鼻咽癌转移淋巴结中EB病毒相关的DNA多聚酶的分离及其特性研究

一种新的DNA多聚酶已从鼻咽癌(NPC)转移淋巴结胞核酶液,通过DEAE-纤维素柱层析而被部分纯化,并可与细胞的α-及β-DNA多聚酶分开。 此酶有下列特点可与细胞DNA多聚酶区分:(1)可放DEAE-纤维素吸附,需用130mMK_2HPO_4缓冲液方可洗脱下来。(2)可被浓盐所激活,150——200mMKCl或75mM(NH_4)_2SO_4可使它显示最高的酶活性。(3)最适pH为8.0。(4)对磷酰甲酸盐的抑制较敏感。(5)能很好地利用某些合成模板,如poly(dA)·oligo(dT)_(10)及poly(dA)·oligo(dT)_(12-18)。但不能利用poly(rA)·oligo(dT)_(10),证明此酶并非细胞的γ-DNA多聚酶,而与巴基特淋巴瘤的EB病毒相关的(EBV)DNA多聚酶性质十分相似。对照的Raji细胞未见此种EBV-DNA多聚酶。 从鼻咽癌淋巴结中分离出此种EBV-DNA多聚酶,将对EB病毒与NPC的发病关系提供新的证据。     

依赖于DNA的RNA聚合酶与体外转录的研究——Ⅱ.酵母变异株20B-12 RNA聚合酶B的纯化及鉴定

从酵母变异株20B-12经过超声波处理、硫酸铵沉淀、DEAE纤维素、磷酸纤维素和DEAE-Sephadex层析等步骤,纯化依赖于DNA的RNA聚合酶B,在聚丙烯酰胺凝胶电泳中呈二条有聚合酶B活力的区带。其中不含有DNase、RNase和蛋白酶活力,无内源DNA。50μg/ml的α-鹅膏蕈碱抑制聚合酶活力达90％以上。最适(NH_4)_2SO_4浓度为40mM。最适Mn~(2 )浓度为2mM。Mg~(2 )对酶B无激活作用。变性DNA对酶B较天然DNA有更高的效率。     

依赖于DNA的RNA聚合酶与体外转录的研究 Ⅰ.酵母变异株20B-12 RNA聚合酶A和C的纯化及鉴定

从酵母变异株20B-12经过超声波处理、硫酸铵沉淀、DEAE纤维素和磷酸纤维素层析等步骤,纯化依赖于DNA的RNA聚合酶A和C,得到聚丙烯酰胺凝胶电泳均一的条带。其中不含DNase、RNase 和蛋白酶活力,无内源DNA。测定了 RNA 聚合酶A和C对α-鹅膏荤碱的敏感性。酶A在α-鹅膏荤碱为400μg/ml时,活性受到抑制,而酶C在该浓度时,几乎不受抑制。(NH_4)_2SO_4对酶A的最适浓度为20mM,对酶C有二个最适浓度,分别为40mM和240mM。无二价金属离子Mn~(2+)或Mg~(2+),酶A和C几乎无活力。两种酶最适Mn~(2+)浓度均为2.5mM,Mg~(2+)浓度均为5mM。两种酶以热变性小牛胸腺DNA为模板,测活性均较天然小牛胸腺DNA为模板时高。     

用试管-玻片培养技术研究铵对木麻黄根瘤形成的影响

用植物试管玻片培养技术研究NH_4~ 对细枝木麻黄及Frankia菌株Co01共生体系建立过程的影响。NH_4~ (100,150ppm(NH_4)_2SO_4)通过阻止菌株Cc01与其宿主细枝木麻黄根毛壁的亲和作用来影响结瘤。但NH_4~ 不能阻抑菌株Cc01中结瘤基因pel和cel的表达及纤维素酶和果胶酶活性,且菌丝一旦侵入宿主皮层细胞,并形成根瘤原基及前根瘤,则NH_4~ (250ppm(NH_4)_2SO_4)就不再阻止原基进一步发育为成熟的根瘤。但在这种情况下,NH_4~ 能抑制根瘤的固氮活性。     

