克雷伯氏菌甘油脱氢酶dhaD的克隆表达、纯化及酶学性质研究

以克雷伯氏菌基因组DNA为模板，扩增得到编码甘油脱氢酶（GDH）的基因dhaD，将其克隆到大肠杆菌表达载体pET-28a(+)上，在E.coliBL21（DE3）中诱导表达，利用表达载体pET-28a(+)上的6·His-Tag标记选用Ni柱亲和层析法纯化表达具有活性的甘油脱氢酶(GDH)，纯化后比酶活达到156U/mg，纯化倍数达4.6倍，回收率为67.4%。并初步研究了该酶的酶学性质，酶反应的最适pH为11.0，在pH7.0～12.0范围内稳定；酶反应的最适温度为30℃，稳定范围为25～45℃; 酶动力学参数以甘油为底物的Km为0.54 mmol/L, Vmax为0.49 μmol/(mL·min)。     

中国林蛙Onconase样核糖核酸酶的基因克隆和表达

Onconase是从美洲豹蛙卵中提取的一种核糖核酸酶,由于其抗肿瘤活性而具有潜在的临床应用价值.以中国林蛙基因组为模板,克隆了一个新的RNase基因,并由此推导出了成熟林蛙RNase的氨基酸顺序.该酶是由103个氨基酸残基组成的,它保留了RNaseA家族成员酶催化活性必须的组氨酸和赖氨酸残基,以及CKXXNTF的序列特征,与Onconase具有73％的氨基酸顺序的相似性.林蛙酶比Onconue少一个氨基酸,成为选今为止发现的RNaseA家族中的最小成员;并且,林蛙酶拥有的精氨酸和酪氨酸残基比Onconase多3个.此外,在利用原核表达系统对林蛙RNase基因进行表达的过程中,表达产物对宿主显示出一定的细胞毒性.     

假单胞菌L-半胱氨酸合成酶的纯化和性质研究

假单胞菌TS-1138的细胞浆液通过硫铵沉淀、SephadexG-75凝胶过滤、DEAE-Cellulose52离子交换、SephadexG 100凝胶过滤等分离纯化手段分别将从L-ATC合成L 半胱氨酸的两个酶——L-ATC水解酶和L-SCC水解酶纯化了 83.9和 90.3倍。SDS-PAGE鉴定均为单一条带 ,两种酶的相对分子质量分别为 37.5和 42.8kD ;酶反应的最适温度均为 35℃ ,最适pH分别为 7.0和 8.0 ;酶的米氏常数分别为 0.67mmol L和 0.15mmol L ,     

排硫硫杆菌D6中硫化氢脱氢酶的纯化及性质

从烟气生物脱硫系统的好氧产硫磁性稳态流化床反应器中,经反复纯化分离出脱硫优势菌排硫硫杆菌菌株D6,采用四步工艺纯化出膜结合型硫化氢脱氢酶。SDS-PAGE测定显示其由α1β1亚基组成,光谱分析表明含有1 mol FAD/mol酶,血红素染色揭示小亚基上结合有1 mol血红素c/mol酶,该酶属于氧还蛋白家族。该酶的最适pH为8.6,对马心细胞色素c和硫化物的表观Km分别为2.5μmol/L和6.1μmol/L,反应计量实验表明其氧化产物为元素硫。硫化氢脱氢酶受到硫和亚硫酸盐的抑制,100μmol/L的氰化钾对该酶抑制率达72%。     

中国林蛙细胞毒性核糖核酸酶的原核表达

目的:利用大肠杆菌以非包涵体的形式表达中国两栖动物林蛙的细胞毒性核糖核酸酶,探索原核表达系统表达细胞毒性核糖核酸酶的条件.方法:利用中国林蛙核糖核酸酶基因及表达质粒pFlag-CTS构建重组表达质粒,转导BL21后用IPTG诱导表达,观察不同诱导温度下细菌的生长情况.利用聚丙烯酰胺凝胶电泳和免疫印迹方法进行重组蛋白的检测.结果:通过观察不同诱导温度下细菌的生长情况,发现利用较低温度可以降低宿主细胞对表达产物细胞毒性的敏感性,成功地得到了这种目的基因的表达产物.结论:低温(20℃)可以有效表达目的蛋白.     

