抗呼吸道合胞病毒免疫核糖核酸免疫活性的研究

本文在小鼠模型系统中,应用ELISA和~(125)IUdR释放试验分别研究抗呼吸道合胞病毒免疫核糖核酸(RSV—i—RNA)诱生血清特异性抗体活性和以细胞毒活性为指标检测其增强细胞免疫的作用。实验结果表明:RSV-i-RNA能诱生血清特异性抗体和提高鼠脾细胞对靶细胞的杀伤作用,且明显高于正常核糖核酸对照组。 RSV-i-RNA的上述作用可被RNase所阻断,但不受DNase和Pronase的影响。     

重组旋毛虫plancitoxin-1-like活性位点突变体蛋白的核酸酶活性

旋毛虫plancitoxin-1-like(Ts-Pt)是旋毛虫125种DNaseⅡ家族蛋白中唯一具有典型DNaseⅡ活性区域HKD基序的核酸酶,且普遍认为,组氨酸位点是DNaseⅡ的活性氨基酸位点。为研究Ts-Pt活性位点突变体蛋白的核酸酶活性,利用重叠PCR方法获得Ts-Pt活性位点突变体片段,以p ET-28a(+)为载体构建重组表达质粒并在大肠杆菌中诱导表达。重组Ts-Pt突变体蛋白经亲和层析纯化后进行SDS-PAGE分析。利用琼脂糖凝胶电泳法和核酸酶酶谱分析重组Ts-Pt突变体蛋白的核酸酶活性。成功构建含Ts-Pt突变体重组质粒的基因工程菌,SDS-PAGE和亲和层析纯化结果显示,重组Ts-Pt突变体蛋白呈包涵体表达。重组蛋白经复性后并没有表现出核酸酶活性,但核酸酶酶谱分析结果显示,包涵体表达的重组Ts-Pt突变体蛋白表现出降解DNA的能力。同时,N端和C端活性位点H及HCK和DHSK突变并不影响Ts-Pt的核酸酶活性,研究结果为进一步研究庞大的DNaseⅡ家族蛋白在旋毛虫发育和感染方面的作用提供一定的参考。     

树突状细胞与CIK细胞共培养诱生的DCCIK细胞群对肿瘤的杀伤作用

由树突状细胞(DC)与细胞因子诱导的同源杀伤细胞(CIK)的共培养诱生的细胞群(DCCIK)对肿瘤细胞的细胞毒活性的研究。DCCIK细胞体外杀伤肿瘤靶细胞A549(MTT法),效靶比为10∶1、5∶1时杀伤率分别为61%、52%。DCCIK细胞诱导培养3周后,效靶比为10∶1、5∶1时杀伤率分别为64%和56%。数据亦表明DCCIK细胞对靶细胞的杀伤优于CIK细胞。动物体内实验分荷瘤A549、BEL7404和A375三组,每组分(A)DCCIK 化疗、(B)单用化疗。治疗20天、35天后测量各组肿瘤消失率。结果显示:DCCIK 化疗的抑瘤效果明显好于单纯化疗。提示DCCIK细胞有临床应用前景。     

几种药物对小鼠腹水癌细胞中一种DNA多聚酶及酶与模板复合体的作用

在小鼠艾氏腹水癌细胞中存在着一种DNA多聚酶及其与DNA模板的复合体。以不同药物对部分纯化的酶蛋白和复合体进行抑制实验,发现Aphidicolin、溴化乙锭、新生霉素和肝素均不同程度地抑制复合体和酶蛋白的活性;但酶蛋白和复合体对双脱氧胸苷三磷酸(ddTTP)不敏感。另外还发现复合体较酶蛋白对抑制剂有较强的抗性。     

