分子伴侣grp75在缺糖损伤细胞恢复中的作用

Glucose-regulated proteins 75(grp75) is a member of hsp70 family. The expression of grp75 is upregulated during glucose starvation (such as ischemia). To evaluate grp75 function, CHL cells were cultured with glucose-free media for 20 h (A) and glucose-free media for 12 h + glucose-containing media for 8 h (ischemia reperfusion) (B). A constructed rat grp75 cDNA expression vector (pcDNA/grp75) was transfected into CHL cells and a cell strain that stably overexpressed grp75 was obtained. The transfected cells and untransfected cells(control group) were cultured with A or B. By MTT, LDH leakage measurement and flow cytometry analysis, growth rate of untransfected cells in B is significantly lower than that in glucose-containing media for 20 h (C) (p < 0.05) and A (p < 0.05). Growth rate of transfected cells is apparently higher than that of control group in B (p < 0.01). LDH liberation percentage of untransfected cells in B is obviously higher than that in C(p < 0.01) and it is not different from A(p > 0.05). LDH liberation percentage of transfected cells is apparently lower than that of control group in B(p < 0.01). Apoptosis of transfected cells is obviously lower by flow cytometry analysis. These results provide evidence for the cytoprotective function of grp75 during glucose starving and ischemia reperfusion.     

L-谷氨酰胺对PC12细胞中grp75的调控作用

条件必需氨基酸谷胺酰胺可上调细胞中热激蛋白(hsp)的表达,为观察谷氨酰胺是否对hsp 家族成员grp75的表达具有调控作用,以PC12细胞为模型用免疫组化、蛋白质印迹法和RT-PCR 等方法检测谷胺酰胺对grp75基因的表达的影响;并以MTT法观察谷氨酰胺对PC12的细胞和grp75低表达的PC12细胞缺糖损伤的保护作用。结果表明谷氨酰胺可以上调grp75的表达,特别是对缺糖细胞的上调作用更显著;但这种上调作用与谷氨酰胺的作用浓度和作用时间并未显示出有明显的关系。MTT检测显示,谷氨酰胺使细胞在缺糖条件下的存活率明显上升;grp75低表达细胞与未转染的细胞相比这种保护效应明显降低,说明谷氨酰胺通过调节grp75的表达对缺糖损伤起到保护作用     

分子伴侣grp75在缺糖损伤细胞恢复中的作用

葡萄糖调节蛋白75(grp75)属于热休克蛋白70家族中的一员,细胞中葡萄糖水平下降时(类似于缺血),grp75表达增高。为研究grp75在缺糖及缺糖再灌注条件下对细胞的作用,本文以中国仓鼠肺细胞株CHL为材料,采用脂质体介导的方法,以grp75表达载体pcDNA3/grp75转染CHL细胞,获得过表达grp75的细胞克隆;置于无糖培养基培养20h及无糖培养12h换含糖培养基继续培养8h(缺糖再灌注)或含糖培养20h,运用MTT法、LDH测定和流式细胞术分析等方法评估细胞损伤程度。MTT测定显示,未转染细胞缺糖再灌流的增殖能力比完全培养20h增殖能力明显降低(p<0.05),且低于无糖培养20h(p<0.05),转染细胞缺糖再灌流的增殖能力明显高于对照组(p<0.01);LDH测定结果显示,未转染细胞缺糖再灌流LDH释放百分比显著高于完全培养20h(p<0.01),与无糖培养20h无明显差别(p>0.05),转染细胞缺糖再灌流LDH释放百分比显著低于对照组(p<0.01);流式细胞术分析表明,转染细胞的凋亡率明显低于对照组。以上结果表明grp75过表达的细胞在缺糖损伤细胞的恢复中具有一定强度的抗损害作用。     

L-谷氨酰胺对PC12细胞中grp75的调控作用

条件必需氨基酸谷胺酰胺可上调细胞中热激蛋白（hsp）的表达,为观察谷氨酰胺是否对hsp家族成员grp75的表达具有调控作用,以PC12细胞为模型用免疫组化、蛋白质印迹法和RT—PCR等方法检测谷胺酰胺对grp75基因的表达的影响：并以MTT法观察谷氨酰胺对PC12的细胞和grp75低表达的PC12细胞缺糖损伤的保护作用。结果表明谷氨酰胺可以上调grp75的表达．特别是对缺糖细胞的上调作用更显著;但这种上调作用与谷氨酰胺的作用浓度和作用时间并未显示出有明显的关系。MTT检测显示,谷氨酰胺使细胞在缺糖条件下的存活率明显上升：grp75低表达细胞与未转染的细胞相比这种保护效应明显降低,说明谷氨酰胺通过调节grp75的表达对缺糖损伤起到保护作用。     

