Analysis of Protein-Protein Interactions in the Arabidopsis Flavonoid Enzyme System

WPCM 2 B P Z Courier 10cpi #| x =V x 6 x @ 8；X @佳能LBP-8III（附加）calb8ad。PRS x @ 0jzx @ #| x 2 zb % x F '佳能LBP-8III（附加）calb8ad。PRS x @ 0jzx @信使10cpi信使脚注16cpi。吗?x x x, x 6 x @ 8；X @ + h h h, s, h 6 X @；@ t" t} r} r 6e t} r 6h t} r。适当的细胞内胆固醇水平的维持依赖于胆固醇外排和脂质成分与血清中细胞外受体结合后摄取之间的动态平衡。为了研究这一现象，开发了一种测定“释放”胆固醇与血清受体分子结合的方法。本研究的目的是扩大目前的测定，以获得更多的信息，其他参数，可以影响胆固醇外排。这些因素包括细胞内胆固醇含量和血清成分的相对活性，而不是那些已知的调节胆固醇稳态的脂蛋白，如卵磷脂胆固醇酯水解酶（LCAT）和胆固醇酯转移蛋白（CETP）。为了开始监测胆固醇进入细胞外受体池后的运动，将开发测定细胞内酰基胆固醇酯转移酶（ACAT）的方法。在许多细胞系统中，ACAT活性的变化与细胞胆固醇有关。有了ACAT酶测定，胆固醇外排值将与血清成分的变化相关，如高密度脂蛋白（HDL）亚类、其他脂蛋白以及LCAT和CETP的标准参数。最后，这种新的检测方法将被用于筛选来自基因工程小鼠的小体积血清样本，这些小鼠有多余的或缺失的血清蛋白基因拷贝，这些血清蛋白在胆固醇代谢中起着至关重要的作用。一种更好的方法将被开发出来来测量血清中循环总胆固醇浓度的变化。这是很难做到的，因为胆固醇与多种不同的循环血清成分结合存在，在这种结合状态下，它不能通过目前使用的普通程序检测到。这种检测系统的发展和血清中更多不同细胞外胆固醇吸收成分的鉴定将为更好地理解脊椎动物的胆固醇代谢提供帮助。抗坏血酸在一些哺乳动物组织细胞中的浓度几乎是血浆中的20倍。在哺乳动物系统中，抗坏血酸以两种状态存在；抗坏血酸，还原形式，可自氧化产生脱氢抗坏血酸（DHAA）。DHAA是有毒的，当它被细胞吸收时，它以还原形式储存在细胞内。DHAA在细胞内还原为抗坏血酸的机制尚不清楚。多种含SH的化合物，如还原性谷胱甘肽、谷胱甘肽与NADPH或单独NADPH，可以非酶性地还原DHAA。然而，在某些细胞中，dha还原为抗坏血酸的速率是依赖于谷胱甘肽的化学还原反应速率的25倍。这表明在某些细胞中可能存在一种特定的dha抗坏血酸还原系统。在这个实验室中，在至少一种类型的组织中检测到独特的还原活性。初步数据表明，DHAA还原活性可归因于至少两种不同的蛋白质；一种蛋白质出现在细胞质中，另一种与膜相关。在目前的建议中，我们将尝试分离和表征这些假定的DHAA还原蛋白的作用和合成调节，并确定它们在正常细胞功能中的作用。抗坏血酸是动物细胞中同样重要的化合物。然而，在进入细胞的过程中，抗坏血酸可能与氧反应，形成一种有毒的衍生物，叫做脱氢抗坏血酸（DHAA）。为了保护自己，当细胞吸收DHAA时，它会转化为抗坏血酸。问题出现了，抗坏血酸是由DHAA通过细胞内的化学反应形成的，还是有一种特殊的酶系统导致了反应的发生。后一种情况可能是正确的，因为一些细胞可以以比普通化学反应更快的速度转化DHAA。本实验室的初步结果表明，在某些细胞中确实存在两种蛋白质，它们可能形成一个将dha转化为抗坏血酸的系统。一种蛋白质存在于细胞质中，另一种似乎与细胞膜物质结合。我们将尝试纯化这两种蛋白，并确定它们是如何将DHAA转化为抗坏血酸的。我们还将试图确定是什么导致细胞形成这些蛋白质，以及它们对细胞是否重要。我们发现了一种新的delta - 12去饱和酶，并开始对这种酶进行生化表征，该酶将第二个双键置于必需脂肪酸亚油酸中。脊椎动物的饮食中需要18:2（n 6）酸，它成为膜的结构成分，是类二十烷酸的前体。直到最近，包括昆虫在内的所有动物都被认为不能合成亚油酸。只有植物、原生动物和一些真菌容易合成脂肪酸。然而，我们实验室的研究表明，许多昆虫物种确实具有δ 12去饱和酶，能够产生亚油酸。昆虫中去饱和酶的发现为研究脂质生物合成途径中关键酶的进化提供了机会。我们研究的长期目标之一是检测不同？并确定delta 12去饱和过程是否是一种原始的代谢活动，随后被大多数动物所丢失，或者这种酶的拥有是否反映了趋同进化的一个例子。第二个目标是在转录和翻译水平上研究去饱和酶表达的调控。本文介绍了从两种具有代表性的昆虫中克隆该酶的研究。一旦昆虫体内这种酶的基因编码被分离出来，未来旨在回答有关这种关键酶的进化和调控的基本问题的实验就可以开始了。亚油酸是一种脂质或脂肪，是动物细胞壁所必需的。脂质分子也是动物合成类二十烷所必需的，类二十烷在细胞内通讯中起作用。起初，人们认为亚油酸只能由植物、原生动物和某些真菌合成，动物必须从饮食中摄取这种脂质分子。然而，该实验室最近在昆虫体内发现了一种合成亚油酸的关键酶，即delta 12去饱和酶。这一发现为进一步了解亚油酸合成途径的进化史提供了一个机会。这可能是在动物细胞进化过程中丢失的古老途径，或者是昆虫最近获得的一种特性。实验将进行，首先克隆和测序昆虫δ 12去饱和酶基因(s)，然后进行

WPCM 2 B P Z Courier 10cpi #| x =V x 6 X @ 8 ; X @ Canon LBP-8III (Additional) CALB8IAD.PRS x @ 0J zX @ #| x 2 Z B % X F ` Canon LBP-8III (Additional) CALB8IAD.PRS x @ 0J zX @Courier 10cpi Courier Footnote 16cpi . ? x x x , x 6 X @ 8 ; X @ + H H H , S , H 6 X @ ; @ t" t } r } r 6e t t } r 6h t } r . s u t a s } r' I ut r PQV 6e L 2 K 9308279 delalleramoya The maintenance of proper intracellular levels of cholesterol is dependent on a dynamic equilibrium between cholesterol efflux and uptake of the lipid component after having bound to an extracellular acceptor in serum. To study this phenomenon, a method was developed to assay the combination of "released" cholesterol with serum acceptor molecules. The objective of the present research is to extend the current assay in order to obtain more information on other parameters that can influence cholesterol efflux. These factors include intracellular cholesterol content and the relative