菠菜种子胰蛋白酶抑制剂对K-562细胞增殖的抑制及促凋亡作用研究

应用MTT法分析了菠菜种子胰蛋白酶抑制剂SOTI对人慢性髓原性白血病K-562细胞生长的影响。结果显示SOTI能够抑制K-562细胞的增殖,其抑制细胞增殖的作用呈现明显的剂量-效应关系;进一步观察了不同浓度的SOTI处理K-562细胞所导致的形态学变化和诱导凋亡作用,证实SOTI具有明显的体外抗癌活性,SO—TI的抗癌活性与其诱导凋亡有关。     

白菜型油菜种子胰蛋白酶抑制剂纯化及部分性质研究

采用热变性、硫酸铵分步盐析及离子交换层析和分子筛层析等方法,从白菜型油菜种子中得到胰蛋白酶抑制剂(BNTI)。SDS-PAGE检测为单一条带,表明纯化的胰蛋白酶抑制剂电泳均一。SDS-PAGE测定其分子量约为14．4kD,等电聚焦测定其等电点约为4．7。BNTI具有较高的热稳定性。本文还考察了温度对溶液中BCH蛋白构象的影响,荧光光谱和测定抑制活力结果表明BNTI中的色氨酸和酪氨酸残基位于疏水部位。     

甘薯和花生胰蛋白酶抑制剂的初步研究

许多植物蛋白制成品均含有抑制动物消化的蛋白酶抑制剂。目前已从某些豆类及蔬菜种子中分离出多种对胰蛋白酶具有抑制作用的活性物质。该实验以花生、甘薯等为原料,通过DEAE-Sepharose4BFF阴离子交换柱层析分离胰蛋白酶抑制剂,以N-苯甲酰-L-精氨酸乙酯(BAEE)为底物测定其对胰蛋白酶的抑制活性;将具有抑制活性的组分通过SDS-PAGE测定蛋白质相对分子质量(Mr);以聚丙烯酰胺凝胶等电聚焦电泳测定蛋白质等电点(pI)。结果显示,甘薯中至少有4种胰蛋白酶抑制剂组分,相对分子质量为20～25kD、等电点在pH5.0～6.6之间;花生中至少有三种胰蛋白酶抑制剂组分,相对分子质量为30～70kD、等电点在pH5.0～5.8之间。     

海芋胰蛋白酶抑制剂的分离纯化及性质研究

利用亲和层析和分子筛凝胶过滤等技术,从海芋根茎中分离纯化到一种胰蛋白酶抑制剂,简称ＡＭＴＩ。经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ鉴定均显示单一条带,经ＳＤＳ－ＰＡＧＥ测定,其分子量为２２０００,经等电聚焦（ＩＥＦ）测定,其等电点为６．２。根据对胰蛋白酶的抑制比可知该抑制剂为单头抑制剂,其抑制活性在６０℃和ｐＨ５￣１１范围内保持稳定。     

野生型和突变型荞麦蛋白酶抑制剂的活性比较及抗肿瘤功能分析

运用定点突变技术研究重组荞麦胰蛋白酶抑制剂(rBTI)的作用位点,先后构建了R45A-aBTI 和R45F-fBTI 两个突变体．抑制活性测定显示,aBTI 和fBTI 均丧失了胰蛋白酶抑制活性,却分别增加了对弹性蛋白酶和胰凝乳蛋白酶的抑制活性,确定Arg⁴⁵为野生型rBTI 的作用位点．稳定性分析表明,rBTI、aBTI 和fBTI 均具有很高的热稳定性及酸碱稳定性．采用MTT 比色法分别检测野生型和突变型抑制剂对肿瘤细胞生长的抑制作用．结果表明,突变前后的3种抑制剂对HL-60 和EC9706 细胞的生长均显示出很强的抑制作用,并且具有明显的浓度依赖性和时间依赖性．通过研究不仅确定了rBTI 的作用位点,而且获得了两种新型蛋白酶抑制剂,且作用位点的改变并不影响其对肿瘤细胞的生长抑制作用．为进一步研究胰蛋白酶抑制剂的结构与功能的关系以及抗肿瘤药物的研制提供了新的思路．     

