Squalene supplementation alters genes associated with liver cholesterol metabolism

Background – Squalene is a component of shark liver oil and has been speculated to have cholesterol reducing properties. High levels of total and LDL cholesterol have been shown to contribute to the development of chronic heart disease. The liver is central to the regulation of cholesterol metabolism and dietary intervention has long been recognized as a primary means to reduce the risks of chronic heart disease and related ailments.
Objectives – To determine the effect of dietary squalene supplementation on gene transcripts associated with liver cholesterol metabolism. Specifically the effect of squalene supplementation on mRNA levels for proteins that
regulate cholesterol biosynthesis (HMDH & ERG1), cholesterol elimination (SRB1), bile synthesis (CP7A1 & CP27A) and cholesterol excretion by the liver into bile (ABCG5 & ABCG8) was investigated.
Design – Rats (n=32) were divided into four groups and supplemented for 12 weeks. Groups one and two were fed a cholesterol rich diet for six weeks followed by six weeks of a cholesterol rich diet plus 1.75mg/day of squalene or 3.5 mg/day. Group three was fed a cholesterol rich diet for 12 weeks and group four was fed standard rat chow for 12 weeks. Blood lipid levels were monitored during the study and liver gene expression was determined at the
conclusion of the feeding trial via RT-PCR.
Outcomes – 3.5 mg/day of squalene lowered total and LDL cholesterol in rats consuming a cholesterol rich diet. This dose of squalene also resulted in constant levels of HMDH and ERG1 whereas the cholesterol rich diet halved mRNA levels of these enzymes. Furthermore 3.5 mg/day of squalene caused a greater than 3.0 fold increase in mRNA levels of the proteins SRB1, CP7A1, CP27A and ABCG5.
Conclusion – Dietary squalene supplementation at a dose of 3.5 mg/day lowers total and LDL cholesterol in rats consuming a cholesterol rich diet. These reductions in cholesterol levels may be due to increased cholesterol
elimination, bile synthesis and cholesterol excretion by the liver into bile mediated by changes in gene expression of key enzymes involved in these metabolic pathways

Squalene does not exhibit a chemopreventive activity and increases plasma cholesterol in a Wistar rat hepatocarcinogenesis model

The eventual chemopreventive effect of squalene (SQ), a triterpene present in olive oil, was evaluated when administered to Wistar rats during a period comprising the initiation and selection/promotion of the resistant hepatocyte (RH) model of hepatocarcinogenesis. During 8 consecutive wk, animals received by gavage SQ (100 or 150 mg/100 g body weight) dissolved in corn oil (CO) daily. Animals treated with only CO and submitted to the RH model were used as controls. Treatments with SQ did not result in inhibition of macroscopically visible hepatocyte nodules (P > 0.05) or of hepatic placental glutathione S-transferase-positive preneoplastic lesions (PNL; P > 0.05). Hepatic cell proliferation and apoptosis indexes were not different (P > 0.05) among the different experimental groups, both regarding PNL and surrounding normal tissue areas. There were no significant differences (P > 0.05) among comets presented by rats treated with the two SQ doses or with CO. on the other hand, SQ increased total plasma cholesterol levels when administered at both doses (P < 0.05). This indicates that the isoprenoid was absorbed. Thus, SQ did not present chemopreventive activity during hepatocarcinogenesis and had a hypercholesterolemic effect, suggesting caution when considering its use in chemoprevention of cancer.