苎麻酶法脱胶研究

Bacillas sp.No.5黄产生果胶酶的最适条件是氮源1％(NH_4)SO_4,碳源5％麸皮,诱导物2％甜菜渣:34℃、培养34h。酶作用最适温度50℃,最适pH9.6;50℃以下、pH8.4以下,酶的稳定性较好。用粗酶液脱胶时,先将苎麻纤维放入稀酸中煮30分钟,再放入80～100ngu/ml(粗酶液pH9.6,保温50℃、4小时,可脱去麻纤维93％的胶质。在脱胶过程中,可用测定还原基团来指示脱胶程度。     

几种氧化酶活性与植物对二氧化硫抗性的研究——植物本底及受污染后酶活性大小与抗性的关系

对27种不同抗性等级植物本底(未经污染处理的正常植物)多酚氧化酶、抗坏血酸氧化酶和过氧化物酶活性测定结果,植物本底多酚氧化酶活性与抗性有呈负相关的趋势,抗坏血酸氧化酶和过氧化物酶活性大小与抗性不具规律性。 对植物受不同浓度SO_2污染后酶活性变化分析结果看出,蚬木(抗性植物)和汗斑草(敏感植物)在浓度达受阈前,多酚氧化酶、抗坏血酸氧化酶和过氧化物酶均有随浓度的升高而酶活性逐渐增大的趋势,仅是不同的酶其活性高峰在不同浓度梯度中出现迟早不同而己。在污染浓度达受伤阈后,随着浓度的继续增大,酶活性逐渐下降。而白蝉(抗性植物)和大猪屎青(敏感植物)有的酶具有规律性,有的酶不具有这种规律性。 用使可见伤害达50％的SO_2污染汗斑草后4小时(一次污染),过氧化物酶活性为0.34(未受污染的为12.69),仅为未受污染的2.68％,降低了97.32％,但24小时后为7.68,为未受污染的60.52％,比受污染后4小时提高了57.84％,72小时后为8.52,为未受污染的67.13％,比受污染后4小时提高了65.45％。多酚氧化酶亦具有这种规律性。说明二氧化硫对这两种酶的抑制作用是可逆的。     

赤霉酸对小麦和大麦糊粉层细胞中异柠檬酸裂解酶形成的作用

小麦无胚半种子在10~(-5)M赤霉酸(GA_3)处理15～17小时开始出现异柠檬酸裂解酶(ICL)活性。激素处理48小时ICL活性达高峰,随后下降。没有外源GA_3则测不到ICL活性。大麦糊粉层没有外源GA_3仍可测到一定水平的ICL活性。50μMABA可以阻止10~(-3)MGA_3对小麦和大麦糊粉层ICL的诱导作用。3′-脱氧腺苷酸(0.3 mM)、6-甲基嘌呤(0.1～1.0 mM)及放线菌素D(50 μg/ml)强烈地阻止GA_3诱导ICL的作用。L-天门冬酰胺和L-谷氨酰胺(3mM)抑制GA_3作用20～30％。抑制剂实验结果表明GA_3对小麦糊粉层细胞ICL的诱导合成可能与酶基因的转录有关。     

小麦离体根K~ 吸收能力的诱导提高与原生质膜-ATP酶活性及膜磷脂/膜蛋白比率变化的关系

小麦离体根在通气的去离子水中或0.1mM CaSO_4溶液中浸洗24小时,显著地增加K~ 的吸收速率。0.1mM CaSO_4溶液浸洗的增益效应大于去离子水。经0.1mM CHI,2mM FPA和0.01mM ABA浸洗24小时的小麦根,K~ 的吸收明显的降低,但它们对未经预处理的小麦根的K~ 吸收没有影响。小麦根匀浆经不连续梯度离心获得的34—45％蔗糖溶液介面处富含原生质膜部分和45％蔗糖浓度溶液中的沉降膜微囊(membrane vesicle),都有Mg~(2 ) K~ -ATP酶活性。从去离子水或0.1mM CaSO_4溶液浸洗过的小麦根获得的两部分膜制剂中,ATP酶的活性高于对照,而经CHI,FPA或ABA预处理过的小麦根的膜制剂内,ATP酶的活性低于对照。这些ATP酶活性的变动与K~ 吸收能力的增减呈正相关,与膜磷脂/膜蛋白的比率呈负的相关性。     