绿豆胰蛋白酶抑制剂┐猪胰蛋白酶复合物的四方晶体结构

本文报道了绿豆胰蛋白酶抑制剂－猪胰蛋白酶（１∶２）复合物的四方晶体的结构测定。晶体属于Ｉ４２２空间群,衍射分辨率为０．２５ｎｍ,共确定了绿豆抑制剂５６个残基的空间位置。与已报道的三方晶体结构相比,其中的抑制剂分子的结构基本相同,并且抑制剂－酶三元复合物在四方晶体中也处于堆积无序状态,即复合物按两种取向进行堆积：Ｔａ∶ＭａＭｂ∶Ｔｂ和Ｔｂ∶ＭｂＭａ∶Ｔａ（Ｔａ,Ｔｂ为胰蛋白酶,Ｍａ为抑制剂结合环Ⅰ,Ｍｂ为抑制剂结合环Ⅱ）。但四方晶体结构比三方晶体结构多测定了一个抑制剂残基Ｐｒｏ１１的位置,同时,四方晶体中的抑制剂不存在膺β－折叠片层结构,其构象与三方晶体中的抑制剂相比有所变化。分析表明抑制剂的两个刚性的结构域之间的连接肽具有一定的柔性,并因此形成不同的晶型。此外,绿豆抑制剂与其他Ｂｏｗｍａｎ－Ｂｉｒｋ抑制剂相比,分子的两个双股的反平行β－折叠片层和抑制活性位点所在的结合环的结构具有很高的保守性     

决明子胰蛋白酶抑制剂COTI对菜青虫生长抑制作用的研究

从决明子中分离得到胰蛋白酶抑制剂COTI,利用其缓冲液浸泡油菜叶片饲喂菜青虫(Pieris rapae L)幼虫后,幼虫体重明显下降,分析显示菜青虫中肠提取物中蛋白酶活性受到COTI的抑制,表明决明子胰蛋白酶抑制剂对菜青虫的生长具有明显的抑制作用。     

决明胰蛋白酶抑制剂基因的克隆及其原核表达载体构建

[目的]克隆决明胰蛋白酶抑制剂全长cDNA序列,并构建原核表达载体。[方法]从决明种子中提取胰蛋白酶抑制剂总RNA,通过RT-PCR得到胰蛋白酶抑制剂cDNA,纯化后与PMD19-T载体连接,转化至大肠杆菌DH5α,获得了决明胰蛋白酶抑制剂基因的全长序列,并将该序列克隆到原核表达载体pET-28a(+)中,构建重组质粒pET-28a(+)/COTI。[结果]决明胰蛋白酶抑制剂基因核苷酸长度为630bp,编码一条长度为209个氨基酸的多肽。决明胰蛋白酶抑制剂与不同植物来源的Kunitz蛋白酶抑制剂有高度的同源性,表明其属于Kunitz蛋白酶抑制剂家族成员。所获重组质粒pET-28a(+)/COTI经过双酶切鉴定,其含有目的片段,且重组质粒构建正确。[结论]克隆了决明胰蛋白酶抑制剂的全长cDNA序列,并成功构建了含有该基因的原核表达载体,这为该基因的进一步表达及功能鉴定奠定了基础。     

豇豆胰蛋白酶抑制剂不同组分的制备方法

以豇豆种子为材料,经脱脂、乙酸钠抽提、硫酸铵分级沉淀得到胰蛋白酶抑制剂粗提物。再经凝胶过滤、离子交换层析和FPLC,分别得到了豇豆蛋白酶抑制剂的不同组分,并进行了SDS-PAGE分析。同时通过明胶活性电泳,证实了各部分均具备专一的胰蛋白酶抑制剂活性。     

响应面法优化荞麦胰蛋白酶抑制剂固定化条件

目的:以大肠杆菌表达的重组荞麦胰蛋白酶抑制剂为原材料,研究其固定化方法及条件。方法:以0.2%聚乙烯醇-3%海藻酸钠溶液为载体,CaCl2为固定剂,用物理包埋法对荞麦胰蛋白酶抑制剂进行固定化;在CaCl2浓度、载体与抑制剂体积比以及固定化时间3个单因素基础上,利用响应面法对荞麦胰蛋白酶抑制剂固定化的影响因素进行优化。结果:建立了响应面法优化固定荞麦胰蛋白酶抑制剂的模型,经优化后得到如下最佳固定化条件:CaCl2浓度为5.5%,载体与抑制剂体积比为1.6∶1,固定化时间为31min。在此条件下实际测得固定化抑制剂抑制率为72.4%,而模型预测此条件下的抑制率为74.3%,实测值与理论值相差很小。结论:所建模型拟合程度较高,用该模型优化荞麦胰蛋白酶抑制剂固定化的工艺条件参数准确可信,可为进一步开发胰蛋白酶的应用提供重要参考。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.