晶状体生化的研究 平阳霉素诱发大鼠白内障的研究

 本文报道间日经皮下注射平阳霉素(Pingyangmycin)(25μg/10g体重)于出生后四日之乳鼠可诱发白内障,其诱发率为84.6％。患白内障时晶状体中可溶性蛋白质含量明显降低。经Sephadex G-200柱层析可见βH,γ晶体蛋白降低,α晶体蛋白则相对升高。经10％聚丙烯酰胺凝胶电泳及等电聚焦电泳可见γ晶体蛋白改变明显,这些结果和我们用半乳糖及亚硒酸钠诱发白内障所得结果一致。     

用蛋白质印迹转移技术分析正常及硒性白内障大鼠晶状体及房水中蛋白质的性质

本文用蛋白质印迹转移技术分析了正常及硒性白内障大鼠晶状体及房水中蛋白质的性质。结果表明,晶状体中的脲溶性蛋白质可被抗α及抗γ晶体蛋白血清识别,提示α及γ晶体蛋白均为脲溶性蛋白质的主要成份。患白内障时房水中的蛋白质含量明显增加,且主要被抗γ血清识别,而被抗α血清识别的成份很少,表明在大鼠硒性白内障形成过程中,有较多低分子量蛋白质漏出到房水中,且其主要成份为γ晶体蛋白。此外,我们还发现正常及硒性白内障大鼠晶状体膜蛋白质与抗α及抗γ血清起反应的程度及分布有所不同,提示晶状体细胞膜与晶体蛋白之间存在着相互作用。     

晶状体生化的研究——Na_2~(75)SeO_2诱发大鼠白内障的同位素示踪研究

我们用Na~(75)_2SeO_3皮下注射诱发大鼠产生白内障后,观察~(75)Se在房水及晶状体中的分布。发现注射后4小时内,~(75)Se迅速进入眼内,产生白内障的大鼠晶状体中~(75)Se较多,而注射后未产生白内障的大鼠透明晶状体中~(75)Se进入很少。经三氯醋酸处理后,~(75)Se主要在沉淀部分,经巯基乙醇处理后,~(75)Se转移到上清液中,经Se-phadex G-200柱层析可见~(75)Se与α、β_H、β_L、γ晶体蛋白都结合。这提示~(75)Se可能是与晶体蛋白质中的半胱氨酸残基的巯基相连,从而使蛋白质交联起来,导致晶状体中高分子量蛋白质的形成,引起光线的散射,而表现为白内障。     

正常与患白内障大鼠晶状体中脲溶性蛋白质性质的研究

本文研究了正常及三种类型白内障大鼠晶状体中脲溶性蛋白质的含量及性质的变化,发现在每种类型白内障晶状体中,水溶性蛋白质均减少,水不溶性蛋白质则都相对增加。经SephadexG-200柱层析及SDS聚丙烯酰胺凝胶电泳发现,晶状体中脲溶性蛋白质主要是由二硫键交联而成的高分子聚合物。经巯基乙醇还原后,绝大部分高分子聚合物可分解成低分子量蛋白质,其分子量与水溶性的γ晶体蛋白相同。这提示晶状体中脲溶性蛋白质的主要成分很可能是以二硫键交联而成的γ晶体蛋白聚合物。此结果与本实验室所得白内障晶状体水溶性蛋白质的变化相吻合。     

大鼠βB2—晶体蛋白的克隆,高效表达及纯化

β－晶体蛋白是含量最高的一组晶状体结构蛋白。它的正常结构对维持晶状体的高折射系数和透明至关重要,而其结构的改变与白内障的形成密切相关。为了获得大量纯的βＢ２－晶体蛋白并用定点突变的方法研究其聚合的机制,建立了大鼠βＢ２－晶体蛋白的细菌表达系统及纯化方法。用ＲＴ－ＰＣＲ的方法克隆了β－晶体蛋白中含量最高的βＢ２－晶体蛋白的ｃＤＮＡ。用苯啶酮酸诱导ｒｅｃＡ启动子后,蛋白质在大肠杆菌中得到了高效表达。β     

对人眼晶状体α－晶体蛋白聚集体的准弹性激光散射研究

以准弹性激光散射技术,研究了人眼晶状体内α－晶体蛋白聚集体的扩散系数和流体力学半径,以及其弥散性随温度和浓度的变化。所研究的α－晶体蛋白用分离的方法分别取自成人和胚胎眼晶状体。研究结果表明,人眼α－晶体蛋白在一定浓度和温度下可形成聚集体,且其聚集体半径随α－晶体蛋白的浓度近乎线性地增大,随温度的增加而变小。对α－晶体蛋白溶液（５０ｍｍｏｌ／Ｌ磷酸脂缓冲液）的弥散度分析表明,溶液中有两种不同粒径的散射体,除开α－晶体蛋白聚集体外,还有一种粒径约为１８．５ｎｍ的估计是α－晶体蛋白单体分子。由于α－晶体蛋白分子的变性聚集在人眼白内障的形成起着重要作用,故本研究的结果对于人眼白内障随年龄及温度变化的发展过程及其机理的认识具有指导意义。     