里氏木霉纤维素酶的分离纯化及酶学性质

通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting凝胶色谱脱盐、Source 15 Q阴离子交换色谱技术,里氏木霉(Rut C-30)纤维素酶主要组分得以初步分开,再经过Source 15 S阳离子交换色谱、HiPrep Sephacryl S-100 HR凝胶过滤色谱、Superdex 75 PrepGrade凝胶过滤色谱进一步分离纯化,得到2个纯化的内切葡聚糖酶组分EGⅡ、EGⅠ和一个外切葡聚糖酶组分CBHⅠ;经过SDS-PAGE电泳鉴定为电泳纯,测得相对分子质量分别为5.22×104,5.62×104和6.90×104。EGⅡ的最适反应pH是5.6,最适反应温度为65℃;EGⅠ的最适反应pH是4.4,最适反应温度为55℃;以羧甲基纤维素(CMC)为底物时,EGⅠ、EGⅡ的米氏常数(Km)分别为2.20 mg/mL、3.38 mg/mL。CBHⅠ的最适反应pH是5.8,最适反应温度为60℃,以对硝基苯基-β-D-纤维二糖苷(PNPC)为底物时,米氏常数(Km)为0.12 mg/mL。     

瘦肉型猪(PIC344)精液酸性磷酸酶部分性质研究

用纱网滤掉瘦肉型猪 (PIC344) 新鲜精液中胶状物得原精液, 该原精液经硫酸铵分段盐析、DEAE Sepharose F F 离子交换柱层析、Sephacryl S 200 凝胶过滤后分离纯化到酸性磷酸酶 (Acid Phosphatase, 简称ACPase)。纯化倍数为22 78, 酶液比活力为15 26U/mg蛋白。纯化酶液经非还原性SDS PAGE检测, 呈现单一蛋白着色带。测得该酶相对分子质量为52 3kD, 等电点为5 1, 米氏常数 (Km 值) 为3 08×10-3mol/L。测得该酶最适pH为3 6, 最适温度为52℃。ACPase在pH 3 5~6 0范围内稳定, 在40℃以下稳定, 50℃保温30min后酶活仍能保持59 2%。     

红栓菌胞外漆酶的诱导,纯化及部分特性研究

红栓菌(pycnoporus cinnabarius)在发酵培养3d后出现胞外漆酶活性峰。木素类似物对红栓菌胞外漆酶活性有诱导作用,阿魏酸、香兰素,愈创木酚和DL-β-苯丙氨酸诱导24h后,发酵液中漆酶活性分别是对照的2.8、4.3、3.5和1.7倍。发酵液经(NH_4)_2S0_4沉淀,Sephadex G-150、DEAE-Sephadex A-25、Sephadex G-25柱层析纯化后,冷冻干燥。高效液相色谱(HPLC)检测为一单峰,分子量为26000,含17种氨基酸,氨基酸总量占酶组成的64.27％。等离子光谱(ICP)分析表明漆酶含有铜。该酶反应的最适温度为30℃,与邻联苯甲胺反应最适pH为4.0,Km值为385μmol/L;与丁香醛连氮反应最适pH为5.8,Km值为833μmol/L。     

红栓菌胞外漆酶的诱导、纯化及部分特性研究

红栓菌(pycnoporus cinnabarius)在发酵培养3d后出现胞外漆酶活性峰。木素类似物对红栓菌胞外漆酶活性有诱导作用,阿魏酸、香兰素,愈创木酚和DL-β-苯丙氨酸诱导24h后,发酵液中漆酶活性分别是对照的2.8、4.3、3.5和1.7倍。发酵液经(NH_4)_2S0_4沉淀,Sephadex G-150、DEAE-Sephadex A-25、Sephadex G-25柱层析纯化后,冷冻干燥。高效液相色谱(HPLC)检测为一单峰,分子量为26000,含17种氨基酸,氨基酸总量占酶组成的64.27％。等离子光谱(ICP)分析表明漆酶含有铜。该酶反应的最适温度为30℃,与邻联苯甲胺反应最适pH为4.0,Km值为385μmol/L;与丁香醛连氮反应最适pH为5.8,Km值为833μmol/L。     

