脱氧核糖核酸内切酶与细胞凋亡

哺乳动物细胞广泛存在细胞凋亡。脱氧核糖核酸内切酶(DNase)在细胞凋亡中发挥重要作用,染色质DNA降解、浓缩及细胞核结构的破坏都与DNase有关。目前已发现多种DNase参与不同类型细胞的凋亡。本重点对DNaseⅠ、DNaseⅡ、NUC18、NUC70、AN34、DFF(DFF40/DFF45)等内源性DNase与哺乳动物细胞凋亡的关系作系统的归纳和分析。     

钙、镁离子在活化HL-60细胞核酸内切酶中的作用不同

EGTA ,EDTA抑制游离HL- 6 0细胞核中核酸内切酶的活化 .EDTA对游离HL -6 0细胞核中核酸内切酶活性的抑制可被外加Ca ²⁺逆转 ,而EDTA对该酶活性的抑制却不能被外加Mg ²⁺逆转 .用CHELEX 1 0 0去除游离核孵育缓冲液中存在的Ca2 ²⁺,Mg ²⁺后 ,外界Ca2 ²⁺( 1～ 1 0mmol/L)单独可诱导游离HL -6 0细胞核中的核酸内切酶活化 ,且强度一致 ;而外加Mg ²⁺则不能诱导该酶活性 .只有在钙存在( 0 1～ 1 0mmol/L)的条件下 ,该酶活性才随Mg ²⁺浓度增高而增高 .这说明HL- 6 0细胞中核酸内切酶的活化必须有Ca ²⁺存在 ,在Ca ²⁺存在的条件下 ,其活性可被镁增强 ,Ca ²⁺和Mg ²⁺在核酸内切酶活化中的作用是不同的 .     

单纯疱疹病毒基因组的限制性核酸内切酶分析

为了获得单纯疱疹病毒(HSV)基因组的限制性核酸内切酶(RE)分析资料和选择合适的RE去研究HSV感染,用11种常用的RE对HSV两个型别的实验室标准毒株的基因组分别作了分析。比较研究的结果表明,BamHI、HpaI和PstI等RE较适于HSV的研究和分型。本研究所采用的小量提取HSV DNA的方法具有快速、简便和实用的特点,值得在HSV感染的诊断、分型和HSV分子流行病学诸研究中推广使用。     

BamHI DNA甲基化酶的纯化及对BamHI限制性核酸内切酶限制作用的保护作用

用硫酸铵盐析、磷酸纤维素柱层析和肝素-Sepharose 4B亲和层析从解淀粉芽孢杆菌(Rocllus amyloliquefaciens)纯化了BamHl DNA甲基化酶。并以λDNA为底物,研究了λDNA被BamHl DNA甲基化酶作用后,对BamHl限制性核酸内切酶的切割产生了明显的阻抗作用。     

BamHI DNA甲基化酶的纯化及对BamHI限制性核酸内切酶限制作用的保护作用

用硫酸铵盐析、磷酸纤维素柱层析和肝素-Sepharose 4B亲和层析从解淀粉芽孢杆菌(Raviflus amyloliquefaciens)纯化了BamHI DNA甲基化酶。并以λDNA为底物,研究了λDNA被BamHIDNA甲基化酶作用后,对BamHI限制性核酸内切酶的切割产生了明显的阻抗作用。     

限制性核酸内切酶（restrictionendonuclease）

利用基因重组技术的研究以及产品的制造得到广泛普及,其中的有功之臣就是限制性核酸内切酶。具有识别特定的碱基序列并加以切割的功能。日本东京大学的小宫山真教授为我们就其有关机理及应用用研究进行阐述。[编者按]     

极端嗜热古菌DNA修复核酸内切酶的研究进展

极端嗜热古菌由于生活在高温环境,其基因组DNA面临着严重的挑战,因此,它们如何维持其基因组稳定是本研究领域最为关注的科学问题之一。极端嗜热古菌具有与常温微生物相似的自发突变频率,暗示着它们比常温微生物具有更加有效的DNA修复体系进行修复高温所造成的基因组DNA损伤。目前,极端嗜热古菌DNA修复的分子机制尚不清楚。核酸内切酶在DNA修复途径中发挥着重要的作用。基因组序列显示极端嗜热古菌编码多种DNA修复核酸内切酶,但是其研究尚处于初期阶段。本文综述了极端嗜热古菌DNA修复核酸内切酶Nuc S、Endo V、Endo Q、XPF和Hjc的研究进展,并对今后的研究提出了展望。     

Localization of Apoptosis (Programmed Cell Death) in Mice by Administration of Lipopolysaccharide

Localization of apoptotic cells by administration of lipopolysaccharide into mice was studied by using the in situ specific labeling of fragmented DNA. This method clearly stained the nuclei of thymocytes at the cortex of the thymus. The nuclei of cells in the bone marrow and in the spleen were also positively stained. It was suggested that the cortex in the thymus is where the LPS-induced programmed cell death occurs.     