Evidence for direct squalene and 2,3-oxidosqualene binding by supernatant protein factor

We present the crystal structures of the SEC14-like domain of supernatant protein factor (SPF) in complex with squalene and 2,3-oxidosqualene. The structures were resolved at 1.75 Å (complex with squalene) and 1.6 Å resolution (complex with 2,3-oxidosqualene), leading in both cases to clear images of the protein/ substrate interactions. Ligand binding is facilitated by removal of the Golgi-dynamics (GOLD) C-terminal domain of SPF, which, as shown in previous structures of the apo-protein, blocked the opening of the binding pocket to the exterior. Both substrates bind into a large hydrophobic cavity, typical of such lipid-transporter family. Our structures report no specific recognition mode for the epoxide group. In fact, for both molecules, ligand affinity is dominated by hydrophobic interactions, and independent investigations by computational models or differential scanning micro-calorimetry reveal similar binding affinities for both ligands. Our findings elucidate the molecular bases of the role of SPF in sterol endo-synthesis, supporting the original hypothesis that SPF is a facilitator of substrate flow within the sterol synthetic pathway. Moreover, our results suggest that the GOLD domain acts as a regulator, as its conformational displacement must occur to favor ligand binding and release during the different synthetic steps.

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis

Squalene epoxidase, a membrane-associated enzyme that converts squalene to squalene 2,3-oxide, plays an important role in the maintenance of cholesterol homeostasis. In 1957, Bloch and colleagues identified a factor from rat liver cytosol termed “supernatant protein factor (SPF),” which promotes the squalene epoxidation catalyzed by rat liver microsomes with oxygen, NADPH, FAD, and phospholipid [Tchen, T. T. & Bloch, K. (1957) J. Biol. Chem. 226, 921–930]. Although purification of SPF by 11,000-fold was reported, no information is so far available on the primary structure or biological function of SPF. Here we report the cDNA cloning and expression of SPF from rat and human. The encoded protein of 403 amino acids belongs to a family of cytosolic lipid-binding/transfer proteins such as α-tocopherol transfer protein, cellular retinal binding protein, yeast phosphatidylinositol transfer protein (Sec14p), and squid retinal binding protein. Recombinant SPF produced in Escherichia coli enhances microsomal squalene epoxidase activity and promotes intermembrane transfer of squalene in vitro. SPF mRNA is expressed abundantly in the liver and small intestine, both of which are important sites of cholesterol biosynthesis. SPF is expressed significantly in isolated hepatocytes, but the expression level was markedly decreased after 48 h of in vitro culture. Moreover, SPF was not detectable in most of the cell lines tested, including HepG2 and McARH7777 hepatomas. Transfection of SPF cDNA in McARH7777 significantly stimulated de novo cholesterol biosynthesis. These data suggest that SPF is a cytosolic squalene transfer protein capable of regulating cholesterol biosynthesis.

Ozone-initiated particle formation, particle aging, and precursors in a laser printer

An increasing number of researchers have hypothesized that ozone may be involved in the particle formation processes that occur during printing, however no studies have investigated this further. In the current study, this hypothesis was tested in a chamber study by adding supplemental ozone to the chamber after a print job without measurable ozone emissions. Subsequent particle number concentration and size distribution measurements showed that new particles were formed minutes after the addition of ozone. The results demonstrated that ozone did react with printer-generated volatile organic compounds (VOCs) to form secondary organic aerosols (SOAs). The hypothesis was further confirmed by the observation of correlations among VOCs, ozone, and particles concentrations during a print job with measurable ozone emissions. The potential particle precursors were identified by a number of furnace tests, which suggested that squalene and styrene were the most likely SOA precursors with respect to ozone. Overall, this study significantly improved scientific understanding of the formation mechanisms of printer-generated particles, and highlighted the possible SOA formation potential of unsaturated nonterpene organic compounds by ozone-initiated reactions in the indoor environment. © 2011 American Chemical Society.

Effect of dietary cholesterol and ubiquinone on isoprene synthesis in rat liver

The effect of dietary cholesterol and ubiquinone on the synthesis of isoprene compounds in the liver, as tested by the incorporation of acetate-1-14C and mevalonate-2-14C, was studied in rats. In cholesterol feeding, there appears to be a second site of inhibition after squalene in addition to the previously known primary site of inhibition at the β-hydroxy-β-methyl glutaryl-CoA reductase. Feeding ubiquinone inhibited at some common step between acetate and mevalonate in the synthesis of both cholesterol and ubiquinone, without affecting the acetate activation or fatty acid synthesis, and also at a step in the synthesis of ubiquinone not common with the synthesis of cholesterol. These results are suggestive of a role for ubiquinone in the regulation of isoprene synthesis.