甘草次酸对甲状腺癌细胞SW579 凋亡的影响及其机制*

目的:研究甘草次酸对甲状腺癌细胞SW579凋亡的影响及其可能的机制。方法:甲状腺癌细胞SW579分成4组,每组设置5个复孔,对照组为含10%胎牛血清的DMEM培养基;低浓度甘草次酸组为含浓度50 μmol/L+ 10%胎牛血清的DMEM培养基;中浓度甘草次酸组为含浓度100 μmol/L+ 10%胎牛血清的DMEM培养基;高浓度甘草次酸组为含浓度200 μmol/L+ 10%胎牛血清的DMEM培养基;各组在5%的二氧化碳培养箱中孵育24 h和48 h后,通过Annexin V / PI双标记流式细胞术检测甲状腺癌细胞SW579的凋亡比例,蛋白质印迹法检测检PI3K、AKT1、p-AKT蛋白的表达。结果:与对照组比较,孵育24 h及48 h后,50 μmol/L甘草次酸组凋亡细胞比例有所升高,AKT1、p-AKT、PI3K蛋白的相对表达量变化不明显,均无显著性差异(P＞0.05);100 μmol/L和200 μmol/L甘草次酸组的凋亡细胞比例均显著升高,AKT1、p-AKT蛋白的相对表达量均明显降低 (P＜0.05)。结论:100 μmol/L和200 μmol/L的甘草次酸可通过抑制AKT蛋白的表达促进甲状腺癌细胞SW579 凋亡。     

Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog)

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, ~50 and ~25%, respectively.     

Synergistic cytotoxicity of Rana catesbeiana ribonuclease and IFN-gamma on hepatoma cells

RC-RNase purified from Rana catesbeiana (bullfrog) oocytes is a pyrimidine-guanine sequence-specific ribonuclease. RC-RNase is derived from the RNase superfamily genes exerting distinct ribonucleolytic activity and possesses cytotoxicity to tumor cells, but rarely to primary cells. In this study, we utilized RC-RNase to function with antiproliferative cytokines. The combination with TNF-alpha or TNF-beta would not aggravate cell death. However, the combination with IFN-gamma could induce synergistic cytotoxicity verified by XTT assays toward three hepatoma cell lines bearing different differentiation stages. The distinct cytotoxicity from RC-RNase or RC-RNase/IFN-gamma on different hepatoma cells was correlated with the differentiation extent but not the proliferation rate of the cells. Despite the synergistic cytotoxicity and severe mitochondrial disruptions in the RC-RNase/IFN-gamma-treated cells, we scarcely detected any significant feature of apoptosis or necrosis by FACS analysis on annexin-V/propidium iodide staining. The mechanisms of cell death triggered by RC-RNase or RC-RNase/IFN-gamma require further investigation.     

抗肿瘤蛋白Onconase的原核细胞表达及细胞毒性检测

本文主要利用pET22b( )表达载体在大肠杆菌中表达一种在两栖类蛙(Ranapipiens)卵细胞内存在的核糖核酸酶-Onconase。通过包涵体复性、蛋白纯化等程序最终获得与天然蛋白活性相似的重组蛋白,并检测Onconase对源于皮肤T细胞淋巴瘤的Hut-78肿瘤细胞的毒性(IC50=0．51μmol／L),证明Onconase用于抗淋巴瘤的可行性。     

The high molecular weight and the low molecular weight acid phosphatases of the frog liver and their phosphotyrosine activity

1. Two molecular weight classes of non-specific acid phosphatases (AcPases) (3.1.3.2) are present in the frog (Rana esculenta) liver: a higher molecular weight (HMW) of Mr 140,560 and a lower molecular weight (LMW) of Mr 38,180 enzyme. 2. The LMW AcPase was described earlier and the HMW AcPase of optimum pH 4.8 is shown to be a L(+)-tartrate sensitive, thermolabile, dimeric glycoenzyme slightly activated by DTT. 3. The HMW and the LMW AcPases exhibit activity for phosphotyrosine which showed similar sensitivity to various effectors as the p-nitrophenyl phosphatase activity; however, both enzymes differed substantially in this respect suggesting that they might be involved in different metabolic steps.     

Rhodanese (thiosulfate:cyanide sulfurtransferase) from frog Rana temporaria

The molecular mass of rhodanese from the mitochondrial fraction of frog Rana temporaria liver, equaling 8.7 kDa, was determined by high-performance size exclusion chromatography (HP-SEC). The considerable difference in molecular weight and the lack of common antigenic determinants between frog liver rhodanese and bovine rhodanese suggest the occurrence of different forms of this sulfurtransferase in the liver of these animals.     

