拟南芥吲哚—3—甘油磷酸合酶免疫分析

吲哚３甘油磷酸合酶（ＩＧＳ,ｉｎｄｏｌｅ３ｇｌｙｃｅｒｏｌｐｈｏｓｐｈａｔｅｓｙｎｔｈａｓｅ,ＥＣ４．１．１．４８）在色氨酸与吲哚乙酸的生物合成途径中,催化生成吲哚３甘油磷酸。研究该基因的表达调控,对于阐明高等植物是如何调控色氨酸及生长素合成是十分重要的。利用已克隆的ＩＧＳｃＤＮＡ,构建了谷胱甘肽转移酶（ＧＳＴ,ｇｌｕｔａｔｈｉｏｎｅＳｔｒａｎｓｆｅｒａｓｅ,ＥＣ２．５．１．１８）与吲哚３甘油磷酸合酶融合蛋白的表达质粒,并将其导入到在异丙基βＤ硫代半乳糖苷（ＩＰＴＧ）诱导下能高效表达的ＩＧＳ基因缺陷菌株ｔｒｐＣ９８００λＫＣ大肠杆菌中。高表达的融合蛋白通过谷胱甘肽琼脂糖（ｇｌｕｔａｔｈｉｏｎｅａｇａｒｏｓｅ）亲和层析和ＳＤＳ聚丙烯酰胺凝胶电泳纯化后,用以免疫兔子制备抗血清。免疫印迹法分析表明拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）四种常用生态型只合成一种分子量约为４０ｋＤ的吲哚３甘油磷酸合酶蛋白。在Ａｇ＋、紫外线等逆境条件下,ＩＧＳ含量都有较大幅度的增加,这说明ＩＧＳ可能与植物的防御反应紧密相关。     

SCAP与胆固醇水平的调节机制

SREBP裂解激活蛋白（SREBP cleavage-activating protein,SCAP）是哺乳动物脂质合成和摄入的中心调节因素。在胆固醇代谢的反馈调节系统中,SCAP与膜结合转录因子胆固醇调节元件结合蛋白（sterol regulatory element binding proteins,SREBPs）等调节因子,共同控制一系列酶编码基因的转录过程,包括胆固醇和脂肪酸生物合成过程中所需的酶。作者介绍了SREBP的结合、分类和功能及其二步蛋白水解释放;SCAP的结合和作用机制及其在胆固醇水平调节中的作用;SCAP基因缺陷型及其胆固醇水平异常,并提出了尚待解决的问题,对SCAP的研究是胆固醇水平调节领域的一个新课题。     

植物3-羟基-3-甲基戊二酰辅酶A还原酶基因研究进展

细胞分裂素、赤霉素、脱落酸、叶绿素、萜类等类异戊二烯物质,是植物中广泛存在的一类代谢产物,在植物生长发育过程中起着非常重要的作用。一些萜类化合物作为药物的合成前体或有效的药用成分在工农业及医药生产上具有重要的经济价值。类异戊二烯物质主要通过甲羟戊酸代谢途径中的一系列酶催化合成,其中,3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)是该代谢途径中的第一个关键限速酶,能够将3-羟基-3-甲基戊二酰辅酶A转化成中间代谢产物甲羟戊酸。对植物HMGR基因的克隆、酶结构和功能分析、基因组织表达及调控等方面进行了综述,旨在为其在重要农作物的遗传改良、代谢产物工程植物创制以及植物亲缘关系分析中的应用等研究提供理论依据。     

微生物异戊烯基转移酶研究进展

类异戊二烯(isoprenoids)是最具化学多样性的一种天然分子家族,参与微生物中类胡萝卜素、甾醇等次生代谢物的合成,这类物质在工业大规模生产中具有广阔的商业前景。异戊烯基转移酶是类异戊二烯合成途径中的关键酶,其活性及编码基因的转录水平参与调节次生代谢物产量,在类异戊二烯化合物生物合成途径中发挥重要作用。本文重点归纳了微生物中异戊烯基转移酶的发现与鉴定,分析其结构特点与链长决定机制,讨论异戊烯基转移酶家族之间的复杂进化,概述酶基因表达调控的应用以及生物合成研究现状,为深入研究异戊烯基转移酶作用机理及各领域中的应用提供思路。     

法尼酯X受体在脂质代谢过程中的调控作用

法尼酯X受体(Farnesoid X Receptor,FXR)属于配体依赖的核转录因子,可被内源性配体胆汁酸激活,通过调节胆汁酸、胆固醇、脂蛋白及脂肪酸代谢维持血浆中脂质的稳态,从而达到调节脂质代谢的目的。最近研究发现FXR在脉管系统中也有表达活性,开辟了FXR调节脂质代谢的新途径。随着新配体及靶基因的发现,研究FXR的作用机制以及寻找对脂质代谢具有调控作用的FXR的配体,对于脂代谢异常和动脉粥样硬化的防治具有重要意义。本文综述了该领域的最新进展。     