grp75：脑缺备性损伤的分子标记

ｇｒｐ７５基因是一种对缺氧、缺葡萄糖敏感的基因,由于它在脑缺血性损伤刺激后呈现－上调表达,故可作为是脑缺血损伤的一种新的分子标记,有意义的是相对于热激蛋白等其他分子标记而言,在以缺葡萄糖为主要特征的脑缺血性损伤中,ｇｒｐ７５上有更高的特异性和敏感性;另一方面,ｇｒｐ７５作为一种保护性蛋白,对于细胞损伤的分子治疗将具有重要的、潜在的应用前景。     

辛酸钠对骨骼肌细胞缺糖缺氧/再灌注损伤的保护作用

该研究探索了辛酸钠对骨骼肌细胞缺糖缺氧/再灌注(OGD/Rep)损伤的保护作用.利用锥虫蓝染色法测定6种不同浓度的辛酸钠培养液对正常培养骨骼肌细胞24h存活率的影响.随后采用缺糖缺氧后复糖复氧的方法构建骨骼肌细胞OGD/Rep损伤模型,将细胞随机分为对照组、OGD/Rep组、0.25 mmol/L辛酸钠组和0.50 m...     

GRP75经由Raf/Mek/Erk1/2信号转导通路抑制缺糖诱导的细胞凋亡

本文旨在探讨促存活信号通路Raf/Mek/Erk1/2是否参与了葡萄糖调节蛋白75(glucose-regulated protein75,GRP75)对缺糖诱导的细胞凋亡的抑制作用。GRP75过表达的PC12细胞给予Raf/Mek/Erk1/2通路抑制剂U0126预处理之后,无糖培养6、12和24h,同时以DMSO预处理的GRP75过表达PC12细胞组为对照。Western blot检测Erk1/2的磷酸化和表达水平,MTT实验检测细胞存活率,Hoechst 33258染色观察凋亡细胞核的形态学改变,流式细胞仪检测细胞亚二倍体峰,免疫荧光检测细胞色素c(cytochrome c,Cytc)向胞浆的弥散情况。结果显示:U0126在没有影响Erk1/2表达水平的前提下,阻断了GRP75对Erk1/2磷酸化水平的维持;U0126处理组的凋亡率明显高于对照组;U0126处理组Cytc从线粒体向胞浆释放的时间明显早于对照组,同时Cytc向胞浆的弥散程度大于对照组。以上结果提示,U0126通过抑制Erk1/2磷酸化,阻断了缺糖状态下GRP75对Cytc释放和细胞凋亡的抑制作用,这表明GRP75是通过Raf/Mek/Er...     

葡萄糖调节蛋白75的研究进展

葡萄糖调节蛋白75(Grp75)是高度保守的热激蛋白家族中的一员,在细胞内主要行使伴侣蛋白的功能,帮助未折叠或错误折叠蛋白质进行正确的折叠,还与细胞内多个因子结合,参与细胞内多个重要的生物学过程。现就Grp75的基因定位、蛋白质分布和功能以及临床研究展开本综述。     

TRPC通道对PC12细胞缺氧缺糖再灌注后氧化损伤的保护作用

瞬时受体电位C通道(transient receptor potential canonical,TRPC)属于瞬时受体电位（TRP）离子通道家族成员之一,与Ca2+ 调控及氧化应激关系密切．然而,TRPC通道在缺血缺氧性脑损伤中的作用仍有争议．本实验采用MTT法、台盼蓝排斥实验、LDH漏出实验联合Annexin/PI染色流式分析等显示,当采用通道阻断剂-SKF96365特异阻断TRPC通道时,PC12细胞对缺氧缺糖再灌注（oxygen-glucose deprivation／reperfusion,OGD-R）引起的损伤变得更敏感,细胞凋亡增加．尽管同时采用SKF96365、NMDA受体、AMPA受体和L-型钙通道阻断剂可引起细胞损伤和死亡减弱,但仍呈现明显的SKF96365浓度依懒性,提示TRPC通道参与细胞对缺氧缺糖再灌注耐受的调节,具有保护作用．钙指示剂Fluo-3AM荧光标记结合激光共聚焦显微镜分析显示,SKF96365可明显降低缺氧缺糖再灌注后细胞内的Ca2+浓度．总之,实验结果提示,TRPC通道对缺氧缺糖再灌注引起的细胞损伤和凋亡具有保护作用,其机制可能涉及TRPC通道对细胞内钙浓度的调节,详尽机制有待进一步研究．     

