二甲亚砜与维甲酸对人卵巢癌细胞COC1的生长抑制及转化生长因子β_1表达的影响

本实验以人卵巢癌细胞株（ＣＯＣ１）为模型,观察诱导分化剂二甲亚砜（ＤＭＳＯ）与维甲酸（ＲＡ）对该细胞生长增殖、ＤＮＡ合成和转化生长因子β１（ＴＧＦβ１）在细胞内表达的影响。结果显示：ＤＭＳＯ与ＲＡ对人卵巢细胞ＣＯＣ１生长有明显的抑制作用,生长曲线表明作用５天后其生长抑制率分别为６２．７％和４２．１％;３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入实验说明ＤＭＳＯ组与ＲＡ组的单位时间计数率（ＣＰＭ）明显低于对照组（Ｐ＜０．０１）。用药３天后百分掺入抑制率分别为６０．４％与３７．９％,表明ＤＭＳＯ与ＲＡ抑制ＣＯＣ１细胞的ＤＮＡ合成;免疫细胞化学反应表明,ＤＭＳＯ或ＲＡ处理５天后,对照组细胞ＴＧＦβ１表达为阳性,定位于胞浆,而处理组细胞ＴＧＦβ１呈阴性或弱阳性反应。以上结果提示ＤＭＳＯ和ＲＡ对人卵巢癌细胞有一定的诱导分化作用。     

血管紧张素Ⅱ对大鼠心肌肌浆网Ca~(2+),Mg~(2+)-ATPase基因转录调节的影响

观察血管紧张素Ⅱ（ＡｎｇⅡ）对心肌肌浆网Ｃａ２＋,Ｍｇ２＋－ＡＴＰａｓｅ基因（ＳＥＲＣＡ２ａ）转录调节的影响,评价ＤＭＰ８１１对此效应的干预作用．６周龄雄性ＳＤ大鼠随机分为３组,每组６只．组１：生理盐水输注;组２：ＡｎｇⅡ输注＋ＤＭＰ８１１管饲（３ｍｇ·ｄ－１·ｋｇ－１）;组３：ＡｎｇⅡ输注（２００ｎｇ·ｍｉｎ－１·ｋｇ－１．１周后称其体重,取心脏并称重,提取心脏总ＲＮＡ后采用Ｎｏｒｔｈｅｒｎｂｌｏｔ的方法检测ＳＥＲ－ＣＡ２ａ的转录水平,采用ＲＴ－ＰＣＲ检测ＡｎｇⅡ１型受体（ＡＴ１）ｍＲＮＡ水平．实验后,组３心重（ＣＷ）、心重／体重（Ｃ／Ｂ）、ＡＴ１受体转录水平均高于组１（分别增加４．７±０．４％,４．９±０．９％和２４．７±３．５％;Ｐ＜０．０１）,而ＳＥＲＣＡ２ａ基因转录水平显著低于组１（降低２０．１±３．０％,Ｐ＜０．０１）,并且ＳＥＲＣＡ２ａｍＲＮＡ水平与ＡＴ１受体ｍＲＮＡ水平呈负相关（ｒ＝－０．７４,Ｐ＜０．０１）．ＡｎｇⅡ导致的上述改变能被ＤＭＰ８１１完全阻断．ＡｎｇⅡ通过其Ⅰ型受体的介导,诱导了ＳＥＲＣＡ２ａ的转录下调     

戊型肝炎病人血清抗—HEV IgG与IgM和HEV RNA的动态变化

利用酶联免疫试验（ＥＩＡ）及逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）,检测了２１０份急性非甲非乙非丙肝炎患者血清和４０例戊型肝炎（戊肝）病人系列血清。在急性非甲非乙非丙肝炎血清中,抗－ＨＥＶ ＩｇＧ、抗－ＨＥＶ ＩｇＭ和ＨＥＶ ＲＮＡ阳性率分别为６２．８６％、４５．２３％和４０．４８％。在戊肝系列血清检测中,抗－ＨＥＶ ＩｇＧ阳性率发病１个月内为９２．５％,发病２￣６个月１００％,１２个月９４．７％,     