晶状体生化的研究——亚硒酸钠诱发大鼠白内障晶状体中巯基、二硫键及维生素C的含量

我们测定了正常及亚硒酸钠诱发的白内障大鼠晶状体中非蛋白质巯基、蛋白质巯基、蛋白质结合巯基和维生素C的含量,发现随着白内障的进展非蛋白质巯基及蛋白质巯基均减少,蛋白质结合巯基在核混浊时增加,而在整个晶状体混浊时下降到与正常对照组相近,在白内障形成过程中二硫交联的蛋白质含量明显增加,而维生素C含量似乎无明显变化。     

先天性与后天性大鼠白内障晶体中游离氨基酸含量变化的研究

用药和的诱发大鼠先天性白内障动物模型,检测了晶体中１８种游离氨基酸含量,并与大鼠硒性和半乳糖性白内障晶体中ＦＦＡ含量进行了比较,发现：仔一代和仔二代先天性白内障晶体中各ＦＡＡ含量接近,先天性白内障晶体内,酪氨酸和羟脯氨酸含量明显高于正常对照组（Ｐ＞０．０５）;谷氨酸、甘氨酸等９种ＦＡＡ明显低于硒性白内障（Ｐ＜０．０５）;而酪氨酸高于半乳糖性白内障组,半胱氨酸等５种ＦＡＡ含量均低于半乳糖性白内障组。     

先天性与后天性大鼠白内障晶体中游离氨基酸含量变化的研究

用药物诱发大鼠先天性白内障动物模型,检测了晶体中１８种游离氨基酸（ＦＡＡ）含量,并与大鼠硒性和半乳糖性白内障晶体中ＦＦＡ含量进行了比较,发现：仔一代和仔二代先天性白内障晶体中各ＦＡＡ含量接近;先天性白内障晶体内,酪氨酸和羟脯氨酸含量明显高于正常对照组（Ｐ＜０．０５）;谷氨酸,甘氨酸等９种ＦＡＡ明显低于硒性白内障（Ｐ＜０．０５）;而酪氨酸高于半乳糖性白内障组,半胱氨酸等５种ＦＡＡ含量均低于半乳糖性白内障组。同时还发现：硒性和半乳糖性白内障晶体内一些ＦＡＡ变化与以往报道不同,这些现象提示先天性与后天性白内障病变过程可能不同。     

亚硒酸钠诱发大鼠白内障晶状体前列腺素生物合成的研究

 用亚硒酸钠诱发大鼠产生白内障后,将晶状体微粒体与外源性花生四烯酸共同孵育,用放射免疫方法测定白内障晶状体前列腺素E_2(PGE_2)及前列腺素F_2α(PG-F_2α)的生物合成情况,并与正常晶状体进行了比较,结果表明大鼠晶状体具有酶促合成PGs的能力。正常晶状体及白内障晶状体合成PGE_2的能力分别为687.75±113.97及1095.00±79.39pg/100mg晶状体湿重/15分钟,PGE_2α则分别为51.45±36.72及158.83±115.94pg/100mg晶状体湿重/15分钟(平均数±S.D.)。这说明大鼠白内障晶状体合成PGs的能力明显增高,与正常晶状体相比有显著性差异(PGE_2P<0.001,PGF_2αP<0.02)。在前2次注射亚硒酸钠后,大鼠白内障晶状体PGs的合成能力逐渐高于正常晶状体,并随注射亚硒酸钠的次数增加和白内障晶状体混浊程度加重,PGs在晶状体内的含量增加。     

金鱼(红龙睛)次黄嘌呤-鸟嘌呤磷酸核糖转移酶的分离及特性初步研究

 从金鱼肝脏分离并部分纯化了HGPRT。初步结果表明,该酶的分子量为78,000左右。由3个亚基组成,每个亚基的分子量约为27,000。金鱼的HGPRT很稳定,在85℃加热10分钟,至少保留80％的酶活性。该酶与PRPP的反应产物在淀粉凝胶电泳图谱上出现两条反应带。     

Further studies on bovine serum amylase. 2. Affinity electrophoresis

The polymorphism of bovine serum amylase, which is controlled by the Ami locus, has previously only been demonstrated by starch gel electrophoresis. The addition of maltose to starch gels has been demonstrated to inhibit any subsequent separation of the Ami isozymes by starch gel electrophoresis. When electrophoresis was conducted in a support medium in the absence of starch no polymorphic variation was detected amongst samples from animals of different Ami phenotypes. The addition of starch to agarose gels has been shown to facilitate the subsequent detection of the Ami polymorphism by agarose/starch gel electrophoresis. The electrophoretic resolution of the Ami isozymes has been demonstrated to depend upon differences in affinity for starch rather than differences in net charge. The starch gel electrophoretic separation of the Ami isozymes is. therefore, another example of affinity electrophoresis. All the Ami amylases have been shown to share a common isoelectric point of pH 3.5.     

