Affinity chromatography-dependent selection (ACDS)of genomic DNA fragments bound specifically to bacterial synthesized Myc/Myn proteins

This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vec-tor respectively Fusion GST-Myc and GST-Myn synthe-sized in E.coli hosts showed affinity to CACGTG E-boxDNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR. A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA. At least two genomic DNA fragments ob- tained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone. Significance of the work and of the technique itself as well as identification of the DNAs are discussed.

Detection and Analysis of an Estrous－associated Oviductal Glycoprotein DNA Fragment from Primates by PCR

DNA was extracted from lymphocytes of primates and PCR was used to amplify the estrous-associated glycoprotein DNA fragment. 537 kb DNA fragments from crab eating macaque were detected. Sequences from chimpanzee, orangutan and human were also amplified, but they were shorter than predicted. We were unable to amplify sequences from spider monkey, salmon, several rodents, bandicoot, sheep and pig . Analysis of the restriction map revealed several conserved RE sites in the amplified sequences of the primates. The results support the view that monkey has more close relationship with human than any other species at molecular level.

盐酸奥布卡因对人角膜内皮细胞影响作用的实验研究

目的:揭示眼科局部麻醉剂盐酸奥布卡因(oxybuprocaine hydrochloride,OBPC-HCl)对体外培养人角膜内皮(HCE)细胞的影响作用,为眼科临床安全用药提供实验依据。方法:用不同浓度OBPC-HCl处理体外培养的HCE细胞,在倒置显微镜下观察细胞的生长和形态变化,用吖啶橙/溴化乙锭(AO/EB)荧光双染色法检测质膜的通透性,用琼脂糖凝胶电泳法检测DNA的断片化,用透射电镜检测细胞的超微结构。结果:OBPC-HCl在62.5mg/L～4g/L的浓度范围内均能不同程度地引起HCE细胞出现细胞皱缩、胞内空泡化、质膜通透性增大、染色质凝缩、凋亡小体和DNA断片化等典型的细胞凋亡特征,并具有浓度和时间依赖性,临床使用浓度4g/LOBPC-HCl对HCE细胞的凋亡诱导作用最大,处理1h后HCE细胞的凋亡率已高达100%。结论:OBPC-HCl在62.5mg/L～4g/L的浓度范围内能显著诱导HCE细胞凋亡,在眼科临床应用中对HCE细胞的毒副作用极大。

噻吗心安对人角膜内皮细胞的影响作用研究

目的:揭示抗青光眼药物噻吗心安(timolol maleate)对体外培养人角膜内皮(HCE)细胞的影响作用,为其眼科临床安全用药提供实验依据。方法:用不同浓度噻吗心安处理体外培养的HCE细胞,在倒置显微镜下观察细胞的生长、增殖和形态变化,利用吖啶橙/溴化乙锭荧光双染色法检测质膜的通透性,利用琼脂糖凝胶电泳法检测DNA的断片化,利用透射电镜观察细胞的超微结构。结果:噻吗心安在0.15625～5g/L的浓度范围内均能不同程度地引起HCE细胞出现不同程度的生长缓慢、数量减少、胞质皱缩、胞内空泡化、变圆脱落、质膜通透性增大、染色质凝缩、DNA断片化和出现凋亡小体等典型的细胞凋亡特征,并具有浓度和时间依赖性。临床使用浓度2.5～5g/L的噻吗心安对HCE细胞的凋亡诱导作用最大,处理28h的凋亡率高达83.23%～96.71%。结论:噻吗心安在0.15625～5g/L的浓度范围内能显著诱导HCE细胞凋亡,临床使用浓度时对HCE细胞的毒性作用极大,在眼科临床中应谨慎使用。

调心方对Aβ和TNFα诱导的SK-N-SH细胞株毒性的保护作用

目的 :观察调心方对由TNFα加Aβ(2 5～ 35 )诱导的SK N SH (神经源性细胞株 )细胞毒性的保护作用。 方法 :用TNFα 5 0ng/ml和Aβ(2 5～ 35 ) 0 1μM处理SK N SH细胞造成细胞损伤模型。以MTT和DNAladder为指标。 结果 :TNFα和Aβ(2 5～ 35 )处理SK N SH细胞 2 4小时细胞生存率减少约 5 0 % ,而调心方含药血清能提高细胞模型的生存率 ,减少细胞DNA的降解。结论 :一定剂量的TNFα加小剂量的Aβ(2 5～ 35 )能造成接近体内病理生理机制的细胞损伤模型 ,调心方对TNFα加Aβ(2 5～35 )