activities of serum components, other than those lipoproteins already known to regulate cholesterol homeostasis such as lecithin cholesterol ester hydrolase (LCAT) and cholesterol ester transfer protein (CETP). To begin to monitor the movement of cholesterol after entry into the extracellular acceptor pool, methods will be developed to assay intracellular acyl cholesterol ester transferase (ACAT). Changes in ACAT activity have been shown to correlate with cell cholesterol in numerous cell systems. With the ACAT enzyme assay in place, cholesterol efflux values will be correlated with changes in serum components such as high density lipoprotein (HDL) subclasses, other lipoproteins, and standard parameters of LCAT and CETP. Finally, the new assay method will be scaled down for screening small volume serum samples from genetically engineered mice, that have extra, or missing copies, of genes for serum proteins that play a crucial role in cholesterol metabolism. %%% A better method will be developed to measure changes in the circulating total concentration of cholesterol in serum. This is difficult to do because cholesterol exists in combination with a variety of different circulating serum components, and in this combined state, it is not detected by ordinary procedures now in use. Development of such an assay system and the identification of more of the different extracellular cholesterol absorbing components in serum will provide a better understanding of cholesterol metabolism in vertebrate animals. *** 0*0*0* 9306526 Bode Ascorbic acid is maintained in some mammalian tissue cells at concentrations almost 20 fold higher than in plasma. In the mammalian system, ascorbate exists in either of two states; ascorbic acid, the reduced form, which may autooxidize to produce dehydroascorbic acid (DHAA). DHAA is toxic, and when it is taken up by cells, it is stored intracellularly in the reduced form. The mechanism of DHAA reduction to ascorbic acid inside cells is not clear. DHAA may be reduced nonenzymatically by a variety of SH containing compounds, such as reduced glutathione, glutathione together with NADPH, or NADPH alone. In some cells, however, the rate of DHAA reduction to ascorbic acid is 2 5 times above the rate of the glutathione dependent chemical reduction reaction. This suggests that a specific DHAA ascorbic acid reduction system may be at work in some cells. In this laboratory, unique reducing activities have been detected in at least one type of tissue. Preliminary data indicate that DHAA reduction activiti es are attributable to at least two separate proteins; one protein appears in the cytosol and the other is membrane associated. In the present proposal we will attempt to isolate and characterize the action and synthetic regulation these presumptive DHAA reduction proteins and determine their role in normal cell function. %%% Ascorbic acid is as important compound in animal cells. However, on the way to the cells, ascorbic acid may react with oxygen and form a toxic derivative called dehydroascorbic acid (DHAA). To protect themselves, when the cells take up DHAA, it is converted to ascorbic acid. The question arises whether ascorbic acid is formed