Apoptosis and DNA fragmentation precede TNF-induced cytolysis in U937 cells.

The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF-mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2-3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1-2 h prior to cell death as defined by trypan blue exclusion or 51Cr release. DNA fragmentation was not a non-specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF-resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF-mediated cytotoxicity. Two agents known to inhibit TNF-mediated cytotoxicity, ZnSO4 and 3-aminobenzamide, were shown to inhibit TNF-induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.     

Apoptosis in Etiolated Wheat Seedlings: 1. Ultrastructure of the Apoptotic Cell

We studied the process of apoptosis in etiolated wheat (Triticum aestivum L.) seedlings. As a result, an integral pattern of the apoptotic plant cell ultrastructure was established. In the apoptotic cells of the coleoptile, we observed chromatin condensation and margination, an increased density and specific cytoplasm fragmentation accompanied by the appearance of unusual cytoplasmic vesicles containing subcellular organelles, mitochondria in particular, in the vacuoles.     

Genotoxic effect of cadmium is associated with apoptotic changes in tobacco cells

This work explores the influence of cadmium on a suspension cell culture of Nicotiana tabacum (TBY‐2) by examining cell morphology, viability and DNA integrity. Changes in these parameters were strikingly dependent on concentration of cadmium in the culture medium: a concentration of 50–100 mmol m ^{? 3} CdSO₄ induced apoptotic changes including DNA fragmentation into oligonucleosomal units, while 1 mol m ^{? 3} Cd^{2 +} showed strong cytotoxicity, but no fragmentation of DNA. Low cadmium concentrations (below 10 mmol m ^{? 3}) affected neither cell viability nor DNA integrity. A detailed kinetic study showed a significant delay in the onset of apoptosis after the application of high concentrations of cadmium. From days 0–3 after the application of 50 mmol m ^{? 3} CdSO₄, the morphology of the cells, their viability and growth were indistinguishable between control and treated cells, and ‘domain’ DNA fragmentation into 50–200 kb fragments was observed at the DNA level. After this (days 4–7), there was a characteristic and rapid decrease in cell viability, distinct changes in cell morphology and oligonucleosomal fragmentation. The results suggest that chronic exposure of plant cells to cadmium can trigger programmed cell death.     

Development of oxidovanadium and oxido-peroxido vanadium-based artificial DNA nucleases via multi spectroscopic investigations and theoretical simulation of DNA binding

Benzimidazole is a neutral ligand which is often used to synthesize bioactive compounds. Two transition metal benzimidazole-based complexes, namely, vanadium (IV) dioxido complex (complex 1) and vanadium (V) oxido-peroxido complex (complex 2) with tridentate benzimidazole ligand, 2,6-di (1H-benzo[d]imidazol-2-yl) pyridine (Byim) have been designed with the intention of developing potential DNA nuclease. Different studies involving biochemical and biophysical techniques along with molecular docking suggest that both the complexes interact with DNA, while the mode of binding is intercalation. The complexes were further used for DNA cleavage activity. Both of them were found to have substantial DNA nuclease activity, but complex 2 was more potent than complex 1 in exhibiting such activity.     

嗜热古菌Archaeoglobus fulgidus RecJ核酸酶的表达纯化及酶学特征

[目的]克隆表达嗜热古菌Archaeoglobus fulgidus(A.fulgidus)来源的RecJ核酸酶基因(ORF编号AF_0699,NCBI数据库基因登陆号为AF_RS03550),对该重组蛋白的核酸酶活性及酶学特征进行鉴定和分析.[方法]将A.fulgidus RecJ(AfuRecJ)核酸酶在大肠杆菌中...     

In vitro apoptotic cell death during erythroid differentiation

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.     

Caspase-dependent and independent cell death in rat hepatoma 5123tc cells

The objective of this study was to establish whether apoptosis in 5123tc rat hepatoma cells required the caspase-3 dependent pathway. Apoptosis was induced by either growth factor deprivation or treatment with a topoisomerase II inhibitor, VM26, in the absence or presence of caspase inhibitors (DEVD-fmk, z-VAD-fmk and BAF). The results indicated that, although these inhibitors at 10 M concentration completely blocked caspase-3 activity, they had no effect on either the rate of cell death or on any other apoptotic features, e.g., chromatin condensation, DNA fragmentation, protein cleavage, suggesting that caspase-3 was not required to mediate nuclear destruction in these hepatoma cells. At higher concentrations, up to 100 M, z-VAD-fmk and BAF, but not DEVD-fmk, did block apoptosis, however, they also caused cell swelling and membrane permeabilization, which are the hallmarks of necrotic cell death. Clearly, high concentrations of these inhibitors must have interfered non-specifically with other metabolic pathways, e.g., z-VAD-fmk at a high concentration blocked protein phosphorylation, and caused cell death by a different mechanism.