Esterolytic activities of rat intestinal mucosa. 2. Purification and properties of a glycerol-ester hydrolase.

A glycerol-ester hydrolase from rat intestinal cells has been purified using chromatography on carboxyhexanoyl-Sepharose-glyceryldioctanoate and preparative gel electrophoresis. The enzyme gives a single band by analytical gel electrophoresis; it is a monomer of molecular weight 68000. The optimum pH for its action on glyceryl tributyrate is between 8.0 and 8.5; the activation energy was calculated to be 8.7 kcal x mol-1 (36.4 kJ/mol). Its substrate specificity is mainly directed against esters of glycerol and of primary monoalcohols. Similarly to pancreatic lipase but contrary to liver esterase, it is inhibited by bile salts; relief of this inhibition by colipase is only observed for pancreatic lipase. The possible role of the glycerol-ester hydrolase in the absorption of short and of medium chain triglycerides is discussed.     

全反式维甲酸下调肝癌细胞HepG2内AFP结合蛋白的表达

当成人肝细胞发生癌变,甲胎蛋白(alpha-fetoprotein,AFP)在血清中的含量会急剧增加.AFP可与细胞表面AFP结合蛋白(AFP binding protein,ABP)结合促使细胞增殖分化.全反式维甲酸(al1-trans retinoic acid,ATRA)通过与特异性维甲酸受体(retinoic acid receptor,RAR)结合发挥抑制肿瘤生长的作用.Western印迹检测肝癌细胞HepG2和HLE中ABP的表达.结果显示,ABP在HepG2细胞中高表达,在HLE细胞中无明显表达.这一结果与2种细胞AFP的表达情况一致.激光扫描共聚焦显微镜定位分析显示,ABP存在于HepG2细胞胞膜和胞浆,用80μmol/L ATRA处理HepG2细胞4 h,可导致RAR入核增加.用不同浓度(20～160μmol/L)ATRA处理HepG2细胞后培养36 h.Western印迹结果表明,细胞ABP的表达随着ATRA浓度的增高而越少,ATRA浓度达80μmol/L时,HepG2细胞的ABP表达减少,ATRA浓度为160μmol/L时,ABP几乎无表达;加入80μmol/L ATRA后,随着作用时间延长,HepG2细胞ABP表达逐渐减少,当作用时间为12 h时,ABP表达明显减少.结果表明,ABP的表达对ATRA的反应呈剂量和时间依赖性.免疫共沉淀结果表明,AFP、ABP及RAR这3种蛋白质具有互相结合的作用.这些结果为进一步深入研究AFP在肝癌发生过程中的作用机制提供了依据.     

Barley aleurone layers secrete a nuclease in response to gibberellic Acid : purification and partial characterization of the associated ribonuclease, deoxyribonuclease, and 3'-nucleotidase activities

Incubation of barley (Hordeum vulgare L. cv Himalaya) half-seeds with gibberellic acid enhances the secretion of ribonuclease and deoxyribonuclease from aleurone tissue (MJ Chrispeels, JE Varner 1967 Plant Physiol 42: 398-406; L Taiz, JE Starks 1977 Plant Physiol 60: 182-189). These activities were over 50-fold greater in medium of half-seeds incubated with gibberellic acid than in control medium. Ribonuclease and deoxyribonuclease activities initially appeared in the medium 24 to 48 hours after hormone induction and increased for up to 96 hours. Both activities had a pH optimum of 6.0 and a temperature optimum of 55°C. When the medium from gibberellic acid-treated half-seeds was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the major ribonuclease and deoxyribonuclease activity bands comigrated. The two enzyme activities remained associated throughout a 2,700-fold purification employing ammonium sulfate fractionation, Heparin-Agarose affinity chromatography, and Reactive Blue 2-Agarose affinity chromatography. Also accompanying the ribonuclease and deoxyribonuclease activities throughout purification was the ability to hydrolyze the 3′-phosphoester linkage of 3′-AMP. The purified protein was composed of a single polypeptide with an apparent molecular weight of 36 kilodaltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is concluded that in response to gibberellic acid, barley aleurone tissue secretes a nuclease having ribonuclease, deoxyribonuclease, and 3′-nucleotidase activities.     