黄杆菌(Flavobacterium sp.)几丁质酶的纯化和性质

黄杆菌(Flavobacteium sp.)在几丁质的诱导下产生几丁质酶.通过(NH_4)_2SO_4沉淀、DEAE纤维素柱层析、Sephacryl 300柱层析及Sephadex G-75柱层析,从Flavobacterium sp.培养上清液中分离纯化了几丁质酶.SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)纯度分析表明,纯化后的几丁质酶达到了均一的程度.用SDS-PAGE测得该酶的分子量约45D00道尔顿.该酶水解几丁质的最适pH为 7.0,最适温度为50℃,-20C贮存两年以上仍有活性.水解几丁质的Km值为5.0mg/ml.金属离子对几丁质酶活性影响较大,Ca^(2+) 、Co^(2+)’和Cu^(2+)对酶有激活作用.而NH_4^-、Ba^(2+)、Mg^(2+)、Mn^(2+)对酶有抑制作用.几丁质酶水解几丁质的产物是几丁质二糖.     

基于4-1BBL和CD3双信号的融合蛋白协同抗肿瘤作用研究

旨在研究4-1BBL/CD20融合蛋白增强抗CD3/抗CD20 diabody介导的特异性靶向杀伤活性。采用亲和层析法纯化本室构建的抗-CD3/抗-CD20 diabody和4-1BBL/CD20融合蛋白可溶性表达产物;采用calcein释放试验测定其介导的体外靶向杀伤活性;采用人B淋巴瘤细胞系Raji裸鼠移植瘤模型测定其介导的体内靶向杀伤活性。纯化4-1BBL/CD20融合蛋白在体外能增强抗-CD3/抗-CD20 diabody介导激活的T细胞杀伤Raji细胞;在人B淋巴瘤细胞系Raji裸鼠移植瘤模型联合人T淋巴细胞4-1BBL/CD20融合蛋白增强抗-CD3/抗-CD20 diabody高效抑制Raji细胞裸鼠移植瘤的生长,明显延长荷瘤裸鼠的生存时间。在体外和体内4-1BBL/CD20融合蛋白均能增强抗-CD3/抗-CD20 diabody介导激活的T细胞杀伤表达CD20抗原的肿增细胞,是一个有望用于B细胞恶性肿瘤临床治疗的特异性融合蛋白。     

Cellular and Epstein-Barr virus specific DNA polymerases in virus-producing Burkitt's lymphoma cell lines.

We have determined the levels of cellular DNA polymerases and Epstein-Barr virus specific DNA polymerase in three Burkitt's lymphoma cell lines producing varying amounts of EBV, one of which was induced by 12-0-tetra-decanoylphorbol-13-acetate (TPA). There was a proportional increase in the level of EBV-DNA polymerase with an increase in the percent of virus-producing cells. However, there was a reciprocal relationship between the levels of EBV-DNA polymerase and DNA polymerase alpha i.e., in cell line containing the highest level of EBV-DNA polymerase, activity of DNA polymerase alpha, but not of DNA polymerase beta, was reduced to an insignificantly low level. TPA does not have any direct effect on activities of either EBV-DNA polymerase or DNA polymerase alpha. EBV-DNA polymerases isolated from cells grown with or without TPA are indistinguishable in their properties such as elution position on phosphocellulose column, molecular weight, mono and divalent cation requirements, pH optimum, and other requirements for optimum activity. Addition of crude extracts of cells grown in presence of TPA to the purified DNA polymerase alpha did not inhibit its activity indicating that the observed loss was not due to any specific inhibitor present in TPA treated cells. Raji, a nonproducer cell line, did not contain EBV-DNA polymerase. There was no induction of EBV-DNA polymerase when Raji cells were grown in presence of TPA. The phenomenon of reduction in the levels of DNA polymerase alpha in cells induced to produce EBV may represent a mechanism by which the host DNA replication is shut off following virus infection.     