传染性法氏囊病病毒中国强毒株A节段cDNA基因的克隆和序列分析

以来自哈尔滨传染性法氏囊病病毒（ＩＢＤＶ） 强毒株（Ｈａｒｂｉｎ 毒株,Ｈ） 的基因组ＲＮＡ为模板,用反转录聚合酶链反应（ＲＴ－ ＰＣＲ） 的方法得到了其Ａ 节段的全长ｃＤＮＡ 片段,分５＇端（１ ６５９ｂｐ） 和３＇端（１ ４４４ｂｐ） 上下两段分别克隆到ｐＧＥＭＢ○Ｒ － Ｔ 载体上,测定了其核苷酸顺序,在长为３ １０１ ｂｐ 中含有两个阅读框ＯＲＦＡ１ 和ＯＲＦＡ２ ,分别编码１ ０１２ 个氨基酸的前体蛋白（ＶＰ２ － ４ －３） 和１４５ 个氨基酸的ＶＰ５,ＯＲＦＡ１ 和ＯＲＦＡ２ 有部分的重叠。将核苷酸序列及推测出的氨基酸序列与已报道的ＩＢＤＶ 血清Ⅰ型和Ⅱ型毒株的相应序列进行了比较,结果表明：Ｈ 毒株与其它血清Ⅰ型毒株之间,在核苷酸水平上存在２５ｂｐ － ２６７ｂｐ 的差异;在氨基酸水平上存在１７ ～４０ 个氨基酸的差异。在ＶＰ２ － ４ － ３ 内比较显示,Ｈ 毒株与Ｐ２ 、Ｃｕ－ １ 之间氨基酸的差异最小为１ ．７％ ,Ｈ 毒株与ＵＫ６６１ 之间氨基酸的差异最大为３ ．９ ％ 。变异主要发生在ＶＰ２ 的可变区（２０６ － ３５０ 位氨基酸） ,在Ｈ 毒株所特有的１２ 个氨基酸当中,该区就占５ 个,代表１ ．７６ ％ 的变异。ＶＰ４、ＶＰ３ 和ＶＰ５区各有     

逆转录-聚合酶链反应技术诊断丙型肝炎病毒感染

本文采用逆转录－聚合酶链反应（ＲＴ-ＰＣＲ）技术对２１２例住院及门诊病人其中肝病患者９８例（慢性肝炎４３例、肝炎后肝硬化４７例、原发性肝细胞癌８例）进行ＨＣＶ－ＲＮＡ检测。结果９８例慢性肝病患者血清中ＨＣＶ-ＲＮＡ-ＰＣＲ阳性２７例（２７．６％）,１１４例非肝病患者血清中ＨＣＶ-ＲＮＡ-ＰＣＲ阳性９例（７．９％）,两组间差异非常显著（Ｐ（０．０１）,各种肝病患者的ＨＣＶ-ＲＮＡ-ＰＣＲ阳性率均高于非肝病组。６８例患者同时进行了ＨＣＶ-ＲＮＡ-ＰＣＲ检测和抗－ＨＣＶ检测,２５例抗－ＨＣＶ阳性的患者中ＨＣＶ－ＲＮＡ-ＰＣＲ,２１例阳性（８４％）,４３例抗－ＨＣＶ阴性的患者中ＨＣＶ-ＲＮＡ-ＰＣＲ,９例阳性（２０．１％）、有输血及血制品史者４８例,其中ＨＣＶ-ＲＮＡ-ＰＣＲ阳性１６例（３３．３％）,１６４倒无输血史者中ＨＣＶ-ＲＮＡ-ＰＣＲ阳性２０例（１２．２％）,两组间差异非常显著（Ｐ（０．０１）。结果表明：１．ＨＣＶ感染与慢性肝病有密切联系,说明ＨＣＶ感染是慢性肝炎、肝硬化、肝癌的致病因素;２．ＨＣＶ-ＰＣＲ法具有特异性好、灵敏度高、简便快速等特点,弥补了抗－ＨＣＶ检测的不足之处,是目前确定ＨＣＶ感染的主要手段;３．ＨＣＶ感染与输血关系密切,因此对献血员进行常规ＨＣＶ检测对预防由输血所致ＨＣＶ感染有着极其重要的临床意义。     