青蒿反义鲨烯合酶基因对烟草的转化

利用反义技术研究生物代谢途径以及对其生物合成进行调控成为植物次生代谢研究领域内一个重要手段之一，并与新兴的RNAi技术一起成为本领域内重要的研究热点。在植物类异戊二烯代谢途径中存在着羟甲基戊二酰辅酶A还原酶(HMGR)、法呢基焦磷酸合酶（FPS）和鲨烯合酶（SQS）等几种关键的分支酶，他们被认为在异戊二烯类的生物合成中发挥着关键的调节作用。其中，鲨烯合酶处于HMGR和FPS的下游，并与倍半萜合酶等利用共同的前体－法呢基二磷酸（FPP），以FPP起始合成一系列的下游产物。因此，FPP成为类异戊二烯途径中的关键调节点之一。本论文基于此目的，利用反义技术研究了FPP合成鲨烯这一途径受到抑制对其他以FPP为生物合成前体的代谢支路的影响。 利用植物双元转化载体pBI121，将青蒿中鲨烯合酶基因的cDNA（约1.5kb）序列插入到pBI121中，取代原有的GUS序列，构建成植物转化载体pBIASS。以根癌农杆菌为介导，将青蒿鲨烯合酶反义基因序列导入到烟草，整合到其基因组中 ，成功获得转基因植株。对转基因烟草进行分子检测表明，外源鲨烯合酶基因的序列已经稳定整合到烟草基因组中，并对内源的烟草鲨烯合酶基因表达产生影响。转基因烟草中检测到内源鲨烯合酶基因的mRNA的水平降低。对鲨烯合酶下游产物之一的胆固醇的含量分析显示，活性减低的鲨烯合酶使胆固醇的生物合成下降约40％左右。同时，另一条以FPP为共同前体的二萜代谢途径产物之一GA3的含量得到了提高，比对照提高约30％。

水稻GA3应答基因表达谱微阵列分析与OsGAS1功能初步研究

利用反义技术研究生物代谢途径以及对其生物合成进行调控成为植物次生代谢研究领域内一个重要手段之一，并与新兴的RNAi技术一起成为本领域内重要的研究热点。在植物类异戊二烯代谢途径中存在着羟甲基戊二酰辅酶A还原酶(HMGR)、法呢基焦磷酸合酶（FPS）和鲨烯合酶（SQS）等几种关键的分支酶，他们被认为在异戊二烯类的生物合成中发挥着关键的调节作用。其中，鲨烯合酶处于HMGR和FPS的下游，并与倍半萜合酶等利用共同的前体－法呢基二磷酸（FPP），以FPP起始合成一系列的下游产物。因此，FPP成为类异戊二烯途径中的关键调节点之一。本论文基于此目的，利用反义技术研究了FPP合成鲨烯这一途径受到抑制对其他以FPP为生物合成前体的代谢支路的影响。 利用植物双元转化载体pBI121，将青蒿中鲨烯合酶基因的cDNA（约1.5kb）序列插入到pBI121中，取代原有的GUS序列，构建成植物转化载体pBIASS。以根癌农杆菌为介导，将青蒿鲨烯合酶反义基因序列导入到烟草，整合到其基因组中 ，成功获得转基因植株。对转基因烟草进行分子检测表明，外源鲨烯合酶基因的序列已经稳定整合到烟草基因组中，并对内源的烟草鲨烯合酶基因表达产生影响。转基因烟草中检测到内源鲨烯合酶基因的mRNA的水平降低。对鲨烯合酶下游产物之一的胆固醇的含量分析显示，活性减低的鲨烯合酶使胆固醇的生物合成下降约40％左右。同时，另一条以FPP为共同前体的二萜代谢途径产物之一GA3的含量得到了提高，比对照提高约30％。

