腺病毒介导的人ING4基因诱导C6鼠胶质瘤细胞凋亡

目的：研究肿瘤抑制基因人ING4 (inhibitor of growth family, member 4)对C6鼠胶质瘤细胞的促凋亡作用。方法：将携有绿色荧光蛋白（GFP）腺病毒空载体Ad及重组腺病毒Ad-hING4-His（由本科室构建）分别感染C6细胞,RT-PCR法检测hING4的转录,Western-blotting法检测目的蛋白的表达。并观测hING4基因表达对C6胶质瘤细胞的作用,用MTT法绘制生长曲线,计算抑瘤率。再取重组腺病毒Ad-hING4-His及空腺病毒Ad作用后的C6细胞分别行激光共聚焦显微镜观察凋亡小体、透射电镜观察亚细胞结构的变化,抽提基因组DNA行琼脂糖凝胶电泳及流式细胞仪检测。结果： Ad-hING4-His感染C6细胞后,RT-PCR及Western-blotting结果提示有目的基因的转录和表达。hING4基因表达可以显着抑制C6细胞生长。激光共聚焦观察可见明显核断裂、透射电镜可见实验组细胞呈凋亡表现、基因组DNA电泳呈现梯形条带,流式细胞仪检测有明显AP峰,凋亡率达18.1％。结论：hING4可以通过促进细胞凋亡作用而显着抑制C6细胞的增殖和生长。     

Degradation of 4-hydroxyphenylacetate by Xanthobacter 124X

Xanthobacter 124X when grom on 4-hydroxyphenylacetate was able to hydroxylate this compound yielding homogenisate. Ring fission of this latter compound gave maleylacetoacetate which was isomerized to fumarylacetoacetate. The isomerase involved resembled maleylacetoacetate isomerases in Gram-negative bacteria in that glutathione was required for activity. Fumarate and acetoacetate were both detected as products of the hydrolysis of fumarylacetoacetate.     

Cap-dependent eukaryotic initiation factor-mRNA interactions probed by cross-linking

Cap-dependent ribosome recruitment to eukaryotic mRNAs during translation initiation is stimulated by the eukaryotic initiation factor (eIF) 4F complex and eIF4B. eIF4F is a heterotrimeric complex composed of three subunits: eIF4E, a 7-methyl guanosine cap binding protein; eIF4A, a DEAD-box RNA helicase; and eIF4G. The interactions of eIF4E, eIF4A, and eIF4B with mRNA have previously been monitored by chemical- and UV-based cross-linking approaches aimed at characterizing the initial protein/mRNA interactions that lead to ribosome recruitment. These studies have led to a model whereby eIF4E interacts with the 7-methyl guanosine cap structure in an ATP-independent manner, followed by an ATP-dependent interaction of eIF4A and eIF4B. Herein, we apply a splint-ligation-mediated approach to generate 4-thiouridine-containing mRNA adjacent to a radiolabel group that we utilize to monitor cap-dependent cross-linking of proteins adjacent to, and downstream from, the cap structure. Using this approach, we demonstrate interactions between eIF4G, eIF4H, and eIF3 subunits with the mRNA during the cap recognition process.     

Catabolism of 2,7-dichloro- and 2,4,8-trichlorodibenzofuran by Sphingomonas sp strain RW1

Due to their physicochemical and toxicological properties, polychlorinated dibenzofurans are regarded as a class of compounds providing reason for serious environmental concern. While the nonhalogenated basic structure dibenzofuran is effectively mineralized by appropriate bacterial strains, its polychlorinated derivatives are not. To elucidate the ability of the strain Sphingomonas sp RW1 to metabolize some of these chlorinated derivatives, we performed turnover experiments using 2,7-dichloro- and 2,4,8-trichlorodibenzofuran. As indicated by the oxygen-uptake rates determined for these two chlorinated dibenzofurans, Sphingomonassp RW1 can catabolize these chlorinated dibenzofurans yielding small quantities of oxidation products, which we isolated and subsequently characterized employing GC/MS and ¹H- as well as ¹³C-NMR spectroscopy. In the case of 2,7-dichlorodibenzofuran, two metabolites accumulated, which we identified as 6-chloro- and 7-chloro-2-methyl-4H-chromen-4-one. The single metabolite isolated from the turnover experiments performed with 2,4,8-trichlorodibenzofuran was unequivocally identified as 6,8-dichloro-2-methyl-4H-chromen-4-one. Received 26 April 1999/ Accepted in revised form 23 July 1999     