纤维连接蛋白对支气管上皮细胞抗氧化损伤保护作用的研究

目的和方法：为验证整合素分子激活对支气管上皮细胞（BEC）的抗氧化性保护作用,本实验用臭氧（O3）攻击培养的兔BEC,测定细胞的^3H释放率、乳酸脱氢酶（LDH）释放活性及脂质过氧化产物丙二醛（MDA）含量,反映细胞损伤程度;观察纤维连接蛋白（Fn）及人工合成的精－甘－天冬氨酸片段（RGD肽）的保护效应。结果：①臭氧攻击使BEC的^3H释放率增高,Fn处理可减少臭氧所致的^3H释放,钙调素抑制剂W7能抑制Fn的这一作用,RGD可减轻臭氧所致的^3H释放;②臭氧攻击后细胞上清液中LDH释放增多,Fn或RGD处理均能降低LDH释放,W7阻断Fn的这一效奕;③臭氧作用后明显提高细胞内MDA含量,Fn或RGD可降低MDA含量;④臭氧攻击使细胞内GSH含量下降,Fn或RGD可增加BEC内GSH的含量;⑤Fn可增强BEC内过氧化氢酶（CAT）活性,但可被W7阻断,RGD则显示有剂量依赖性促进作用。结论：Fn及其特异识别片段与BEC的整合素分子结合后,可减轻臭氧对BEC细胞的损伤,其机理与经钙调素途径上调BEC抗氧化能力有关。     

A Sericin-Derived Peptide Protects Sf9 Insect Cells from Death Caused by Acute Serum Deprivation

Sericin is the silk protein enveloping fibroin fibers in cocoons. Sericin hydrolysate protects cultured Sf9 insect cells from death caused by serum deprivation; the activity depends on the repeats of 38 amino acids. A partial peptide from the 38 residues, SGGSSTYGYS, inhibited serum-deprivation death as well. Cell viabilities in the presence of 10% (v/v) foetal calf serum, no additives and 1 mM SGGSSTYGYS were 96, 12 and 31% on the third day after inoculation, respectively. Aromatic residues seemed to be important because SGGSSTWGWS had the same activity as SGGSSTYGYS but SGGSSTAGAS had no activity.     

The squamous cell carcinoma cell line OM-1 retains both p75-dependent stratified epithelial progenitor potential and cancer stem cell properties

The low-affinity nerve growth factor receptor p75 is a stratified epithelial stem/progenitor marker of human epithelia. We found OM-1, a human squamous cell carcinoma (SCC) cell line, showed distinct cells with p75 cluster, especially located at the center of a growing colony in a monolayer culture. A cell with p75 cluster was surrounded by cytokeratin 14- and cytokeratin 13-expressing cells that settled at the outer margin of the colony. OM-1 cells were also capable of forming tumor spheres in a cell suspension culture, an ability which was attenuated by the inhibition of p75-signaling. Intriguingly, we also found a p75-negative cell population from a growing culture of OM-1 that re-committed to become p75-clustering cells. These results indicated the possibility that SCC with epithelial multi-layering capacity can exploit the p75-dependent stratified epithelial progenitor property for the cancer stemness.     

Ubiquitin-independent proteasomal degradation of endoplasmic reticulum-localized connexin43 mediated by CIP75

Connexin43 (Cx43) is a transmembrane protein that forms gap junction channels. Regulation of Cx43 turnover is one mechanism to control the level of intercellular communication that occurs through gap junction channels. Proteasomal degradation of Cx43 is regulated in part through CIP75, a ubiquitin-like and ubiquitin-associated domain containing protein. CIP75 interacts with endoplasmic reticulum-localized Cx43, as demonstrated through co-immunoprecipitation and immunofluorescence microscopy experiments. CIP75 also binds to free monoubiquitin and lysine 48-linked tetraubiquitin chains in vitro and binds to ubiquitinated proteins in cellular lysates. However, analysis of Cx43 that immunoprecipitated with CIP75 demonstrated that the Cx43 associated with CIP75 was not ubiquitinated, and a mutant form of Cx43 that lacked lysines capable of ubiquitination retained the capacity to interact with CIP75. These results suggest that although CIP75 can interact with ubiquitinated cellular proteins, its interaction with Cx43 and stimulation of Cx43 proteasomal degradation does not require the ubiquitination of Cx43.     

Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150^Glued subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.     