CHARACTERIZATION OF PATHOGENIC FUNGI GENOMES USING PULSED FIELD GEL ELECTROPHORESIS

Pulsed field gel electrophoresis(PFGE) has been firstly introduced in characterization of the pathogenic fungi Penicillium marne f fei and Exophiala dermatitidis genomes.The numbers and sizes of their chromosomes have been detected.Polymorphism was identified on the smallest chromosome of E.dermatitidis.The result shows that PFGE for characterization of large molecular DNA pathogenic fungi is very suitable,it is more simple and more efficacy.The result also shows the diversity of pathogenic fungi is relative common even in rare occurred pathogenic fungi such as E.dermatitidis.

Cloning and characterization of a novel gene encoding a putative seven-span transmembrane protein localized in endoplasmic reticulum

Objective: To clone and analyze the structure of a novel gene, named EST 1 (endoplasmic reticulum localized seven span transmembrane protein 1) and to analyze the expression pattern and intracellular location of EST 1. Methods: The cDNA library was screened to isolate novel cDNA fragment. The structure of novel gene was analysed by computer software. Expression of EST 1 was analyzed by dot blot and Northern blotting. Intracellular localization was observed after EST 1 enhanced green fluorescence protein (EGFP) fusion gene was transfected into mammalian cells. Results: The full length cDNA of mouse EST 1 was 1 802 bp, with a 1 293 bp open reading frame encoding 431 amino acids. It was predicated that protein encoded by EST 1 contained a signal peptide sequence at the N terminus, seven putative transmembrane domains, and an ER retaining signal at the C terminus. EST 1 EGFP fusion protein showed an ER like intracellular distribution in mammalian cells. Expression pattern analysis showed that EST 1 is expressed in all tissues examined. Conclusion: EST 1 is encoding a putative seven span transmembrane protein localized in endoplasmic reticulum. EST 1 was expressed in all tissues examined, suggesting an essential function of EST 1 in cells.

Identification of TM9SF2 as a Candidate of the Cell Surface Marker Common to Breast Carcinoma Cells

OBJECTIVE We aimed identification of cell surface molecules, which might serve as diagnostic biomarkers or useful targets for therapies, in breast cancer. METHODS We developed unique DNA microarray coupled with spherical self-organizing map （sSOM） analysis to characterize cells and tissues by the cell surface markers. In the microarray 1,797 probes for human genes coding membrane bound proteins were spotted. With this microarray the gene expression profiles of eight breast carcinoma cell lines were compared to identify the genes that were commonly expressed in breast carcinomas but not in normal cells. RESULTS The gene expression profiles of sSOM from the eight breast carcinoma cell lines were successfully distinguished from that of normal breast tissue derived cells suggesting the presence of genes of interest, sSOMon the data extensively filtered revealed several candidate genes, of which expression was significant in carcinoma cells but low in normal cells. Finally, TM9SF2 was nominated through validations of PCR procedures together with CD24 and ErbB3, which are known breast carcinoma markers. TMgSF2 expression was further confirmed by immunological staining. Interestingly, TMgSF2 was found to be expressed in all the cell lines evaluated while CD24 and ErbB3 were not in all of the carcinoma cells, supporting their relationship in sSOM. Although physiological significance of TMgSF2 is unknown yet, siRNA treatment significantly inhibited the growth of MDA- MB-231 cells. CONCLUSION We propose TM9SF2 as a novel and useful diagnostic marker as well as a potential molecular target specific to breast carcinoma cells covering wide range of breast cancer.

