A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 m guanidine hydrochloride (Gdm·HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm·HCl with a G25 column and simultaneously dissolved in 8 m urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding. Revisions requested 31 October 2005; Revisions received 20 December 2005     

Citrus auraptene targets translation of MMP-7 (matrilysin) via ERK1/2-dependent and mTOR-independent mechanism

Matrix metalloproteinase (MMP)-7 is considered to play essential roles in cancer progression. We examined the efficacy of auraptene, a citrus coumarin derivative, for suppressing MMP-7 expression in the human colorectal adenocarcinoma cell line HT-29. Auraptene remarkably inhibited the production of proMMP-7 protein, without affecting its mRNA expression level. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), showed similar results, suggesting that auraptene suppresses mTOR-dependent proMMP-7 translation. Interestingly, however, auraptene showed no effects on the activation of Akt/mTOR signaling, whereas the phosphorylation levels of 4E binding protein (4EBP)1 and eukaryotic translation initiation factor (eIF)4B were substantially decreased. In addition, auraptene remarkably dephosphorylated constitutively activated extracellular signal-regulated kinase (ERK)1/2. Transfection of ERK1/2 siRNA led to a significant reduction of proMMP-7 protein production as well as of the phosphorylation of eIF4B. These results demonstrate that auraptene targets the translation step for proMMP-7 protein synthesis by disrupting ERK1/2-mediated phosphorylation of 4EBP1 and eIF4B.     

外周血NETs、TP53、STAT3表达与弥漫性大B细胞淋巴瘤临床病理及预后的关系分析

摘要 目的：探究外周血中性粒细胞胞外诱捕网（NETs）、TP53、信号转导与转录因子3（STAT3）表达与弥漫性大B细胞淋巴瘤（DLBCL）临床病理及预后的关系。方法：选取2020年3月-2021年12月收治的71例DLBCL患者作为研究对象,抽取患者外周静脉血,采用R-CHOP方案进行治疗,记录患者外周血NETs、TP53、STAT3表达情况并分析DLBCL患者外周血NETs、TP53、STAT3表达与其临床病理及预后的关系。结果：髓细胞组织增生蛋白（MYC）阳性在TP53阳性中的占比显著高于TP53阴性,差异有统计学意义（x²=28.844,P＜0.001）;Hans分型生发中心B细胞（GCB）在STAT3阳性中的占比显著高于STAT3阴性（x²=4.331,P=0.037）,其余差异无统计学意义（P>0.05）;随访截止至2022年6月,随访时长8~28个月,71例患者中共53例缓解DLBCL患者,其余18例为R/R DLBCL患者;NETs阳性、TP53阳性、STAT3阳性患者无进展生存期（PFS）显著低于NETs阴性、TP53阴性、STAT3阴性患者,差异有统计学意义（P<0.05）且NETs阳性、TP53阳性、STAT3阳性患者存活率均低于NETs阴性、TP53阴性、STAT3阴性患者（P<0.05）;单因素分析结果显示Ann Arbor分期、NETs、TP53、STAT3为DLBCL患者的影响因素（P<0.05）;以患者预后情况（R/R DLBCL=1,缓解DLBCL=0）为因变量,将Ann Arbor分期、NETs、TP53、STAT3单因素分析有统计学意义的因素纳入COX回归模型中,结果显示：NETs、TP53、STAT3为DLBCL患者预后的危险因素（P<0.05）。结论：TP53、STAT3表达与DLBCL临床病理存在一定相关性,临床应对DLBCL患者TP53、STAT3表达情况引起重视;NETs、TP53、STAT3表达为DLBCL预后的危险因素,可作为DLBCL患者不良预后的预测指标。     

JNK相互作用蛋白通过JNK途径影响鼻咽癌的增殖和凋亡

EB病毒编码的瘤蛋白潜伏膜蛋白（LMP1）所介导的活化蛋白（AP-1）信号转导途径在细胞增殖、分化、转化与凋亡方面发挥着重要作用.越来越多的证据表明,AP-1信号转导通路中上游激酶JNK在鼻咽癌的发生发展过程中起着重要作用.最近克隆出来的JNK相互作用蛋白(JIP-1)是一种能抑制JNK核移位的胞浆锚蛋白.为探讨JIP在LMP1调控AP-1信号通路中的作用机制,采用间接免疫荧光法和报告基因法,发现JIP通过有效地抑制磷酸化的JNK从胞浆移位入核,从而抑制LMP1上调的AP-1活性.同时,JIP导入鼻咽癌细胞中,MTT法发现JIP能够明显抑制鼻咽癌细胞的生长.进一步发现转染JIP后细胞的集落形成率与对照组相比大约降低了53.6%,也抑制了细胞. 提示JIP可明显抑制细胞的增殖作用.进一步采用流式细胞术分析,结果发现JIP引起细胞G1/S期细胞阻滞,说明JIP是抑制细胞增殖的重要调节子.进一步采用流式细胞术定量发现,转染JIP后细胞的24 h凋亡百分率由1.25%上升到8.25%,上升约6.6倍,48 h由1.04%上升到31.45%,上升约30倍. 采用激光共聚焦显微镜发现,转染JIP后细胞核发生显著变化,核质由均匀状态固缩成高凝集状态,形成了典型的胞膜体.提示JIP可有效地促进细胞凋亡.结果表明,JIP可通过抑制活化的JNK核移位,降低LMP1所介导的AP-1信号通路.并进一步发现JIP可有效地抑制细胞增殖和细胞凋亡,从而提示JIP可作为新的治疗肿瘤潜在靶分子.     

链霉菌C-3662产生的纤溶活性蛋白酶的纯化与理化性质

 从链霉菌 C- 3662发酵上清液中 ,通过硫酸铵沉淀 ,CM- Sepharose Fast Flow和 Phenyl-Sepharose Fast Flow等层析色谱 ,分离纯化得到了具有纤溶活性的蛋白酶 CGW- 3,反向 HPLC鉴定纯度为 90 % ;每立升发酵上清液可得到 8mg纯品 ,活性回收率 46% ,CGW- 3为一单肽链蛋白 ,分子量 2 2 72 1 ,对丝氨酸蛋白酶抑制剂 PMSF敏感 ,对 EDTA不敏感 ;其 N端 1 5个氨基酸的顺序为 VVGGTRAAQGEFPFM,与微生物来源的胰蛋白酶类丝氨酸蛋白酶有较高的同源性 . CGW- 3的等电点 p I9.0 ,纤溶活性的最适 p H为 7.5～ 8.0 ,对温度比较敏感 .CGW- 3不仅具有直接降解纤维蛋白作用 ,而且能够激活纤溶酶原     

人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化

 本文报道了培养的人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化方法。Bowes株人黑色素瘤细胞的分泌产物,经CM-Sephadex C--50层析,赖氨酸-Sepharose 4B,苯甲眯-sepharose 4B亲和层析后,即可得到纯化470倍的蛋白纯品。样品经聚丙烯酰胺凝胶电泳鉴定为均一单带,测得其分子量约为72kD。纯化的t-PA与尿激酶相比较,发现前者有更高亲和纤维蛋白的能力。     

The Saccharomyces cerevisiae phosphotyrosyl phosphatase activator proteins are required for a subset of the functions disrupted by protein phosphatase 2A mutations

In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaDelta mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21Delta genetic background. Moreover, ypaDelta mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypaDelta mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.