抗白血病药物三尖杉酯碱对L1210细胞着丝粒蛋白含量及CenpB基因表达的影响

以小鼠白血病细胞系L1210为对象,探讨了抗白血病药物三尖杉酯碱(Harringtonine,HT或Har)对细胞内着丝粒蛋白含量及着丝粒蛋白CenP基因表达的影响。间接免疫荧光(IIF)检测结果显示,随HT作用时间延长,L1210细胞着丝粒荧光斑点减弱;免疫印迹(Western blot)检测结果显示,所用的抗着丝粒抗血清(ACA 血清)能够识别8种不同分子量的着丝粒蛋白：140、80、70、56、37、34、32和17kD。受HT作用,细胞中这些着丝粒蛋白的含量不同程度地降低。在L1210细胞中识别17、80 and l40kD蛋白质的ACA抗体也分别与分子量相当的已知为CenpA、CenpB和CenpC的3种蛋白发生交叉反应。Northern和Dot blot显示,HT的抑制作用使细胞中CenpB mRNA表达水平较之对照细胞下降。结果表明,HT可(通过抑制基因mRNA的表达)降低细胞中某些着丝粒蛋白的含量;HT对细胞的杀伤及诱导凋亡作用可能与CenpB等着丝粒蛋白基因的表达抑制有关。     

Exocytosis in Single Chromaffin Cells: Regulation by a Secretory Granule-Associated Go Protein

1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, Go has beenfound associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells.2. Microinjection of GTPS and mastoparan strongly inhibits the amperometric response to either nicotine or high K⁺.3. The presence of mastoparan in the cell incubation medium had no effect on K⁺-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane.4.Microinjection of anti-Go antibodies potentiates by more than 50% the K⁺-evoked secretion, whereas anti-Gi_1/2 antibodies have no effect.5. Thus an inhibitory Go protein, probably associated with secretory granules, controls exocytosis in chromaffin cells. The intracellular proteins controlling organelle-associated G proteins are currently unknown. The neuronal cytosolicprotein GAP-43 stimulates Go in purified chromaffin granule membranes and inhibits exocytosis in permeabilized cells. We show here that microinjection of a synthetic peptide corresponding to the domain of GAP-43 that interacts with Go inhibits secretion. We suggest that GAP-43 or a related cytosolic protein controls the exocytotic priming step in chromaffin cells by stimulating a granule-associated Go protein.     

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.     

HCV结构蛋白在昆虫细胞中的表达及病毒样颗粒的组装

本文利用Bac-to-Bac杆状病毒表达系统构建了含有丙型肝炎病毒(Hepatitis C Virus,HCV)结构蛋白编码基因的重组杆状病毒vAcHCVspl,并获得了HCV结构蛋白在昆虫细胞Sf21中的表达。HCV mRNA转录和蛋白质表达时相分析表明,感染后16h HCV结构蛋白编码基因开始转录,72h达最高峰;蛋白质表达则是在感染后48h开始,72h达到高峰。电镜观察表明vAcHCVspl感染的Sf21细胞96h时在细胞质中可见很多空泡,空泡中可见50nm的球形颗粒,为HCV结构蛋白组装的病毒样颗粒。     

p75 Regulates Purkinje Cell Firing by Modulating SK Channel Activity through Rac1

p75 is expressed among Purkinje cells in the adult cerebellum, but its function has remained obscure. Here we report that p75 is involved in maintaining the frequency and regularity of spontaneous firing of Purkinje cells. The overall spontaneous firing activity of Purkinje cells was increased in p75^−/− mice during the phasic firing period due to a longer firing period and accompanying reduction in silence period than in the wild type. We attribute these effects to a reduction in small conductance Ca²⁺-activated potassium (SK) channel activity in Purkinje cells from p75^−/− mice compared with the wild type littermates. The mechanism by which p75 regulates SK channel activity appears to involve its ability to activate Rac1. In organotypic cultures of cerebellar slices, brain-derived neurotrophic factor increased RacGTP levels by activating p75 but not TrkB. These results correlate with a reduction in RacGTP levels in synaptosome fractions from the p75^−/− cerebellum, but not in that from the cortex of the same animals, compared with wild type littermates. More importantly, we demonstrate that Rac1 modulates SK channel activity and firing patterns of Purkinje cells. Along with the finding that spine density was reduced in p75^−/− cerebellum, these data suggest that p75 plays a role in maintaining normalcy of Purkinje cell firing in the cerebellum in part by activating Rac1 in synaptic compartments and modulating SK channels.     

Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3

It is well known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box-binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a VEGF receptor and PI3K/Akt-dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Overexpression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization, and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3, and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.