TOR信号转导通路介导的翻译控制

TOR(target of rapamycin)真核生物中高度保守的一种大分子的Ser/Thr激酶,是免疫抑制剂雷帕霉素的在体内的靶物质.TOR能够对营养状况和生长因子等因素的变化做出应答反应,通过介导磷酸化反应调节蛋白激酶4E-BP1,S6K,eEF2和磷酸酶等的活性,控制下游翻译因子的磷酸化水平,调节核糖体发生,蛋白质合成等生理过程,在细胞的生长,增殖的综合调控中起到中枢作用.     

Persistent ultrastructural changes in pancreatic acinar cells induced by 4-acetylaminofluorene

Male Leeds rats were fed a diet containing 0.05% of the non-carcinogen 4-acetylaminofluorene (4-AAF) for 8–10 months. They were then returned to a normal diet and their pancreatic tissues examined by electron microscopy at intervals between 2 and 12 months after the end of 4-AAF treatment. 4-AAF was found to induce a persistent alteration in the morphology of the granular endoplasmic reticulum, involving distortion and dilatation of the cisternae. In some respects this lesion resembles that which is induced by the carcinogenic isomer, 2-acetylaminofluorene (2-AAF).     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

疏水层析纯化人重组白细胞介素－4

应用疏水层析对大肠杆菌表达的人重组白细胞介素－４（ｒｈＩＬ－４）进行了纯化,含有ｒｈＩＬ－４的包涵体,经洗涤、变性、复性后,以Ｂｕｔｙｌ－Ｓｅｐｈａｒｏｓｅ层析,得到了高纯度的ｒｈＩＬ－４．纯度达９７％;回收率为３２％;比活性为２×１０~７Ｕ／ｍｇ,讨论了ｒｈＩＬ－４疏水层析的条件,并对不同的方法纯化白细胞介素－４进行了比较．     

Purification of rabbit lacrimal gland plasma membranes by aqueous two-phase affinity partitioning

We describe the purification of lacrimal gland plasma membranes by affinity partitioning using a two-phase system containing polyethylene glycol and dextran in which wheat germ agglutinin conjugated to dextran is used as affinity ligand. When partitioning a microsomal fraction, the plasma membrane marker 5′-nucleotidase was obtained in the affinity ligand-containing bottom phase, whereas the endoplasmic reticulum marker NADH-ferricyanide reductase remained in the top phase. The affinity partitioning behaviour of components involved in exocytosis and cellular signalling was also examined.     

ErbB-4受体介导卵巢癌细胞增殖、侵袭等生物学行为的实验研究

探讨ErbB- 4受体在卵巢癌细胞增殖、侵袭等生物学行为中的作用.免疫组化检测ErbB- 4受体在卵巢癌细胞株OVCAR-3表达;噻唑蓝比色法(MTT)测定不同浓度的ErbB- 4单克隆抗体(Ab-3)对体外培养的卵巢癌OVCAR-3细胞增殖的影响;流式细胞仪检测细胞周期和凋亡;侵袭实验观察Ab-3对OVCAR-3侵袭能力的影响.结果表明,ErbB- 4在OVCAR-3细胞株中呈阳性表达;不同浓度(0.625～10 μg/ml)的Ab-3均能抑制OVCAR-3细胞生长,且具有浓度和时间依赖性,其作用72 h的中效浓度为4.48 μg/ml.流式细胞仪检测发现Ab-3(2.0 μg/ml)作用24、48及72 h后,OVCAR-3细胞出现S期阻滞,肿瘤细胞的凋亡率逐渐增加;抗体处理组穿透侵袭小室滤膜的细胞数(78.0±6.1)明显少于对照组(132.0±17.2)(P<0.01).因此,Ab-3可抑制内源性ErbB- 4表达的OVCAR-3细胞株的增殖、侵袭能力,诱导细胞凋亡.     