反义鲨烯合酶基因表达对青蒿素生物合成的影响

青蒿素是从中药青蒿中提取的新型抗疟药物，然而，青蒿素在青蒿中的含量非常低。近年来，随着青蒿素生物合成途径相关酶基因的克隆，基因工程成为提高青蒿素含量的有效途径之一。在对青蒿进行遗传转化过程中，高效稳定的丛生芽诱导体系是青蒿转化成功的关键。然而，随着继代次数的增多，青蒿丛生芽诱导能力存在退化现象。本文首先研究了滤纸对青蒿丛生芽诱导的影响和在遗传转化中的应用，进而研究了反义鲨烯合酶基因表达对青蒿素生物合成的影响。主要结果如下： 研究了在丛生芽诱导培养基上加铺滤纸对青蒿丛生芽诱导的影响，结果发现，加铺滤纸后青蒿丛生芽诱导率显著提高，丛生芽诱导率能够达到97%左右。在此高效丛生芽诱导体系的基础上，我们进一步探讨了滤纸在青蒿遗传转化中的应用。结果表明，在筛选培养基上加铺一层滤纸，青蒿的抗性丛生芽诱导率能够达到59.7%，其中在12.5%的抗性丛生芽中能够得到抗性生根植株，生根植株PCR检测均为阳性，在部分PCR检测阳性的植株中检测到了GUS的稳定表达。 利用上述改进的青蒿遗传转化体系，我们得到了反义鲨烯合酶基因的青蒿转化植株。PCR检测和Southern杂交检测结果证明了反义鲨烯合酶基因已经整合到青蒿基因组中。RT-PCR检测发现，在转基因株系ASQ3和ASQ5中鲨烯合酶基因在mRNA水平上得到部分抑制，鲨烯含量比对照降低了20%左右;青蒿素的含量分别提高了23.2%和21.5%，结果表明抑制鲨烯合酶表达能够有效促进青蒿中青蒿素的生物合成。