IL-2假单孢菌外毒素嵌合蛋白有极高的细胞毒性

单克隆抗体、植物凝集素或多肽激素与细菌或植物毒素化学偶联后,可引导毒素对特异真核细胞的杀伤作用。Lorberboum—Galski 实验室曾用假单孢菌外毒素(PE)制备了杀伤特异靶细胞的制剂。PE 由三个功能区组成。功能区Ⅰ负责细胞识别作用;功能区Ⅱ负责位易,使毒素跨过质膜进入细胞;功能区Ⅲ负责蛋白质合成,延长因子2的 ADP-核糖基化,这是关系细胞存亡的关键步骤。缺失功能区Ⅰ的外毒素 PE_(40),其 ADP-核糖基化作用完好,但杀伤细胞的活性极低,因其不能识别靶细胞。将 PE_(40)与特定基因融合成嵌合基因,产生嵌合蛋白,利用特定基因产物的导向作用杀伤靶细胞。     

真核细胞DNA聚合酶δ的结构与功能

DNA聚合酶δ(Polδ)在真核细胞的DNA复制过程中具有核心酶的作用,同时还参与DNA的修复。Polδ是一种由多个亚基组成的复合体,目前已从哺乳动物、裂殖酵母和芽殖酵母等多种真核生物细胞中分离出,并对它们的亚基组成进行了分析,但还未得到确切一致的结果。Polδ在DNA复制中的具体作用已基本了解,它参与催化整个前导链的复制以及一些或大部分滞后链的复制。此外,Polδ还参与DNA的修复,此酶的这一功能可减少DNA的变异,但目前对其作用机理还知之较少。在Polδ活性调控方面,主要研究了一些相关蛋白因子对Polδ活性的调控作用以及转录因子对催化亚基表达的调控作用。     

穿孔素和颗粒酶的研究进展

肿瘤组织中的浸润淋巴细胞（TIL）的数量和质量与肿瘤的预后密切相关。TIL杀伤肿瘤的最主要的机制是PFP/GrB途径。穿孔素（PFP）通过在靶细胞膜上形成活性孔道使靶细胞渗透压改变而溶解,或者与颗粒酶协同作用而诱导靶细胞凋亡,本文概述了穿孔和颗粒酶的一般性质、作用机制、作用途径的证据以及应用研究的最新进展。     

失重条件对小鼠NK细胞体外生物活性的影响

建立在失重环境下NK细胞的培养体系,探讨失重培养条件对NK细胞活性的影响。取C57BL/6小鼠脾,分离NK细胞,在正常重力和失重状态下旋转细胞两种培养体系中培养NK细胞48 h。以Yac-1细胞为靶细胞,采用MTT法检测其杀伤活性;半定量PCR分析穿孔素、颗粒酶的转录水平。与正常重力培养对照相比,失重培养的NK细胞杀伤活性显著降低,其穿孔素和颗粒酶B的转录水平均显著低于正常重力培养组,穿孔素/GAPDH结果为失重培养组0.625±0.042;正常重力培养组1.054±0.036(P<0.01);得到颗粒酶B/GAPDH结果为失重培养组0.700±0.042;正常重力培养组1.068±0.058(P<0.01)。成功建立了NK细胞体外培养体系,在失重环境下NK细胞杀伤活性降低。     

奥美拉唑诱导非小细胞肺癌细胞对新型分子靶向药物仑伐替尼的耐受

目的:检测奥美拉唑对新型分子靶向药物仑伐替尼杀伤非小细胞肺癌(NSCLC)的影响并阐明其分子机制。方法:体外培养人NSCLC细胞系A549和H460,用奥美拉唑处理细胞,检测奥美拉唑对芳香烃受体(AhR)转录因子活性及下游基因表达的影响;在此基础上用奥美拉唑及仑伐替尼处理A549、H460细胞,检测奥美拉唑对仑伐替尼杀伤A549、H460细胞的影响;培养A549、H460细胞接种免疫缺陷裸鼠后,灌胃给予动物奥美拉唑和仑伐替尼,确定奥美拉唑对仑伐替尼抑制A549、H460细胞皮下肿瘤形成的影响。结果:奥美拉唑能够在A549、H460细胞中上调AhR的转录因子活性及下游基因表达;仑伐替尼能够剂量依赖地杀伤A549、H460细胞,用奥美拉唑处理A549、H460细胞能够显著下调仑伐替尼对细胞的杀伤作用;裸鼠成瘤实验显示,口服给予裸鼠奥美拉唑能够下调仑伐替尼对A549、H460细胞在裸鼠皮下的成瘤的杀伤作用;分子机制实验显示,奥美拉唑以AhR依赖的方式诱导A549、H460细胞对仑伐替尼的耐受作用。结论:奥美拉唑诱导非小细胞肺癌细胞A549、H460对分子靶向药物仑伐替尼的耐受。     