Covalent modifications of aminophospholipids by 4-hydroxynonenal

Lipid oxidation is implicated in a wide range of pathophysiological disorders, which leads to reactive compounds such as aldehydes. Among them 4-hydroxynonenal (4-HNE) reacts strongly with the NH₂ groups of amino acids and forms mainly Michael adducts and minor Schiff-base adducts. Such reactions occur also with compounds containing thiol groups. No data are available describing 4-HNE interactions with amino-phospholipids. To investigate such a possibility, 4-HNE was incubated with either phosphatidylethanolamine (PE) or phosphatidylserine (PS) in an aqueous-organic biphasic system and the resulting products were identified by liquid chromatography-mass spectrometry (LC-MS). Our study points out the potential capacity of 4-HNE to react with phospholipids containing amino groups and particularly PE. The main resulting compounds found were a Michael adduct plus a minor Schiff base adduct, which was partly cyclized as a pyrrole derivative via a loss of water. Its stabilization as a pyrrole derivative allows to differentiate 4-HNE from the other aldehydes generated via lipid oxidation (e.g., malondialdehyde, 2-nonenal) that lack the 4-hydroxyl group. Their formation seems not to be affected when the pH varies from 6.5 to 8.5. Surprisingly, PS reacted poorly producing only a small amount of Michael adduct, the Schiff-base adduct being nondetectable. We conclude that such adducts, if they are formed in cell membranes, could alter the phospholipase-dependent cell signaling.     

化学修饰单克隆抗体模拟谷胱甘肽过氧化物酶

化学修饰具有底物谷胱甘肽（ＧＳＨ）结合部位的单克隆抗体（４Ａ４）,使其结合部位上的丝氨酸（Ｓｅｒ）转变成谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团硒代半胱氨酸（Ｓｅ－Ｃｙｓ）,因而产生高活力的含硒抗体酶（Ｓｅ－ａｂｚｙｍｅ）．突变的４Ａ４（ｍ４Ａ４）的ＧＰＸ活力达到了天然酶活力的１９％,并对ｍ４Ａ４的酶学性质和动力学性质进行了研究;硒代谷胱甘肽（ＧＳｅＨ）连到４Ａ４结合部位,其ＧＰＸ活力由３．８６Ｕ／μｍｏｌ提高到５９８．９Ｕ／μｍｏｌ用黄嘌呤氧化酶／次黄嘌呤为中心的心肌线粒体自由基损伤模型证明Ｓｅ－ａｂｚｙｍｅ（ｍ４Ａ４）可减轻活性氧对线粒体的损伤。     

C-RAF activation promotes BAD poly-ubiquitylation and turn-over by the proteasome

BAD, a member of the BCL2 family, exhibits an original mode of regulation by phosphorylation. In the present report, we examine the role of the kinase C-RAF in this process. We show that the inducible activation of C-RAF promotes the rapid phosphorylation of BAD on Serine-112 (Ser-75 in the human protein), through a cascade involving the kinases MEK and RSK. Our findings reveal a new aspect of the regulation of BAD protein and its control by the RAF pathway: we find that C-RAF activation promotes BAD poly-ubiquitylation in a phosphorylation-dependent fashion, and increases the turn-over of this protein through proteasomal degradation.     

