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1.
人DFF45蛋白多克隆抗体制备及其初步应用 总被引:2,自引:1,他引:1
目的:表达、纯化带GST标签的DNA裂解因子45(DNA fragmentation factor 45 ,DFF45)融合蛋白并制备多克隆抗体。方法:构建pGEX-5X-1/DFF45原核表达质粒,转化大肠杆菌BL21,用IPTG诱导融合蛋白表达,经纯化后免疫日本大耳白兔得到多克隆抗体并用CNBr-activated Sepharose? 4B进行纯化。用间接ELISA法检测抗体效价,Western blot鉴定抗体特异性,同时用免疫荧光染色鉴定抗体特异性并观察DFF45的细胞定位。进一步检测DFF45在人类几种细胞系的表达差异情况。结果:成功构建原核表达质粒,表达、纯化DFF45蛋白并免疫动物后得到多克隆抗体。间接ELISA法显示抗体效价达1:20 000,Western blot确定抗体具有高度特异性。应用该抗体检测发现DFF45在人类几种细胞系中表达存在差异并观察到其细胞定位。结论:DFF45多克隆抗体的成功制备及其在人类细胞系的差异表达,为进一步研究DFF45基因与肿瘤及其相关疾病的关系奠定了基础。 相似文献
2.
Cidea蛋白调节脂肪代谢,在机体能量平衡过程中起重要作用,在转录和翻译后水平受到严格调控,但在翻译水平的调节还不清楚.通过对CIDEA基因敲除小鼠模型研究,鉴定了小鼠棕色脂肪组织内源性表达Cidea蛋白N端缺失异构体mCidea-22.定点突变等研究表明其产生机制为选择性起始翻译.并且,在异位表达时,N端缺失异构体和全长异构体的比例呈现细胞系特异性.此外,蛋白质稳定性实验表明mCidea-22半衰期很短.亚细胞定位研究显示mCidea-22是内质网和脂滴定位蛋白.为深入理解Cidea蛋白的功能和精细调节提供了新的思路和方向. 相似文献
3.
Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells 总被引:3,自引:0,他引:3
Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption. 相似文献
4.
利用两种不相容质粒在大肠杆菌中共表达DFF45和DFF40 总被引:1,自引:0,他引:1
DNA断裂因子(DNA fragmentation factor,DFF)是细胞凋亡过程中起重要作用的蛋白质之一,它由分子量为45kD和40kD的两个亚基构成,分别称为DFF45和DFF40。利用RT-PCR技术从人宫颈癌细胞系HeLa的总RNA中扩增了DFF45和DFF40的cDNA,分别克隆到到卡那霉素抗性表达载体pET-28a( )中,构建了pET28a-DFF45和pET28a-DFF40。用它们单独转化大肠杆菌BL21(DE3)后,经IPTG诱导都可获得高效表达。表达的重组蛋白质各自约占菌体总蛋白质的56%和22%。再将DFF45的cDNA克隆到氨苄青霉素抗性表达载体pET-21a( )中,得到了pET21a-DFF45。利用二者的不同抗性,将pET28a-DFF40和pET21a-DFF45共同转化大肠杆菌BL21(DE3),工程菌经IPTG诱导后实现了DFF45和DFF40的共表达,表达产物各约占菌体总蛋白质的30%和17%。为了研究这两种不相容质粒在细菌中共存的稳定性,我们将共转化子在同时含有卡那霉素和氨苄青霉素的液体培养基中连续培养14h,发现此时仍有75%以上的细菌可同时耐受两种抗生素,即同时含有pET28a-DFF45和pET28a-DFF40,说明利用两个具有不同抗性的不相容载体进行蛋白质共表达的方法是可行的。 相似文献
5.
Muhammad Umer Ramanathan Vaidyanathan Nam-Trung Nguyen Muhammad J.A. Shiddiky 《Biotechnology advances》2018,36(4):1367-1389
Circulating tumor cells (CTCs) and their clusters, also known as circulating tumor microemboli (CTM), have emerged as valuable tool that can provide mechanistic insights into the tumor heterogeneity, clonal evolution, and stochastic events within the metastatic cascade. However, recent investigations have hinted that CTM may not be mere aggregates of tumor cells but cells comprising CTM exhibit distinct phenotypic and molecular characteristics in comparison to single CTCs. Moreover, in many cases CTM demonstrated higher metastatic potential and resistance to apoptosis as compared to their single cell counterparts. Thus, their evaluation and enumeration may provide a new dimension to our understanding of cancer biology and metastatic cancer spread as well as offer novel theranostic biomarkers. Most of the existing technologies for isolation of hematogenous tumor cells largely favor single CTCs, hence there is a need to devise new approaches, or re-configure the existing ones, for specific and efficient CTM isolation. Here we review existing knowledge and insights on CTM biology. Furthermore, a critical commentary on current and emerging trends in CTM enrichment and characterization along with recently developed ex-vivo CTC expansion methodologies is presented with the aim to facilitate researchers to identify further avenues of research and development. 相似文献
6.
