含子区域内检测到5个多态性位点，未检测到致病的基因突变。结论：CRYGA、CRYGB、CRYGC和CRYGD基因编码区域的变异不是引起此全白甲家系的致病基因突变。

关注先天性白内障的疾病相关基因研究

先天性白内障是儿童期视力损害的首要病因之一.随着分子生物学技术的发展,对遗传性先天性白内障相关基因的研究从致病基因定位及突变的筛查,逐渐发展到致病基因突变的机制探索.围绕先天性白内障相关基因研究的主题,本文着重对晶状体蛋白、缝隙连接蛋白、主要内源性蛋白基因等常见候选基因对白内障影响的机制进行归纳,并对先天性白内障相关基因在眼球发育过程、年龄相关性白内障、表观遗传学等其他领域的拓展研究进行阐述与展望.

硫化镍对16HBE细胞中基因的影响

目的 检测结晶型硫化镍 (NiS)对K -ras基因和P15基因的改变及基因组不稳定性的影响 ,从而进一步探讨镍化合物致癌的分子机制。方法 采用限制性片段长度多态性分析和聚合酶链反应 -单链构象多态性分析方法探查结晶型NiS在诱导 16人气管上皮细胞 (HBE)恶变过程中的K -ras基因Exonl第 12密码子及第 12密码子以外的密码子改变情况。采用PCR -SSCP分析方法探查结晶型NiS在诱导 16HBE细胞恶变过程中的P15基因Exon2存在状况。采用随机扩增多态性技术来对结晶型NiS在诱导 16HBE细胞恶变过程中的基因组不稳定性进行分析。结果 K -ras基因Exon1和P15基因Exon2未发生改变。本实验所选用的 7条随机引物均能扩增出清晰、明显的条带。其中P4、P5、P7两条引物扩增的片段在实验组和对照组之间无差异 ,其余 4条引物均有差异。对于同一随机引物他们都具有特异的带型。结论 P15基因第 2外显子和K -ras基因第 1外显子 (包括第 12、13密码子 )可能不是结晶型NiS作用的靶部位。在结晶型NiS诱发细胞恶性转化过程中 ,基因组变得逐渐不稳定。

α晶状体蛋白的研究进展

α晶状体蛋白包括αA和αB晶状体蛋白,属于小分子热休克蛋白家族成员。α晶状体蛋白在晶状体中为主要的屈光介质,同时具有分子伴娘的功能。αA晶状体蛋白主要存在于晶状体,在脾和胸腺有微量表达。αB晶状体蛋白在许多器官、组织、细胞均有结构性表达,在抵御内、外环境应激发挥重要作用。本文对α晶状体蛋白的分子量及结构,在晶状体内、外中的作用,与神经系统疾病的关系及α晶状体蛋白基因敲除研究等方面作一综述。