青蒿素生物合成相关基因的功能分析及遗传转化研究

从中国传统药用植物青蒿（Artemisia annua L.）中提取的青蒿素及其半合成衍生物如蒿甲醚等是一类新型的抗疟特效药，特别是对抗氯喹的恶性疟疾和脑型疟疾有很好的疗效。由于青蒿素在植物中的含量极低，使得其价格很高，特别是对于亚非拉等第三世界国家来说。因此如何提高青蒿素的产量成为近年来研究的热点。各种传统的育种、生理生化手段和细胞培养技术均未取得较好的结果，因此，利用植物基因工程技术提高青蒿素产量已成为研究的重点之一。 本论文围绕青蒿素的生物合成途径开展了以下的工作： 一、中药青蒿紫穗槐二烯合酶的大肠杆菌表达、纯化与功能鉴定 利用RT-PCR方法，从中药青蒿高产株系001中克隆到的中药青蒿紫穗槐二烯合酶(ADS) cDNA, 其推测编码蛋白与前人报道的有两个位点的突变。将其开放阅读框插入到原核表达载体pET30a(+)的BamHⅠ和XhoⅠ酶切位点之间，构建N端携带有HIS6表达标签的紫穗槐二烯合酶重组表达载体pETADS。将pETADS转入大肠杆菌BL21(DE3)， IPTG (Isopropyl-beta -D-thiogalactoside)诱导重组紫穗槐二烯合酶的表达。表达产物经镍琼脂糖柱纯化。纯化蛋白加入酶促反应体系（含FPP），GC-MS分析酶促反应体系的正己烷萃取物，结果显示重组紫穗槐二烯合酶可以催化FPP向紫穗槐二烯的转化。体外酶促动力学分析表明，两个位点的氨基酸突变，并没有影响到青蒿紫穗槐二烯合酶的催化活性。基因组DNA杂交表明，紫穗槐二烯合酶基因在001株系基因组中至少有4个拷贝。 二、中药青蒿鲨烯合酶的大肠杆菌表达、纯化与功能鉴定 将经RACE方法克隆到的中药青蒿鲨烯合酶cDNA(AF302464) 开放阅读框的3'末端截短99 bp，插入到原核表达载体pET30a(+)的NcoⅠ和BamHⅠ酶切位点之间，构建N端和C端均携带有HIS6表达标签的鲨烯合酶重组表达载体pETSSA。将pETSSA转入大肠杆菌BL21(DE3)， IPTG (Isopropyl-beta-D-thio galactoside)诱导重组鲨烯合酶的表达。表达产物经镍琼脂糖柱纯化。纯化蛋白加入酶促反应体系（含FPP和NADPH），GC-MS分析酶促反应体系的正己烷萃取物，结果显示重组鲨烯合酶可以催化FPP向鲨烯的转化。青蒿鲨烯合酶的功能鉴定，为进一步利用反义或RNAi技术限制甾类生物合成，从而提高青蒿中的青蒿素含量提供了基础。 三、中药青蒿法呢醇合酶原核表达、纯化与功能鉴定 将经RACE方法克隆到的中药青蒿倍半萜合酶cDNA ( AF304444) 开放阅读框插入到原核表达载体pET30a(+)的NcoⅠ和BamHⅠ酶切位点之间，构建N端和C端均携带有HIS6表达标签的重组表达载体pET30SESQ。将pET30SESQ转入大肠杆菌BL21(DE3)， IPTG (Isopropyl-beta-D-thioga lactoside)诱导蛋白表达，表达产物经镍琼脂糖柱纯化。纯化蛋白加入酶促反应体系（FPP），GC-MS分析酶促反应体系的正己烷萃取物，结果显示此重组酶可以催化FPP向法呢醇的转化。 四、中药青蒿FPS、ADS双功能酶基因的构建、表达与功能鉴定 将青蒿素生物合成途径中催化两步连续反应的酶：法呢基焦磷酸合酶和紫穗槐二烯合酶的基因进行融合，经大肠杆菌表达后鉴定融合蛋白的功能，结果表明融合蛋白具有了双功能酶活性。进一步将融合酶基因转入酿酒酵母中，发酵后检测紫穗槐二烯的含量，并与同时转入法呢基焦磷酸合酶和紫穗槐二烯合酶单个基因的酵母、单独转入紫穗槐二烯合酶基因的酵母进行了比较，结果表明，转入双功能酶的酵母发酵获得的紫穗槐二烯含量要比两个对照酵母高，这表明，获得的双功能酶的催化效率要比两个单独酶的催化效率高。 五、过量表达青蒿紫穗槐二烯合酶对青蒿中青蒿素及其前体物含量的影响 利用根癌农杆菌介导，将青蒿紫穗槐二烯合酶转入青蒿株系001，分子检测证明，紫穗槐二烯合酶整合到了青蒿基因组中并在mRNA水平得到了高效表达。部分转基因青蒿的青蒿素含量有明显增加，最多的比001株系提高了41%。青蒿酸和二氢青蒿酸含量测定表明，转基因青蒿株系的青蒿酸和二氢青蒿酸含量最多的比对照分别提高了47%和79%。这些结果表明，紫穗槐二烯合成在青蒿素生物合成途径中是一个限速步骤，同时，也显示青蒿酸或二氢青蒿酸的进一步转化也可能是青蒿素生物合成中下游的限速步骤。