Cytostatic and Cytotoxic Properties of Amphinase: A Novel Cytotoxic Ribonuclease from Rana pipiens Oocytes

Onconase (Onc), is a novel amphibian cytotoxic ribonuclease with antitumor activity, and is currently in a confirmatory phase III clinical trial for the treatment of malignant mesothelioma. It was recently reported that Rana pipiens oocytes contain still another ribonuclease, named Amphinase (Amph). Amph shows 38 – 40 % amino acid sequence identity with Onc; presents as four variants varying between themselves from 87 to 99 % in amino acid sequence identity and has a molecular mass ~ 13,000. In the present study we describe the effects of Amph on growth of several tumor cell lines. All four variants demonstrated cytostatic and cytotoxic activity against human promyelocytic HL-60-, Jurkat T-cell- and U-937 monocytic leukemia cells. The pattern of Amph activity to certain extent resembled that of Onc. Thus, cell proliferation was suppressed at 0.5 – 10.0 µg/ml (40 – 80 nM) Amph concentration with distinct accumulation of cells in G1 phase of the cell cycle. In addition, the cells were undergoing apoptosis, which manifested by DNA fragmentation (presence of “sub-G1” cells, TUNEL-positivity), caspases and serine proteases activation as well as activation of transglutaminase. The cytotostatic and cytotoxic effects of Amph required its ribonuclease activity: the enzymatically inactive Amph-2 having histidine at the active site alkylated was ineffective. The effectiveness and cell cycle specificity was generally similar for all four Amph variants and at the equimolar concentrations was somewhat more pronounced than that of Onc. The observed cytostatic and cytotoxic activity of Amph against tumor cell lines suggests that similar to Onc this cytotoxic ribonuclease may have antitumor activity and find an application in clinical oncology.     

2-12烷基-6-甲氧基环己基-2,5-二烯-1,4-二酮(DMDD)抗弥漫大B淋巴瘤的作用及机制*

目的:探讨2-12烷基-6-甲氧基环己基-2,5-二烯-1,4-二酮(DMDD)抗弥漫大B淋巴瘤(DLBCL)的作用及分子机制。方法:动物实验取4周龄BALB/C小鼠,分5组,20只/组,腹股沟注射DLBCL细胞株OCI-LY19细胞1 × 10⁷ cells/ml 每只0.1 ml,两天后分别灌胃0、1、5、25、125 mg/kg剂量的DMDD,1次/2天,给药的第18日,杀10只小鼠,取瘤组织称重,记录剩余小鼠的生存期。细胞实验取OCI-LY19细胞加入96孔培养板,每孔100 μl 1×10⁵ cells/ml,分别加入100 μl DMDD使其终浓度分别为0、1、5、25和125 μmol/L,作用0、24、48和72 h,设三复孔,MTS法检测细胞增殖活性;根据细胞增殖实验结果,选择0 μmol/L、5 μmol/L和25 μmol/L的DMDD作为后续用药浓度作用OCI-LY19细胞24 h,流式细胞仪分析凋亡率,hoechst染色观察细胞核型,JC-1染色观察线粒体膜电位,LDH释放实验评估药物细胞毒性,qPCR、Western blot分析基因转录和表达水平。结果:动物实验表明:与0 mg/kg用药组比,1～125 mg/kg DMDD能抑制小鼠瘤组织生长并延长其生存期(P＜0.01)。细胞实验表明:DMDD用药组OCI-LY19细胞增殖活性明显降低、凋亡水平显著增加(P＜0.01),细胞核出现碎裂、凝集和凋亡小体及线粒体膜电位下降,LDH释放率显著增加(P＜0.01),细胞内caspase-3和bax基因的转录表达和IκBα的磷酸化水平显著上调,bcl-2、bcl-xL、jak2和stat3基因的转录和蛋白表达水平明显受抑(P＜0.01)。结论:DMDD通过抑制JAK2/STAT3和NF-κB信号通路中JAK2、STAT3和p-IκBα的表达,下调BCL-2/BAX、活化Caspase-3,最终激活OCI-LY19细胞线粒体凋亡的内源性通路而促进了DLBCL细胞凋亡,抑制了OCI-LY19细胞增殖,具有抗DLBCL的作用。