Analysis of cell death inducing factors from periodontopathic bacteria.

1.杀细胞毒素的作用在鳞状细胞癌、上皮和造血细胞系中，B可剂量依赖性地诱导细胞死亡。采用WST-1法对红花超声波提取物进行评价。这些结果提示该因子可能通过共同受体或非特异性地诱导细胞死亡。细菌提取物处理后HCT 116细胞的超微结构改变扫描电镜观察到细胞膜严重褶皱（这不是凋亡的特征），透射电镜观察到细胞核碎裂、聚集，胞浆空泡化、肿胀，质膜完整性丧失.杀细胞毒素B的纯化。经DEAE-Sepharose柱层析和分子排阻层析纯化后，在28和400 kDa以上的两个峰级分中检测到了杀细胞活性。比较254 nm处的吸光度表明，该毒素可能与细菌DNA相互作用。所以一间 ...更多信息 在DNA酶I处理后，观察到杀细胞毒性明显转移到28 kDa部分。由于这些结果强烈表明该毒素是DNA结合蛋白，因此通过肝素柱进行进一步纯化。抗杀细胞毒素单克隆抗体的制备用B免疫小鼠。从肿胀的淋巴结中收集白藜芦醇提取物和活化的淋巴细胞。淋巴细胞立即与小鼠骨髓瘤细胞系融合。从杂交瘤中分离出具有抗MA 1细胞杀伤活性的克隆。纯化的毒素处理的MA 1细胞与抗人p53和小鼠抗人Cyclin B1抗体共同孵育，分别用Cy-5和FITC标记的抗体染色。对染色的细胞进行流式细胞术。结果表明，该毒素作用于细胞后，G2期细胞中p53和Cyclin B1表达增加，提示该毒素是CDT家族的一个新成员。少

1. The effects of the cytocidal toxinIn squamous cell carcinoma, epithelial, and hematopoietic cell lines, cell death was induced dose-dependently by B. forsythus ultrasonic extract by the evaluation of WST-1 assay. These results suggest that this factor might induce dell death through a common receptor or non-specifically.2. The ultrastructural change in HCT 116 cells treated with bacterial extractSevere membrane ruffling was observed in SEM analysis (this is not a characteristic feature of apoptosis), Fragmentation and aggregation of nuclei, cytoplasmic vacuolization and swelling, and loss of plasma membrane integrity were demonstrated in TEM analysis.3. Purification of the cytocidal toxinB. forsythus extract was purified by using DEAE Sepharose column and size-exclusion chromatography and cytocidal activity was detected in two peak fractions corresponding to 28 and over 400kDa. Comparing the absorbance at 254 nm suggest that this toxin may interact with bacterial DNA. Therefore, An … More apparent shift of cytocidal toxicity to 28 kDa fraction was observed following DNase I treatment. Since these results strongly suggest that this toxin is a DNA binding protein, further purification was carried out by heparin column.4. Preparation of anti-cytocidal toxin monoclonal antibodiesA mouse was immunized with B. forsythus sonic extract, and the activated lymphocytes were collected from a swollen lymphatic node. The lymphocytes were immediately fused with mouse myeloma cell line. The clones with anti-cytocidal activity against MA1 cells were isolated from hybridomas.5. The effects of cell cycle of MA1 cellsMA1 cells treated with purified toxin were incubated with anti-human p53 and mouse anti-human Cyclin B1 antibodies, and were stained with Cy-5 and FITC labeled antibodies, respectively. Stained cells were subjected to flowcytometry. High levels of p53 and Cyclin B1 were observed at G2 phase in cells treated with this toxin.These results suggest that this toxin is a novel member of CDT family. Less