颗粒酶B：有力的细胞免疫杀伤因子

丝氨酸蛋白酶家族成员颗粒酶B是细胞毒淋巴细胞介导的颗粒胞吐免疫效应中有力的杀伤因子。近年来,颗粒酶B与穿孔素协同作用的靶细胞摄取机制受到质疑,“受体内化”及“静电交换”等新模型被相继提出。颗粒酶B在靶细胞内能够诱导caspase依赖及非依赖性的凋亡,还能直接作用于死亡底物发挥细胞毒作用,同时具有基质重塑等重要的细胞外功能。颗粒酶B与自身免疫疾病及抗病毒感染密切相关,在恶性肿瘤细胞杀伤方面的应用价值正日益受到关注。     

The exonuclease TREX1 is in the SET complex and acts in concert with NM23-H1 to degrade DNA during granzyme A-mediated cell death

Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features of apoptosis. Single-stranded DNA damage is initiated when the endonuclease NM23-H1 becomes activated to nick DNA after granzyme A cleaves its inhibitor, SET. SET and NM23-H1 reside in an endoplasmic reticulum-associated complex (the SET complex) that translocates to the nucleus in response to superoxide generation by granzyme A. We now find the 3'-to-5' exonuclease TREX1, but not its close homolog TREX2, in the SET complex. TREX1 binds to SET and colocalizes and translocates with the SET complex. NM23-H1 and TREX1 work in concert to degrade DNA. Silencing NM23-H1 or TREX1 inhibits DNA damage and death of cells treated with perforin (PFN) and granzyme A, but not of cells treated with perforin and granzyme B (GzmB). After granzyme A activates NM23-H1 to make single-stranded nicks, TREX1 removes nucleotides from the nicked 3' end to reduce the possibility of repair by rejoining the nicked ends.     

Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis,and the nucleosome assembly protein SET is its inhibitor

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.     

Crystal structure of the apoptosis-inducing human granzyme A dimer

Granzyme A (GzmA) belongs to a family of trypsin-like serine proteases localized in cytoplasmic granules of activated lymphocytes and natural killer (NK) cells. In contrast to the related granzyme B (GzmB), GzmA forms a stable disulfide-linked homodimer and triggers target-cell death in a caspase-independent way. Limited proteolysis of a high-molecular-mass complex containing SET (also named putative HLA-associated protein II or PHAPII), PHAPI (pp32, leucine-rich acidic nuclear protein) and HMG2 by GzmA liberates NM23-H1, a Mg2+-dependent DNase that causes single-stranded breaks in nuclear DNA. By analyzing the dimeric GzmA structure at a resolution of 2.5 A, we determined the substrate-binding constraints and selective advantages of the two domains arranged as a unique functional tandem. The active sites of the two subunits point in opposite directions and the nearby noncatalytic surfaces can function as exosites, presenting substrates to the active site region of the adjacent partner in a manner analogous to staphylokinase or streptokinase, which present plasminogen to the cofactor-plasmin and cofactor-plasminogen complexes.     

Granzyme A activates an endoplasmic reticulum-associated caspase-independent nuclease to induce single-stranded DNA nicks

The cytotoxic T lymphocyte protease granzyme A (GzmA) initiates a novel caspase-independent cell death pathway characterized by single-stranded DNA nicking. The previously identified GzmA substrate SET is in a multimeric 270-420-kDa endoplasmic reticulum-associated complex that also contains the tumor suppressor protein pp32. GzmA cleaved the nucleosome assembly protein SET after Lys(176) and disrupted its nucleosome assembly activity. The purified SET complex required only GzmA to reconstitute single-stranded DNA nicking in isolated nuclei. DNA nicking occurred independently of caspase activation. The SET complex contains a 25-kDa Mg(2+)-dependent nuclease that degrades calf thymus DNA and plasmid DNA. Thus, GzmA activates a DNase (GzmA-activated DNase) within the SET complex to produce a novel form of DNA damage during cytotoxic T lymphocyte-mediated death.     