The location of structural difference between ovotransferrin types A and B in hens

A comparison of ovotransferrin types A and B showed that in starch gel electrophoresis both types consisted of one major and one minor component. Both types have a similar amino acid composition as do the fragments from each type. Starch gel electrophoresis shows that the cause of the difference in the elec-trophoretic mobilities between ovotransferrin types A and B lies in the C-termi-nal half of the molecule. No physiological difference was found between types A and B, both types donate iron to chicken embryo red cells at equal rate.     

聚丙烯酰胺凝胶电泳配合荧光增白剂显色检测木霉几丁质酶同工酶谱

为提高木霉几丁质酶检测方法的准确性和灵敏度,建立一种快速检测几丁质酶同工酶的方法。采用活性凝胶电泳、变性凝胶电泳、原位显色凝胶电泳结合荧光增白剂（Calcofluor white M2R）显色从绿色木霉LTR-2发酵产物中检测几丁质酶同工酶。活性凝胶电泳在粗酶液浓缩5倍时显示两条活性谱带,变性凝胶电泳在浓缩10倍时显示一条活性谱带,原位显色凝胶电泳在浓缩20倍时显示两条不清晰的活性谱带,SDS-PAGE显示这两条活性谱带的分子量分别为65kDa和42kDa。结果表明活性聚丙烯酰胺凝胶电泳和Calcofluor white M2R显色相结合的方法在几丁质酶上样量为0.47U时具有较好的分辨能力,是检测木霉几丁质酶同工酶的有效的方法。     

Gradient flattening of sodium dodecyl sulfate (SDS) and isoelectric focusing (IEF) gels

In addition to our previously reported versatile methods for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [1] and isoelectric focusing [IEF]-gel [2], I have achieved molecular weight gradient flattening of the SDS-polyacrylamide gel and pH gradient flattening of the IEF gel at any segment using the same electrophoresis system. Any crowded gel segment where congregated components are not separated well can easily be widened for good separation and any dispersed gel segment where components are too far can easily be narrowed. Therefore, every gel segment can be used effectively and meaningfully because the gradient curve can be ajusted to any distribution of the components. In the crowded area, any small spots of components which could not be detected previously because of nearby heavy staining or strong radioactivity of an abundant component can be sufficiently separated from the nearby spots in a small gel without sacrificing other areas.     

Cost‐effective media for the rapid and high resolution of small DNA fragments using polyacrylamide‐based electrophoresis

Current Tris‐based solutions for DNA electrophoresis produce a positive feedback loop between current and temperature at high voltage, resulting in long running times for the separation of even small DNA fragments. We optimized the separation of small DNA fragments (90–300 bp) in polyacrylamide‐based electrophoresis at high voltages (200volts/cm) by substituting Tris with low concentration alkali salts (e.g. 1 mm LiCl and CsCl). These media reduced the heat produced during electrophoresis, enhanced the DNA fragment resolution, and allowed gels to be run at higher voltages, reducing gel running times by 25%. In addition, the elimination of Tris and EDTA from the buffer reduced material costs approximately 10‐fold.     

快速提取、纯化叶绿体4.5SrRNA的方法

我们采用植物叶与热缓冲液、苯酚直接混合(约65℃)匀浆,离心抽提和乙醇沉淀后,得到植物叶总RNA。经聚丙烯酰胺凝胶电泳分离、纯化,即可得到叶绿体4.5S rRNA,此法不仅操作简单,而且得率高。 同时,经过对同一植物的不同组织或不同细胞组分,如根、细胞质、叶绿体和叶绿体核糖体小分子RNA的提取与鉴定,以简便的方法证明了4.5S rRNA是叶绿体核糖体成份,也证明了我们所采用的提取、纯化4.5SrRNA方法的可靠性。     

玉米酯酶同工酶和过氧化物酶同工酶的分子量研究

本试验利用聚丙烯酰胺凝胶梯度电泳分步染色法直接对玉米苗期酯酶同工酶和过氧化物酶同工酶各酶带的分子量进行了比较测定。酯酶同工酶 E_1、E_2、E_3~F、E_3~S、a、b、c 各酶带的分子量分别为<20000,35200、33000、38500、29900、28500、34000道尔顿过氧化物酶同工酶 PX_4~F和 PX_4~S酶带的分子量分别为131000和149000道尔顿。根据酶带在均匀胶和梯度胶中的位置变化对各酶带的生化性质作了初步分析,发现 E_3~F和 E_3~S、PX_4~F 和 PX_4~S 在迁移率上的差异主要是分子量的差异。本文为同工酶的分子量测定提供了一个简便的方法。     

Analysis of mitogenic activity of proteins after separation by gel electrophoresis

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions. This revised version was published online in July 2006 with corrections to the Cover Date.