Production of 4-hydroxybutyric acid by metabolically engineered Mannheimia succiniciproducens and its conversion to γ-butyrolactone by acid treatment

γ-Butyrolactone (GBL) is an important four carbon (C4) chemical, which has a wide range of industrial applications. GBL can be produced by acid treatment of 4-hydroxybutyric acid (4-HB), which is a derivative of succinic acid. Heterologous metabolic pathways were designed and established in succinic acid overproducing Mannheimia succiniciproducens LPK7 (ldhA pflD pta ackA mutant) by the introduction of heterologous genes that encode succinyl-CoA synthetase, CoA-dependent succinate semialdehyde dehydrogenase, and either 4-hydroxybutyrate dehydrogenase in LPK7 (p3S4CD) or succinate semialdehyde reductase in LPK7 (p3SYCD). Fed-batch cultures of LPK7 (p3S4CD) and LPK7 (p3SYCD) resulted in the production of 6.37 and 6.34 g/L of 4-HB (molar yields of 0.143 and 0.139), respectively. Finally, GBL was produced by acid treatment of the 4-HB obtained from the fermentation broth with molar yield of 0.673. This study demonstrates that 4-HB, and potentially other four carbon platform chemicals, can be produced by the engineered rumen bacterium M. succiniciproducens.     

The utilization of formate by human erythrocytes

The incorporation of radioactive formate into an acid-stable non-volatile form by human erythrocytes is dependent upon the addition of 5-amino-4-imidazolecarboxamide riboside. The formate-incorporating activity of human erythrocytes varies widely among normal individuals and the values obtained are characteristic of the erythrocytes obtained from these individuals. The variation is unrelated to the total folate levels of the erythrocytes as measured by the growth response of Lactobacillus casei but is roughly correlated with the quantity of folate forms in the erythrocytes which support the growth of Steptococcus faecalis. The activities of several enzymes involved in the metabolism of the folate coenzymes has also been measured in extracts of erythrocytes. Extracts from all the individuals contained 10-formyltetrahydrofolate synthase, 5-amino-4-imidazolecarboxamide ribotide transformylase, and 5,10-methylenetetrahydrofolate dehydrogenase. None of the extracts contained detectable quantities of either 5,10-methylenetetrahydrofolate reductase or 5-methyltetrahydrofolate-homocysteine methyltransferase. These data support the conclusion that 5-methyltetrahydrofolate is not in metabolic equilibrium with the other forms of folate in the erythrocyte and the uptake of formate by intact erythrocytes is a function of those forms of the folate coenzymes which can be converted to tetrahydrofolate.     

Aerobic biodegradation of 4-methylpyridine and 4-ethylpyridine by newly isolated Pseudonocardia sp. strain M43

A filamentous bacterium capable of utilizing 4-methylpyridine and 4-ethylpyridine as the sole source of carbon, nitrogen and energy was isolated from sludge. The organism, designated as strain M43, clustered most closely with members of the genus Pseudonocardia by 16S rRNA gene sequence analysis. During the degradation of 4-methylpyridine and 4-ethylpyridine, c. 60% of nitrogen in the pyridine ring was released as ammonia. Metabolite analyses showed that 2-hydroxy-4-methylpyridine and 2-hydroxy-4-ethylpyridine were transiently accumulated during the degradation of 4-methylpyridine and 4-ethylpyridine, respectively. Strain M43 was also able to degrade pyridine, 3,4-dimethylpyridine, 4-carboxypyridine and 2-hydroxy-4-methylpyridine. The results indicate that degradation of 4-methylpyridine and 4-ethylpyridine by strain M43 proceeded via initial hydroxylation.     

Strand breaks of mammalian mitochondrial DNA induced by carcinogens.

Closed circular mitochondrial DNA in mammalian cells was degradated to the open circular form by exposure of the cells to the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline 1-oxide (4NQO). MNNG caused more strand scission of mitochondrial DNA than 4NQO at the same concentration. The action of the carcinogens on mitochondrial DNA did not parallel that with nuclear DNA which was damaged by 4NQO more markedly than by MNNG. Mitochondrial DNA damaged by carcinogens was not repaired during 4-20 h of post-treatment incubation of the cells. Incorporation of labeled thymidine into the closed circular mitochondrial DNA, decreased by the treatment of cells with carcinogens, recovered during post-treatment incubation.