Granzyme B proteolyzes receptors important to proliferation and survival, tipping the balance toward apoptosis

Granzyme B is critical to the ability of natural killer cells and cytotoxic T lymphocytes to induce efficient cell death of virally infected or tumor cell targets. Although granzyme B can cleave and activate caspases to induce apoptosis, granzyme B can also cause caspase-independent cell death. Thirteen prospective granzyme B substrates were identified from a cDNA expression-cleavage screen, including Hsp70, Notch1, fibroblast growth factor receptor-1 (FGFR1), poly-A-binding protein, cAbl, heterogeneous nuclear ribonucleoprotein H', Br140, and intersectin-1. Validation revealed that Notch1 is a substrate of both granzyme B and caspases, whereas FGFR1 is a caspase-independent substrate of granzyme B. Proteolysis of FGFR1 in prostate cancer cells has functionally relevant consequences that indicate its cleavage may be advantageous for granzyme B to kill prostate cancer cells. Therefore, granzyme B not only activates pro-death functions within a target, but also has a previously unidentified role in inactivating pro-growth signals to cause cell death.     

Role of Bid-induced mitochondrial outer membrane permeabilization in granzyme B-induced apoptosis

Cytotoxic lymphocytes (CL) induce death of their targets by granule exocytosis. During this process, enzymes contained within cytotoxic granules (granzymes) are delivered to the target cell where the enzymes trigger the cell death by cleaving specific substrates. Granzyme B is the only granzyme that has been shown to induce cell death by apoptosis, but the exact pathway by which this is achieved has been the subject of hot debate. Furthermore, several other death-inducing granzymes have been identified; therefore, the exact contribution of granzyme B to CL-induced death is unclear. In this study, we discuss our recent findings on granzyme B-induced cell death and discuss the potential relevance of this pathway to CL-induced death of viral-infected and transformed cells.     

Induction of rapid histone degradation by the cytotoxic T lymphocyte protease Granzyme A

The cytotoxic T lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-strand nicking is observed instead of oligonucleosomal fragmentation. Granzyme A is a specific tryptase that concentrates in the nucleus of targeted cells and synergistically enhances DNA fragmentation induced by the caspase activator granzyme B. Here we show that granzyme A treatment of isolated nuclei enhances DNA accessibility to exogenous endonucleases. In vitro and after cell loading with perforin, GrnA completely degrades histone H1 and cleaves core histones into approximately 16-kDa fragments. Histone digestion provides a mechanism for unfolding compacted chromatin and facilitating endogenous DNase access to DNA during T cell and natural killer cell granule-mediated apoptosis.     

Nuclear translocation of granzyme B in target cell apoptosis

Granzyme B is the prototypic member of a family of serine proteases localized to the cytolytic granules of cytotoxic lymphocytes. Together with another granule protein, perforin, granzyme B is capable of inducing all aspects of apoptotic death in target cells. A number of granzyme B substrates have been identified and it has been demonstrated that granzyme B is responsible, directly or indirectly, for the morphological nuclear changes observed in target cell apoptosis, including DNA fragmentation. In an earlier study, we showed that granzyme B binds to a nuclear protein in a manner dependent on its enzymatic activity. Here, we demonstrate that granzyme B is translocated rapidly to the nucleus in cells that have been induced to undergo apoptosis by a granzyme-dependent process, and that translocation is dependent on caspase activity. Appearance of granzyme B in the nucleus of target cells precedes the detection of DNA fragmentation. Although not directly responsible for DNA fragmentation, these data suggest a nuclear role for granzyme B in target cell apoptosis. c-Abl nuclear functions.