Induction of cellular DNA polymerases in a Burkitt lymphoma-derived cell line by treatment with 5-iododeoxyuridine

Cellular DNA polymerases of a Burkitt lymphoma-derived cell line (P3HR-1) were found to be greatly induced by treatment of the cells with 5-iododeoxyuridine (IUdR) at a concentration which induces Epstein-Barr virus (EBV) early antigen (EA) expression. The activities of all the DNA Polymerases alpha, beta and gamma in P3HR-1 cells increased 7-9 fold by exposure of the cells to IUdR (25 micrograms/ml) for 3 days, while the EBV-coded DNA polymerase activity in the cell remained undetectable under the assay conditions employed. Under the same culture conditions with IUdR, EA-positive P3HR-1 cells increased to 16.6% which was much higher than that of the non-treated control cells (0.32%). On the other hand, another Burkitt lymphoma cell line, Raji, had very low incidence (1.27%) of EA induction by IUdR-treatment and the level of DNA polymerase activities remained almost unchanged. From these results it seems that the increase in DNA polymerase activity during the treatment of P3HR-1 cells with IUdR is closely related to high incidence of EA expression in these Burkitt lymphoma cells. Also, the finding has revealed yet unknown effect of IUdR on cultured cells and provides a useful tool to obtain a large quantity of the induced cellular DNA polymerases from the P3HR-1 and KB cells.     

Distinguishing cytomegalovirus, mycoplasma, and cellular DNA polymerases.

Cytomegalovirus-induced DNA polymerase can be distinguished from infected-cell enzymes by activity in 100 mM (NH4)2SO4. Virus polymerase is stimulated to 145% of control, whereas mock-infected cell polymerase is inhibited to 12% of control without added salt. Mycoplasmas induce a DNA polymerase in cell extracts that is stimulated to 130 to 180% by 25 mM (NH4)2SO4. Mycoplasma DNA polymerase may be mistaken for a virus-induced polymerase when virus stocks are contaminated. Identification of virus, cellular, and mycoplasma DNA polymerases in total cell extracts is described using sedimentation rate and effect of inhibitors on DNA polymerase activities.     

Rapid purification of overexpressed T4 DNA polymerase

A method for purifying T4 DNA polymerase from cells harboring overexpression plasmids is described. T4 DNA polymerase is precipitated from induced, lysed cells with polyethyleneimine, then extracted and fractionated further with (NH4)2SO4 before chromatography on a column of single-stranded DNA cellulose. This procedure can be completed in three days and consistently provides enzyme preparations which are at least 98% pure. When necessary, one further chromatography step provides T4 DNA polymerase suitable for recombinant DNA applications.     

人精液对RAJI细胞中EPSTEIN-BARR病毒早期抗原的诱导

在大戟科、瑞香科、豆科和其它科植物中,发现有些植物含有能诱导人伯基特淋巴瘤细胞——Raji细胞中EB病毒早期抗原的物质,并已证明其中有的是很强的促癌物质,如12-0-十四烷酰巴豆醇-13乙酸酯(TPA)、桐油提纯的HHPA、芫花和黄芫花等。某些人的精液也含有能激活EB病毒的物质。本文报告中国人的精液,以及人精液与从宫颈癌病人分离的细菌培养液,对EB病毒早期抗原的诱导作用和协同诱导作用。     

Biological differences between epstein-barr virus (EBV) strains with regard to lymphocyte transforming ability, superinfection and antigen induction

EB virus (EBV) preparations derived from various producing lymphoblastoid cell lines (LCL) differed in their biological properties, as judged by the following four tests: (1) cord blood lymphocyte (CBL) transformation into EBV-carrying LCL; (2) early antigen (EA) induction in Raji cells; (3) inhibition of Raji cell growth; (4) induction of the EBV-determined nuclear antigen (EBNA) in CBL. B95-8 virus transformed and induced EBNA in CBL but did not induce EA in Raji cells, nor did it inhibit their growth. P3HR-1 virus did not transform CBL, induced no EBNA or EA in CBL, but induced EA in Raji cells and inhibited their growth. EBV isolated from the QIMR-WIL, 833L, F137 and cb-8-7 LCL resembled the B95-8 virus with regard to its biological activity (CBL transformation, EA induction in and growth inhibition of Raji cells). Transformation of CBL as contrasted to EA induction in, and growth inhibition of Raji cells thus appear as mutually exclusive viral functions.     