from DHAA by a chemical reaction inside cells, or whether there is a specific enzyme system the causes the reaction to occur. The latter case may be correct, because some cells can convert DHAA at a faster rate than can be explained by just an ordinary chemical reaction. Preliminary results in this laboratory suggest that two proteins do exist in certain cells, which may form a system to change DHAA to ascorbic acid. One protein exists in the cell cytoplasm and the other one seems to be bound to cell membrane material. We will try to purify these two proteins and determine how they operated to change DHAA into ascorbic acid. We will also try to determine what causes the cell to form these proteins and whether they are important for the cell to have. *** 0*0*0* 9306820 Borgeson We have discovered a novel delta 12 desaturase and have begun the biochemical characterization of this enzyme that places the second double bond in the essential fatty acid linoleic acid. Vertebrates require the 18:2(n 6) acid in their diet, where it becomes a structural component of membranes and is a precursor for eicosanoids. Until recently, all animals, including insects, were considered unable to synthesize linoleic acid. Only plants, protozoa and some fungi readily synthesize the fatty acid. Studies in our laboratory demonstrated, howev er, that a number of insect species do possess a delta 12 desaturase and are able to produce linoleic acid. The discovery of the desaturase in insects presents an opportunity to examine the evolution of a key enzyme in the lipid biosynthetic pathway. One of the long term goals of our research is to examine the delta 12 desaturase in different ? species, and to determine if the process of delta 12 desaturation is a primitive metabolic activity, subsequently lost by most animals, or whether possession of this enzyme reflects an example of convergent evolution. A second goal is to examine the regulation of desaturase expression at the transcriptional and translational level. Studies are described in the present proposal to clone the enzyme from two representative insect species. Once the gene coding for the enzyme in insects is isolated, future experiments designed to answer fundamental questions about the evolution and regulation of this key enzyme can begin. %%% Linoleic acid is a type of lipid, or fat, that is required in the walls of animal cells. The lipid molecule is also required in animals for the synthesis of eicosanoids, which operate in intracellular communication. Originally it was believed that linoleic acid was only synthesized by plants, protozoa and certain fungi, and that animals had to ingest the lipid molecule in their diet. However, a key enzyme in the synthesis of linoleic acid, the delta 12 desaturase, was recently found by this laboratory in insects. This discovery provides an opportunity to learn more about the evolutionary history of the linoleic acid synthetic pathway. Could it be an ancient pathway lost during the evoulation of the animal cell, or is it a property that has been acquired more recently by the insects. Experiments will be undertaken, first to clone and sequence the insect delta 12 desaturase gene(s), and th