Desharnais P Dupéré-Minier G Hamelin C Devine P Bernier J 《Apoptosis : an international journal on programmed cell death》2008,13(2):197-212
CD45 is a type I transmembrane molecule with phosphatase activity which comprises up to 10% of the cell surface area in nucleated
haematopoietic cells. We have previously demonstrated the absence of nuclear apoptosis in CD45-negative T cells after chemical-induced
apoptosis. The aim of this study was to characterize the role of CD45 in nuclear apoptosis. In contrast to wild type CD45-positive
T cells, the CD45-deficient T cell lines are resistant to the induction of DNA fragmentation and chromatin condensation following
tributyltin (TBT) or H2O2 exposure, but not to cycloheximide-induced apoptosis. CD45 transfection in deficient cell lines led to the restoration of
chromatin condensation and DNA fragmentation following TBT exposure. In both CD45-positive and negative T cell lines, TBT
exposure mediates intracellular calcium mobilization, caspase-3 activation and DFF45 cleavage. Moreover, DNA fragmentation
was also induced by TBT in cells deficient in expression of p56lck, ZAP-70 and SHP-1. Subcellular partitioning showed a decrease
in nuclear localisation of caspase-3 and DFF40. Together, these results demonstrate for the first time, that CD45 expression
plays a key role in internucleosomal DNA fragmentation and chromatin condensation processes during apoptosis. CD45 activity
or its substrates’ activity, appears to be located downstream of caspase-3 activation and plays a role in retention of DFF40
in the nucleus.
Philippe Desharnais and Geneviève Dupéré-Minier have contributed equally to this work. 相似文献
7.
8.
Iglesias-Guimarais V Gil-Guiñon E Gabernet G García-Belinchón M Sánchez-Osuna M Casanelles E Comella JX Yuste VJ 《The Journal of biological chemistry》2012,287(10):7766-7779
Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. 相似文献
9.
The apoptotic endonuclease DFF40/CAD is inhibited by RNA, heparin and other polyanions 总被引:1,自引:0,他引:1
Widlak P Garrard WT 《Apoptosis : an international journal on programmed cell death》2006,11(8):1331-1337
DFF40/CAD, the major apoptotic nuclease, is specific for double-stranded DNA. However, RNA and single-stranded DNA, though
not substrates for the enzyme, compete with double-stranded DNA and inhibit its cleavage by the nuclease. In addition, other
anionic polymers, like poly-glutamic acid and heparin also inhibit DFF40/CAD, the latter one being highly effective at nanomolar
concentrations. The inhibitory poly-anions bind to the nuclease and impair its ability to bind double-stranded DNA. We propose
that such poly-anions bind to the positively charged surface formed by α4 helices of the DFF40/CAD homodimer. This surface has been proposed recently to bind to either the major groove of DNA or
poly (ADP-ribose), another inhibitor of the nuclease. 相似文献
10.
Yonezawa T Lee JW Hibino A Asai M Hojo H Cha BY Teruya T Nagai K Chung UI Yagasaki K Woo JT 《Biochemical and biophysical research communications》2011,(2):260-265
Sepsis, the systemic response to infection, is the leading cause of death in the intensive care units worldwide. Septic patients can succumb through the development of early refractory hypotension or late multiple organ dysfunction. Misregulation of apoptosis during sepsis may contribute to cellular dysfunction and multiple organ dysfunction. Utilizing a tissue culture model which mimics the human disease, we demonstrate that the addition of sera derived from septic patients induces apoptosis in human fibroblast cells. Addition of septic sera to 2fTGH cells induced apoptosis by activating caspase 8, caspase 3 and DNA fragmentation factor 40 (DFF 40). Interestingly, the addition of septic sera to cells which lack STAT1 (U3A cells) did not activate DFF 40. U3A cells were also shown to be resistant to septic serum induced apoptosis. These data suggest that DFF 40 mediated apoptosis plays a significant role in mediating sepsis induced cellular dysfunction. 相似文献