Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was ...Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.展开更多
Here we report a family with a clinical spectrum of Pachyonychia Congenita Tarda (PCT) encompassing two generations via a balanced chromosomal translocation between 4q26 and 12p12.3. We discuss the effects of chromoso...Here we report a family with a clinical spectrum of Pachyonychia Congenita Tarda (PCT) encompassing two generations via a balanced chromosomal translocation between 4q26 and 12p12.3. We discuss the effects of chromosomal translocations on gene expression through involved breakpoints and structural gene abnormalities detected by array CGH. We believe that the family we present gives further insight to the better understanding of molecular and structural basis of keratin disorders, and to the late onset and genetic basis of PCT through the possible role of C-type lectins and human epithelial membrane protein1 (EMP1). Better understanding of the molecular basis of keratin disorders is the foundation for improved diagnosis, genetic counseling and novel therapeutic approaches to overcome the current treatment limitations related to this disease.展开更多
Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C...Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.展开更多
背景与目的EB病毒(Epstein-Barr virus,EBV)与多种肿瘤包括非霍奇金淋巴瘤(non-Hodgkin'slymphoma,NHL)的发生有着密切关系,其中潜伏膜蛋白1(latent membrane protein 1,LMP1)基因被认为是EBV的致癌基因。近年的研究发现,LMP1基因...背景与目的EB病毒(Epstein-Barr virus,EBV)与多种肿瘤包括非霍奇金淋巴瘤(non-Hodgkin'slymphoma,NHL)的发生有着密切关系,其中潜伏膜蛋白1(latent membrane protein 1,LMP1)基因被认为是EBV的致癌基因。近年的研究发现,LMP1基因的多态性尤其C末端30bp的缺失(del-LMP1)与其致瘤性密切相关,可能在肿瘤的发生发展中起着重要作用。本研究观察del-LMP1基因在非霍奇金淋巴瘤患者和健康人中的表达情况,以探讨缺失型LMP1基因是否与NHL的发生及临床预后相关。方法应用特异性引物,采用聚合酶链反应扩增NHL和健康人中LMP1基因片段(产物包括30bp缺失部分),根据扩增产物的大小判断其是否存在30bp的缺失,并随机抽取PCR产物进行测序。观察缺失型LMP1基因在两者之间的构成比,并分析缺失型LMP1基因与NHL临床预后的关系。结果(1)48例NHL中有23例LMP1基因片段扩增阳性,其中携带缺失型者18例(78%),原型5例(22%);60例健康人有32例LMP1基因片段扩增阳性,其中携带缺失型者13例(41%),原型19例(59%);NHL患者和健康人中30bp缺失型LMP1基因的表达有显著性差异(P<0.05)。(2)LMP1基因片断扩增阳性的NHL中,IPI≥3者为15例,其中缺失型14例(93%),原型1例(7%);IPI<3者为8例,其中缺失型4例(50%),原型4例(50%)。展开更多
文摘Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.
文摘Here we report a family with a clinical spectrum of Pachyonychia Congenita Tarda (PCT) encompassing two generations via a balanced chromosomal translocation between 4q26 and 12p12.3. We discuss the effects of chromosomal translocations on gene expression through involved breakpoints and structural gene abnormalities detected by array CGH. We believe that the family we present gives further insight to the better understanding of molecular and structural basis of keratin disorders, and to the late onset and genetic basis of PCT through the possible role of C-type lectins and human epithelial membrane protein1 (EMP1). Better understanding of the molecular basis of keratin disorders is the foundation for improved diagnosis, genetic counseling and novel therapeutic approaches to overcome the current treatment limitations related to this disease.
基金Supported by the National Natural Science Foundation of China(Nos.30700827 and 30871301)Jilin Provincial Science & Technology Department of China(Nos.20070719 and 20080731)Northeast Normal University,China(Nos.20070401,NENU-STC07005)
文摘Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.
文摘目的:探讨上皮膜蛋白1(epithelial membrane protejn-1,EMP1)在人结直肠癌(colorectal carcinoma,CRC)组织中的表达水平及其过表达对CRC SW-480细胞增殖、凋亡和侵袭的影响。方法:免疫组织化学、Western blotting方法检测63例CRC组织及31例癌旁组织中EMP1的表达水平,分析EMP1表达与CRC临床病理参数的关系。慢病毒介导将pLenti6-EMP1质粒转染SW-480细胞,建立EMP1过表达细胞,转染plenti6/V5-DEST空白质粒为对照。Real-time PCR及Western blotting检测转染后SW-480细胞株中EMP1的表达,MTT、流式细胞术及Transwell实验分别检测EMP1过表达对SW-480细胞增殖、凋亡及侵袭能力的影响。结果:EMP1蛋白在人CRC组织的表达显著低于癌旁组织(0.257±0.022 vs 0.863±0.086,P<0.05),并且其表达水平在不同T分期、有无淋巴结转移、临床分期以及组织分级组间表达有显著差异(P<0.05),而与患者年龄、性别、肿瘤部位无关(P>0.05)。成功构建EMP1过表达的LeEMP1细胞。与LeEmpty细胞相比,LeEMP1细胞的增殖能力显著下降[(60.94±4.04)%vs(100.00±0.00)%,P<0.05],凋亡率显著升高[(12.10±1.30)%vs(3.10±0.60)%,P<0.05]、侵袭转移能力显著降低[穿膜细胞数:(87.00±12.00)vs(178.00±21.00)个,P<0.05]。LeEMP1细胞Caspase-9表达显著高于LeEmpty细胞(0.764±0.073 vs 0.231±0.029,P<0.05),VEGFC表达显著降低(0.185±0.022 vs 0.663±0.065,P<0.05)。结论:人CRC组织中EMP1蛋白表达明显减低,过表达EMP1能够抑制CRC SW-480细胞的恶性生物学行为,其可能通过调控Caspase-9和VEGFC的表达来发挥作用。
文摘背景与目的EB病毒(Epstein-Barr virus,EBV)与多种肿瘤包括非霍奇金淋巴瘤(non-Hodgkin'slymphoma,NHL)的发生有着密切关系,其中潜伏膜蛋白1(latent membrane protein 1,LMP1)基因被认为是EBV的致癌基因。近年的研究发现,LMP1基因的多态性尤其C末端30bp的缺失(del-LMP1)与其致瘤性密切相关,可能在肿瘤的发生发展中起着重要作用。本研究观察del-LMP1基因在非霍奇金淋巴瘤患者和健康人中的表达情况,以探讨缺失型LMP1基因是否与NHL的发生及临床预后相关。方法应用特异性引物,采用聚合酶链反应扩增NHL和健康人中LMP1基因片段(产物包括30bp缺失部分),根据扩增产物的大小判断其是否存在30bp的缺失,并随机抽取PCR产物进行测序。观察缺失型LMP1基因在两者之间的构成比,并分析缺失型LMP1基因与NHL临床预后的关系。结果(1)48例NHL中有23例LMP1基因片段扩增阳性,其中携带缺失型者18例(78%),原型5例(22%);60例健康人有32例LMP1基因片段扩增阳性,其中携带缺失型者13例(41%),原型19例(59%);NHL患者和健康人中30bp缺失型LMP1基因的表达有显著性差异(P<0.05)。(2)LMP1基因片断扩增阳性的NHL中,IPI≥3者为15例,其中缺失型14例(93%),原型1例(7%);IPI<3者为8例,其中缺失型4例(50%),原型4例(50%)。