青蒿素生物合成相关基因对烟草遗传转化的研究

　　青蒿素是存在于中药青蒿(Artemisia annua L.)中的一种含有过氧桥的倍半萜内酯化合物，是中国科学家研发出的当今最有潜力的抗疟药剂，较传统抗疟药很少或无毒副作用，因此青蒿素的生产备受人们关注。目前，青蒿素的生产主要以植物提取为主，但由于青蒿植株中青蒿素的含量很低（约占干重的0.01%～0.8%），从而导致青蒿素价格昂贵，使许多贫困地区的疟疾患者无法得到医治，故提高青蒿植株中青蒿素的含量或扩大青蒿素的来源，降低生产青蒿素的成本具有重要的意义。　　 　　本论文基于扩大青蒿素的来源和提高青蒿植株中青蒿素含量的目的，开展了以下两方面的工作： 一、紫穗槐二烯在烟草中组合生物合成的研究 　　紫穗槐二烯合酶（amorpha-4,11-diene synthase，ADS）是青蒿素生物合成的关键酶之一，为了能在烟草中合成青蒿素的前体，本研究将青蒿的紫穗槐二烯合酶基因置于CaMV 35S启动子控制下，通过根癌农杆菌介导转入烟草（Nicotiana tobacum L.），并获得了转ADS基因烟草植株。经PCR及Southern杂交分析表明，ADS基因已经整合到转基因烟草基因组中；RT-PCR及对转基因烟草中ADS酶活性和产物中紫穗槐二烯和植物甾醇的测定分析，进一步证明整合的ADS基因在转录、翻译水平上均已经表达。上述结果表明，利用基因工程将青蒿素生物合成途径的关键酶基因导入植物，转基因植物中能够合成青蒿素的前体，这一研究结果为利用转基因植物生产青蒿素或其前体奠定了基础。 二、青蒿鲨烯合酶双链干涉基因对烟草的遗传转化研究 　　鲨烯合酶（squalene synthase, SQS）是甾醇类生物合成分支途径的关键酶之一，利用RNA干扰技术(RNA interference，RNAi)抑制目标基因表达的技术已日趋成熟。本文根据植物中hpRNA(hairpin RNA)的原理，在与烟草SQS同源性高达80％的青蒿ASQS序列的5/端保守区选择622 bp作为构建RNAi的序列，借助中间克隆载体，经过三次亚克隆，最后形成含ASQS－RNAi表达盒的双元表达载体pART27－ASQS，并转入农杆菌EHA105。采用农杆菌介导的烟草叶盘转化法，共获得了12棵转基因植株。转基因植株经过PCR和PCR-Southern blotting 检测，证实外源ASQS基因已经导入烟草中，并已经成功整合到烟草基因组中；通过RT-PCR分析说明，转基因烟草中SQS基因的表达已被成功抑制，部分转基因植株中内源SQS的干扰效果高达90％以上。对SQS的直接产物鲨烯和最终产物植物甾醇的检测显示，转基因烟草的植物甾醇和鲨烯的含量明显低于对照。本实验的结果为下一步将此RNA干扰载体导入青蒿，抑制青蒿中ASQS基因的表达，从而提高青蒿素的含量提供了可能。

Hymeniacidon perleve associated bioactive bacterium Pseudomonas sp NJ6-3-1

Among marine bacteria isolated from the cytotoxic sponge Hymeniacidon perleve, one strain NJ6-3-1 classified as Pseudomonas sp. showed both cytotoxic and antimicrobial activities. Fatty acid analysis indicated that the bacterial strain consists mainly of C16:1, C16:0, C18:1, C18:0, C15:0, C14:0. One unusual 9,10-cyclopropane-C17:0 fatty acid and C26:0 also constitute major components, as well as the existence of squalene, the precursor of triterpenoids. The major metabolites in the culture broth were identified as alkaloids, including diketopiperazines and indole compounds, namely 3,6-diisopropylpiperazine-2,5-dione, 3-benzyl-3-isopropylpiperazine-2,5-dione, 3,6-bis-(2-methylpropyl)-piperazine-2,5-dione, indole-3-carboxaldehyde, indole-3-carboxylic acid methyl ester, indole-3-ethanol, and quinazoline-2,4-dione.