NA和5－HT对小脑脑片浦肯野细胞自发及诱发电活动的影响

在大鼠小脑脑片上观察了ＮＡ和５－ＨＴ对浦肯野细胞（ＰＣ）的自发放电活动及由白质刺激所引起的诱发放电活动的影响。结果表明：（１）ＮＡ使ＰＣ产生抑制、兴奋和双相反应,以抑制反应为主（７９．８％）;５－ＨＴ引起ＰＣ兴奋和抑制反应,以兴奋反应略多（５７．８％）。（２）先后灌流ＮＡ和５－ＨＴ对同一个ＰＣ自发放电的影响主要为抑制（ＮＡ）－兴奋（５－ＨＴ）（５３．８％）。（３）ＮＡ对ＰＣ的诱发复杂锋电位（ＣＳ）和简单锋电位（ＳＳ）反应,主要产生增强效应（５７．１％和６２．８％）;５－ＨＴ对ＰＣ诱发ＣＳ和ＳＳ反应则主要产生压抑作用（６０．０％和６８．２％）。（４）先后灌流ＮＡ和５－ＨＴ对同一个ＰＣ的诱发ＣＳ和ＳＳ反应,主要表现为ＮＡ对这两种诱发反应的增强和５－ＨＴ的压抑效应（６０．０％和５２．９％）。这些结果提示,ＮＡ能和５－ＨＴ能传入纤维可以通过释放ＮＡ和５－ＨＴ调节ＰＣ的兴奋性水平并改变ＰＣ对爬行纤维和苦状纤维突触传入的反应敏感性,影响小脑皮层神经元网络的感觉运动整合过程。     

亚洲棉GAE6—3A上游序列的分离及其在烟草中的表达

根据Ｅ６基因保守域设计引物,ＰＣＲ扩增出亚洲棉（Ｇａｓｓｙｐｉｕｍ ａｒｂｏｒｅｕｍ Ｌ．）ＧＡＥ６基因长约４００ｂｐ片段,序列分析表明该片段与海棉（Ｇ．ｂａｒｇｂａｄｅｎｓｅ）Ｅ６基因同源性达９６．８％。进一步合成２个反向引物协助进行ＰＣＲ－９６孔板筛库分离到亚洲板棉ＧＡＥ６－３Ａ克隆。酶切鉴定其插入片段长约８．０ｋｂ,序列测定及分析结果表明其上游和约１．５ｋｂ,将ＧＡＥ６－３Ａ上游序列克 含有     

核酶对bcl－2mRNA的体外切割

通过微机对ｂｃｌ－２ＲＮＡ二级结构的分析,设计针对ｂｃｌ－２片段５＇ＣＧＣＧＡＣＣＣＧＧＵＣＧＣＣＡＧＧＡＣＣＵＣＧ３＇的“锤头状”（Ｈａｍｍｅｒｈｅａｄ）核酶（Ｒｉｂｏｚｙｍｅ,ＲＤ）基因,平端连接于ｐＧＥＭ－３Ｚｆ（－）ＨｉｎｃⅡ位点,克隆后经测序表明序列正确,ｂｃｌ－２和Ｒｉｂｏｚｙｍｅ基因经体外转录,５０℃作用２ｈ,从１６５６－１６５７（Ｃ－Ｇ）位之间切断ｂｃｌ－２ＲＮＡ片段．     

显示基因表达差异的PAGE—银染技术

ｄｄＲＴ－ＰＣＲ（ｄｉｆｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙ　ｒｅｖｅｒｓｅｔｒａｎ－ｓｃｒｉｐｔｉｏｎ－ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）技术是Ｌｉａｎｇ和Ｐａｎｄｅｅ~（［１］）于１９９２年首先建立的一种在ＲＮＡ水平上显示基因表达差异,进而分离目的基因的有效方法。与其它分离基因的方法相比,ｄｄＲＴ－ＰＣＲ方法具有许多优点~（［２］）,目前已经成为筛选未知差异表达基因的最为有效的方法之一。ｄｄＲＴ－ＰＣＲ是３种技术的有机结合：（１）通过错定引物Ｔ１２ＭＮ（Ｍ＝Ａ、Ｔ、Ｃ、Ｇ,Ｎ＝Ａ、Ｃ…     

蝴蝶兰根段的组织培养

１　植物名称　蝴蝶兰（ＰｈａｌａｅｎｏｐｓｉｓＭｅｌｌｅｒＧｏｌｄ“ＮＦＳ”）。２　材料类别　根段。３　培养条件　（１）愈伤组织的诱导及分化培养基：Ｂ５＋ＮＡＡ１．５ｍｇ·Ｌ－１（单位下同）＋ＫＴ０．２＋ＣＭ１５０ｍｌ·Ｌ－１＋３％蔗糖;（２）原球茎增殖培养基：Ｂ５＋ＧＡ０．０５＋ＣＨ１２０＋３％蔗糖;（３）小苗生长培养基：１／２ＭＳ＋２０％香蕉泥＋２％蔗糖;（４）诱导生根培养基：１／２ＭＳ＋ＩＢＡ０．３＋２％蔗糖。上述培养基均加０．２％活性炭,０．５８％琼脂粉,ｐＨ为５．５;培养基在１２１℃高…     

The enhancement of PCR amplification by low molecular-weight sulfones.