胆固醇氧化酶基因的克隆与表达

胆固醇是人体中主要的固醇类化合物,人体内胆固醇主要来源于自身的合成,也有一部分来自食物。胆固醇代谢和输送的异常往往与动脉粥样硬化相关联,能够引起心肌梗死、中风、动脉瘤和胆石症等疾病。胆固醇的一些代谢产物如类甾醇和类甾烷酮有致癌作用。所以检测血浆中和食物中胆固醇的含量在临床上具有重要的意义［１,２］。 胆固醇氧化酶可以和胆固醇脂酶和过氧化氢酶复合在一起,作为血浆总胆固醇含量的诊断试剂。也可将这３种酶固定在膜上,做成酶电极,这样使用起来更方便。胆固醇氧化酶能够降低食品中的胆固醇含量,也可以将胆固醇氧…     

高密度培养大肠杆菌YK537/pSBHL-11生产重组人细胞白介素

在 Ｂ． Ｂｒａｕｎ Ｅ Ｓ１０ 型１５ Ｌ 和 Ｎ Ｂ Ｓ Ｂｉｏ Ｆｌｏ ３０００ 型５ Ｌ 发酵罐中,利用补料分批培养技术高密度表达培养含重组质粒ｐ Ｓ Ｂ Ｈ Ｌ１１ 的大肠杆菌 Ｙ Ｋ５３７ ,生产重组人白细胞介素３（ Ｉ Ｌ３） ,发现在发酵过程中,限制性流加甘油,控制溶解氧在３０ ％ ～４０ ％ 左右、３０ ℃生长１１ｈ ,４２ ℃诱导培养４ｈ ,能将发酵液中最终菌体密度从 Ｏ Ｄ１６６００ 提高到 Ｏ Ｄ５３６００（ 相当于每升发酵液含１０６ 克湿菌体） ,并且保持了白细胞介素３ 的表达量,占菌体总蛋白的３０ ％ 左右,含量超过３３ ％ ｇ／ Ｌ,使 Ｉ Ｌ３ 包涵体产量从湿重２２ｇ／ Ｌ 提高到８５ ｇ／ Ｌ,纯化步骤比较简单,超声破菌后经两次洗涤纯度就达到７０ ％ 以上。     

胰岛素对葡萄糖-6-磷酸酶基因转录的调控

葡萄糖－６－磷酸酶（Ｇｌｕｃｏｓｅ－６－ｐｈｏｓ－ｐｈａｔａｓｅ,Ｇ６Ｐａｓｅ,Ｅ．Ｃ．３．１．３．９）是一种膜结合酶,主要存在于肝和肾细胞中的内质网膜及核膜上,其生物功能是催化葡萄糖异生和糖原分解两个代谢途径中由葡萄糖－６－磷酸到葡萄糖的水解反应,是调节生物体内血糖水平的关键酶之一。胰岛素（Ｉｎｓ）通过调控Ｇ６Ｐａｓｅ而调节血糖水平,近年的研究表明,Ｉｎｓ可以通过调控相关酶的基因转录来实现其相应的生理功能。１．Ｇ６Ｐａｓｅ的分子生物学研究肝微粒体Ｇ６Ｐａｓｅ酶系包括活性部分位于内质网腔表面的Ｇ…     

白细胞介素对大鼠离体垂体前叶细胞增殖的影响

本工作采用大鼠垂体前叶（ＡＰ）细胞原代培养方法,以３ＨＴｄＲ掺入率反映细胞增殖水平,研究了ＩＬ１和ＩＬ６对ＡＰ细胞增殖的影响。结果表明：（１）ＩＬ１（１－１００ｎｇ／ｍｌ）促进雄性大鼠和雌性大鼠ＡＰ细胞的增殖。（２）低浓度的ＩＬ６（０．１ｎｇ／ｍｌ）抑制雄性大鼠的ＡＰ细胞的增殖,而较高浓度的ＩＬ６（１－１０ｎｇ／ｍｌ）则表现为刺激作用。（３）ＩＬ６（０．１－１０ｎｇ／ｍｌ）促进雌性大鼠ＡＰ细胞的增殖。上述结果说明ＩＬ１和ＩＬ６除直接调控ＡＰ细胞的分泌外,也参与调节ＡＰ细胞增殖活动。     

HIV—1gp41基因的高效表达及表达产物的纯化

为了获得高效表达的人类免疫缺陷病毒（ＨＩＶ１）ｇｐ４１蛋白,从而为ＨＩＶ１基因工程诊断抗原的国产化打下基础,用ＰＣＲ的方法从ＨＩＶ１全基因序列中扩增出编码ｇｐ４１Ｎ端的６９０ｂｐ片段。经酶切后,克隆到ｐＥＴ２８ａ载体中,再将重组质粒转化到表达宿主菌ＢＬ２１（ＤＥ３）中,经ＩＰＴＧ诱导,高效表达出ｇｐ４１蛋白。间接ＥＬＩＳＡ、Ｗｅｓｔｅｒｎｂｌｏｔ、ＳＤＳＰＡＧＥ电泳证实,该表达产物具有良好的抗原性和特异性,且表达量约占总菌体蛋白的４５％。重组蛋白经金属鏊合纯化,纯度达９９％。     

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.     

Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin

Plant isoprenoids represent a large group of compounds with a wide range of physiological functions. In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate pathway. In this pathway, farnesyl diphosphate (FPP) occupies a central position, from which isoprene units are dispatched to the different classes of isoprenoids, with sterols as the major end products. The present work deals with effects of squalestatin (SQ) on the metabolism of FPP in proliferating and synchronized cultured tobacco cv. Bright Yellow-2 cells. SQ is a potent inhibitor of squalene synthase (SQS), the first committed enzyme in the sterol pathway. At nanomolar concentrations, SQ severely impaired cell growth and sterol biosynthesis, as attested by the rapid decrease in SQS activity. At the same time, it triggered a several-fold increase in both the enzymic activity and mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase. When SQ was added to cells synchronized by aphidicolin treatment, it was found to block the cell cycle at the end of G(1) phase, but no cell death was induced. Tobacco cells were also fed exogenous tritiated trans-trans farnesol, the allylic alcohol derived from FPP, in the presence and absence of SQ. Evidence is presented that this compound was incorporated into sterols and ubiquinone Q(10). In the presence of SQ, the sterol pathway was inhibited, but no increase in the radioactivity of ubiquinone was observed, suggesting that this metabolic channel was already saturated under normal conditions.     

Is the Reaction Catalyzed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase a Rate-Limiting Step for Isoprenoid Biosynthesis in Plants?

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The rate-limiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMGR activity could be limiting in plants, we introduced a constitutively expressing hamster HMGR gene into tabacco (Nicotiana tabaccum L.) plants to obtain unregulated HMGR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMGR gene led to a 3- to 6-fold increase in the total HMGR enzyme activity. Total sterol accumulation was consequently increased 3- to 10-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. The level of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMGR activity, these results indicate that the activity of one or more later enzyme(s) in the pathway must also be involved in determining the relative accumulation of end-product sterols. The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.     

Improved fruit α‐tocopherol,carotenoid, squalene and phytosterol contents through manipulation of Brassica juncea 3‐HYDROXY‐3‐METHYLGLUTARYL‐COA SYNTHASE1 in transgenic tomato

3‐Hydroxy‐3‐methylglutaryl‐coenzyme A synthase (HMGS) in the mevalonate (MVA) pathway generates isoprenoids including phytosterols. Dietary phytosterols are important because they can lower blood cholesterol levels. Previously, the overexpression of Brassica juncea wild‐type (wt) and mutant (S359A) BjHMGS1 in Arabidopsis up‐regulated several genes in sterol biosynthesis and increased sterol content. Recombinant S359A had earlier displayed a 10‐fold higher in vitro enzyme activity. Furthermore, tobacco HMGS overexpressors (OEs) exhibited improved sterol content, plant growth and seed yield. Increased growth and seed yield in tobacco OE‐S359A over OE‐wtBjHMGS1 coincided with elevations in NtSQS expression and sterol content. Herein, the overexpression of wt and mutant (S359A) BjHMGS1 in a crop plant, tomato (Solanum lycopersicum), caused an accumulation of MVA‐derived squalene and phytosterols, as well as methylerythritol phosphate (MEP)‐derived α‐tocopherol (vitamin E) and carotenoids, which are important to human health as antioxidants. In tomato HMGS‐OE seedlings, genes associated with the biosyntheses of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, brassinosteroids, dolichols, methylerythritol phosphate, carotenoid and vitamin E were up‐regulated. In OE‐S359A tomato fruits, increased squalene and phytosterol contents over OE‐wtBjHMGS1 were attributed to heightened SlHMGR2, SlFPS1, SlSQS and SlCYP710A11 expression. In both tomato OE‐wtBjHMGS1 and OE‐S359A fruits, the up‐regulation of SlGPS and SlGGPPS1 in the MEP pathway that led to α‐tocopherol and carotenoid accumulation indicated cross‐talk between the MVA and MEP pathways. Taken together, the manipulation of BjHMGS1 represents a promising strategy to simultaneously elevate health‐promoting squalene, phytosterols, α‐tocopherol and carotenoids in tomato, an edible fruit.     

The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity.

The mevalonate diphosphate decarboxylase is an enzyme which converts mevalonate diphosphate to isopentenyl diphosphate, the building block of isoprenoids. We used the Saccharomyces cerevisiae temperature-sensitive mutant defective for mevalonate diphosphate decarboxylase previously described (C. Chambon, V. Ladeveve, M. Servouse, L. Blanchard, C. Javelot, B. Vladescu, and F. Karst, Lipids 26:633-636, 1991) to characterize the mutated allele. We showed that a single change in a conserved amino acid accounts for the temperature-sensitive phenotype of the mutant. Complementation experiments were done both in the erg19-mutated background and in a strain in which the ERG19 gene, which was shown to be an essential gene for yeast, was disrupted. Epitope tagging of the wild-type mevalonate diphosphate decarboxylase allowed us to isolate the enzyme in an active form by a versatile one-step immunoprecipitation procedure. Furthermore, during the course of this study, we observed that a high level of expression of the wild-type ERG19 gene led to a lower sterol steady-state accumulation compared to that of a wild-type strain, suggesting that this enzyme may be a key enzyme in mevalonate pathway regulation.