Amplification of Epstein-Barr virus (EBV) DNA by superinfection with a strain of EBV derived from nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) from a nasopharyngeal carcinoma (NPC) hybrid cell line (NPC-KT) lacking defective viral DNA molecules superinfected Raji cells and induced EBV early antigens (EA), as did virus from P3HR-1 cells, which contained defective molecules. The EBV polypeptides induced by NPC-KT appeared to be identical to those induced by P3HR-1 virus. The ability of NPC-KT virus to induce EA was enhanced more than 10-fold by treatment of superinfected cells with dimethyl sulfoxide; however, dimethyl sulfoxide treatment did not enhance superinfection by P3HR-1 virus. After infection, DNA synthesis of both the superinfecting NPC-KT virus and the resident Raji viral genome was induced. In addition to amplified Raji EBV episomal DNA, a fused terminal fragment of NPC-KT viral DNA was detected. The detection of fused terminal DNA fragments suggests that the superinfecting virion DNA either circularizes or polymerizes after superinfection and is possibly amplified through circular or concatenated replicative intermediates.     

Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in rat liver after protein restriction.

Rats were fed for 6 days on a diet containing either 3 or 20% high-quality protein. Nuclei were isolated from liver and DNA-dependent RNA polymerases (EC 2.7.7.6) extracted with 1 M-(NH4)2SO4. The proteins were then precipitated with 3.5 M-(NH4)2SO4 and after dialysis applied to a DEAE-Sephadex column. The column was developed with a gradient of (NH4)2SO4. Polymerase I separated well from alpha-amanitin-sensitive polymerase II. The enzyme activities were compared between the two dietary groups. Rats that had received 3% protein showed a lower polymerase I activity per g wet wt. of liver, per mg of DNA and per mg of protein. Polymerase II was lower in activity per g wet wt. of liver and per mg of DNA, but was higher per mg of protein. Polyacrylamide-gel electrophoretograms showed a higher proportion of contaminating proteins in polymerase II fractions isolated from 20%-protein-fed rats. The data explain the lower activity obtained per mg of protein in these rats. It is concluded that a decrease in dietary protein content from 20 to 3% induces a fall in content and specific activity of RNA polymerase I and II in liver.     

Binding studies of the antiglucocorticoid RU38486 in Daudi and Raji lymphoma cells

The activity of RU38486 has been studied in Burkitt's lymphoma cells which are Epstein-Barr virus (EBV) positive. The early antigens (EA) of the virus are induced by dexamethasone (DXM) in Daudi but not in Raji cells, whereas a growth factor (transforming growth factor-beta, TGF-beta) induces the EA in both cell lines. RU38486 blocks the EA induction obtained by DXM or by TGF-beta in either cell line. In order to understand the interaction of RU38486, we considered its binding to specific receptors. We first investigated the binding of the antagonist in whole cells at 22 degrees C. A number of specific binding sites higher for RU38486 than for DXM was found, suggesting that RU38486 may bind to the glucocorticoid receptor and also to other cellular structures which we called the antiglucocorticoid binding sites ("AGBS"). To support this hypothesis, competition experiments have been conducted between RU38486 and other steroid hormones (progesterone and testosterone) since it is known that RU38486 is also able to interact with their cognate receptors. Binding studies of RU38486 in vitro at 4 degrees C in the presence of cytosolic extracts from Daudi and Raji cells led to conclusions similar to those drawn from the whole cell experiments: more complexes were formed with RU38486 than with DXM. Finally, the steroid-receptor complexes were incubated with DNA-cellulose. Since the binding measured for RU38486 was higher than for DXM, we suspect that sites different from the classical glucocorticoid receptor sites are also able to interact with DNA. The blockage exerted by RU38486 on the EA induced by glucocorticoids or by non-steroidal molecules and the lack of responsiveness to glucocorticoids in Raji cells are discussed in the light of the present findings.