贝特舒对人角膜上皮细胞凋亡诱导作用的实验研究

为了揭示常用青光眼药物贝特舒（betaxol01）对人角膜上皮（HCEP）细胞的毒性及其作用机理，使用不同质量浓度贝特舒对体外培养IqCEP细胞进行了处理，并利用倒置显微镜跟踪观察了细胞的生长和形态，用吖啶橙／溴化乙锭（AO／EB）荧光双染色法检测了质膜的通透性，用琼脂糖凝胶电泳法检测DNA的断片化，用透射电镜检测细胞的超微结构。发现在0．17500—2．80000g／L的质量浓度范围内，贝特舒对HCEP细胞具有显著的毒性。并随着质量浓度的提高和处理时间的延长而逐渐增大，处理24h即可使大部分细胞皱缩死亡；进一步的研究结果发现，质量浓度为0．17500—2．80000g／L的贝特舒能显著提高HCEP细胞的质膜通透性，并使其出现DNA断片化、染色质凝缩和形成凋亡小体等凋亡细胞的结构变化。可见，在贝特舒0．17500—2．80000g／L的质量浓度范围内对HCEP细胞具有显著的毒性，并具有质量浓度和时间依赖性，其毒性作用的发挥是通过诱导细胞凋亡实现的，在眼科I旌床应用中毒副作用极大。

Securinine induced apoptosis in human leukemia HL-60cells

目的：研究一叶秋碱能否诱导ＨＬ６０细胞凋亡．方法：用ＭＴＴ法检测一叶秋碱对细胞增殖影响；应用流式细胞仪检测凋亡细胞数；采用琼脂糖凝胶电泳法观测ＤＮＡ碎片；透射电镜观察凋亡的形态改变．结果：一叶秋碱５－８０ｍｇ·Ｌ－１能诱导ＨＬ６０细胞凋亡．电镜观察到典型的凋亡形态学改变，电泳呈现出阶梯状条带，流式细胞仪检测到凋亡率随剂量的增高而升高．ＭＴＴ法示一叶秋碱抑制ＨＬ６０细胞增殖，并且呈时间、剂量依赖性，药物作用１２ｈ的ＩＣ５０（９５％可信区间）分别为２７（１５－４７）ｍｇ·Ｌ－１．

利多卡因对人角膜上皮细胞的毒性作用及其机理研究

利用不同浓度利多卡因（ｌｉｄｏｃａｉｎｅ）处理体外培养的人角膜上皮（ＨＣＥＰ）细胞系细胞，利用光镜观察、ＭＴＴ、荧光染色、ＤＮＡ电泳、ＴＵＮＥＬ、流式细胞仪和透射电镜方法研究了利多卡因对 ＨＣＥＰ细胞的毒性作用及其机理。光镜观察和 ＭＴＴ检测结果显示，质量浓度 １２５～１０００ｇ／Ｌ的利多卡因对 ＨＣＥＰ细胞具有显著的毒性作用，并具有浓度和时间依赖性；ＡＯ／ＥＢ荧光双染色结果显示，质量浓度 ０６２５～１００００ｇ／Ｌ的利多卡因可引起 ＨＣＥＰ细胞的质膜通透性显著提高，细胞凋亡率也具有浓度和时间依赖性；ＤＮＡ电泳和 ＴＵＮＥＬ检测结果显示，利多卡因能引起 ＨＣＥＰ细胞发生 ＤＮＡ断片化；ＴＥＭ观察结果显示，利多卡因能引起 ＨＣＥＰ细胞的超微结构出现了凋亡细胞的形态结构特征，如胞质空泡化、染色质浓缩、线粒体膨胀且嵴的结构紊乱、出现凋亡小体等；ＡｎｎｅｘｉｎＶ／ＰＩ染色的流式细胞仪检测结果显示，利多卡因能引起 ＨＣＥＰ细胞质膜中的磷脂酰丝氨酸（ＰＳ）发生外翻变化；ＥＬＩＳＡ检测结果显示，利多卡因还能引起 ＨＣＥＰ细胞中胱冬肽酶3、8、9、10表达量的增加，表明利多卡因确能引起 ＨＣＥＰ细胞发生细胞凋亡，而不是细胞坏死。由此可见，利多卡因在质量浓度大于 0.625ｇ／Ｌ时对 ＨＣＥＰ细胞具有显著的细胞毒性，并具有浓度和时间依赖性，且其毒性作用的发挥是通过诱导细胞凋亡实现的，在眼科临床应用中具有很大的毒副作用，应谨慎使用。