DNA amplification by polymerase chain reaction (PCR) is frequently complicated by the problems of low yield and specificity, especially when the GC content of the target sequence is high. A common approach to the optimization of such reactions is the addition of small quantities of certain organic chemicals, such as dimethylsulfoxide (DMSO), betaine, polyethylene glycol and formamide, to the reaction mixture. Even in the presence of such additives, however, the amplification of GC-rich templates is often ineffective. In this paper, we introduce a novel class of PCR-enhancing compounds, the low molecular-weight sulfones, that are effective in the optimization of high GC template amplification. We describe here the results of an extensive structure-activity investigation in which we studied the effects of a series of six different sulfones on PCR amplification. We identify two sulfones, sulfolane and methyl sulfone, that are especially potent enhancers of high GC template amplification, and show that these compounds often outperform DMSO and betaine, two of the most effective PCR enhancers currently used. We conclude with a brief discussion of the role that the sulfone functional group may play in such enhancement.     

FMR-1基因中CGG重复序列扩增条件的优化

目的:优化PCR扩增条件,建立一种有效检测脆性X综合征的方法。方法:在常规PCR的基础上,采用耐高温酶替代法、碱基替代法,同时加入有机溶剂DMSO等,对表型正常的人群进行FMR1基因CGG重复序列检测。结果:改良PCR法可以提高G C富集区扩增效率,并取得了较好的效果。结论:建立了一种扩增FMR-1基因中CGG重复序列的可行方法。     

Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates

ABSTRACT: BACKGROUND: While being a standard powerful molecular biology technique, applications of the PCR to the amplification of high GC-rich DNA samples still present challenges which include limited yield and poor specificity of the reaction. Organic solvents, including DMSO and formamide, have been often employed as additives to increase the efficiency of amplification of high GC content (GC > 60%) DNA sequences. Bovine serum albumin (BSA) has been used as an additive in several applications, including restriction enzyme digestions as well as in PCR amplification of templates from environmental samples that contain potential inhibitors such as phenolic compounds. FINDINGS: Significant increase in PCR amplification yields of GC-rich DNA targets ranging in sizes from 0.4 kb to 7.1 kb were achieved by using BSA as a co-additive along with DMSO and formamide. Notably, enhancing effects of BSA occurs in the initial PCR cycles with BSA additions having no detrimental impact on PCR yield or specificity. When a PCR was set up such that the cycling parameters paused after every ten cycles to allow for supplementation of BSA, combining BSA and organic solvent produced significantly higher yields relative to conditions using the solvent alone. The co-enhancing effects of BSA in presence of organic solvents were also obtained in other PCR applications, including site-directed mutagenesis and overlap extension PCR. CONCLUSIONS: BSA significantly enhances PCR amplification yield when used in combination with organic solvents, DMSO or formamide. BSA enhancing effects were obtained in several PCR applications, with DNA templates of high GC content and spanning a broad size range. When added to the reaction buffer, promoting effects of BSA were seen in the first cycles of the PCR, regardless of the size of the DNA to amplify. The strategy outlined here provides a cost-effective alternative for increasing the efficiency of PCR amplification of GC-rich DNA targets over a broad size range.     

Novel sulfoxides facilitate GC-rich template amplification

Certain organic solvents, such as DMSO and betaine, have been reported to enhance PCR amplification, particularly for hard-to-amplify high-GC templates. As a result of extensive structure-activity studies between two groups of compounds--amides and sulfones--we have recently discovered several other potent PCR enhancers. Here we describe the effects of a series of different sulfoxides on GC-rich template amplification and report several of these to be exceptionally effective, often outperforming DMSO. We introduce them as novel PCR enhancers. We identify tetramethylene sulfoxide as the most potent sulfur-oxygen compound in the enhancement of PCR amplification and as one of the most potent PCR enhancers currently known.     

Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA of Terminalia arjuna

Owing to the presence of higher amount of polyphenolic and polysaccharide compounds, Terminalia arjuna (Roxburgh) is a significant medicinal plant in the Indian primeval medicine system. Polyphenolic and polysaccharide compounds also acts as inhibitors during Genomic DNA isolation from young leaves of T. arjuna, resulting in recovery of low quality genomic DNA, which affects downstream applications like PCR, restriction digestion’s, etc. In this study, nine different methods of genomic DNA isolation were used, out of which two methods were modified CTAB based methods, third one was HEPES based method and remaining six methods was FTA Plant Saver Card based. Out of the six FTA card based methods, in the first method, leaves were directly pressed inside the circle of FTA card. In the second and third methods, the leaves were homogenized with PBS and DNase RNase free water and the sample was applied on the FTA card. In the fourth and fifth methods: finally recovered DNA from two modified CTAB based methods was directly applied to the FTA card. In the sixth method, DNA precipitated after first phenol:chloroform:isoamyl alcohol precipitation of modified CTAB based methods dissolved in DNase RNase free water and applied to FTA Card. To optimize the PCR conditions, BSA (400 ng/μl), formamide (2.5%), DMSO (5% and 10%) and glycerol (5%, 10%, 15%, and 20%) was added into the PCR mix as enhancement agents for amplification of low quality genomic DNA (A₂₆₀/A₂₈₀ – 1.27 ± 0.090) of T. arjuna recovered using the HEPES Based method. It was found that the BSA was the best among them followed by 10% glycerol. In addition of BSA to the PCR mixture, it specifically enhances the amplification of the low quality DNA. It reduces the noise in-between the amplified bands and increases the intensity of PCR product.     

Enhancer aided in vitro permeation of atenolol and prazosin hydrochloride through mice skin

Effect of penetration enhancers were studied on the permeation of antihypertensive drugs prazosin hydrochloride and atenolol through full thickness skin of swiss albino mice. Atenolol was delivered to skin from saturated alcoholic solution containing 5% of 1-decanol and alcohol alone, while prazosin hydrochloride was saturated in dimethyl formamide(DMF, 5% v/v in water) and dimethyl sulfoxide(DMSO, 5% v/v in water). Atenolol permeation was augmented significantly in decanolic solution and also in pure alcohol. In case of prazosin hydrochloride, significant enhancement of permeation was shown by DMSO but not by DMF.     

酶抗体应用实现热启动PCR

通过制备酶抗体,形成抗原抗体复合物,使得酶在较高的温度95℃,1 min,得以释放发挥聚合活性,实现热启动PCR.建立的热启动PCR体系灵敏度高,可以检测到1个拷贝的DNA分子,较普通PCR体系提高了3个数量级,并且具有较好的线性关系.这种通过形成抗原抗体复合物达到热启动PCR目的的方法的建立,具有着重要的意义,是提高PCR灵敏度和特异性的重要方法之一.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Reevaluation of the exact CAG repeat length in hereditary cerebellar ataxias using highly denaturing conditions and long PCR

Hereditary cerebellar ataxias, including spinocerebellar ataxia type I (SCA1), dentato-rubro-pallidoluysian atrophy (DRPLA), and Machado-Joseph disease (MJD), have been associated with unstable CAG repeats. The length of the CAG repeat is a major factor in determining the age of onset of these diseases. In electrophoresis through acrylamide gels with formamide, the CAG repeat length following the polymerase chain reaction (PCR) coincides with the sequence-determined repeat length after subcloning. However, without formamide, PCR products with long CAG repeats appear 1–4 repeats shorter than when electrophoresed with formamide, and the repeat lengths are variable. In addition, the larger the CAG repeats are, the more difficult are the PCR reactions. A mixture containing thermostable Taq and Pwo DNA polymerases (so-called “long PCR”) is much more sensitive than that with Taq polymerase alone in detecting expanded CAG repeats. Therefore, highly denaturing conditions, especially formamide gel electrophoresis, and the “long PCR” protocol should be used to evaluate the exact CAG repeat length. We have used these principles to detect unstable CAG repeats. The normal ranges are 14–34 repeats for MJD, 6–31 repeats for DRPLA, and 21–32 repeats for SCA1. Received: 29 August 1995 / Revised: 12 October 1995