HEPARIN CHEMISTRY REGULATING LIVER GENE EXPRESSION

肝脏特异基因的表达，特别是其mRNA的合成 发现依赖于多肽激素和肝素的协同作用 (HPS)。比较了40多种完好的、原生的 糖胺多聚糖(GAG)和PGS表明，只有HPS或PGS合并Hp- 与Gag链一样，可以与多肽激素协同调节mRNA 组织特异性基因的合成。相比之下，硫酸肝素(HSS)， 在所有浓度和所有来源的情况下，被证明是不活跃的甚至 抑制性的。所有被测试的PG都观察到转录后效应， 对于所有种类的HPS和弱含地甘膦硫酸盐的口香糖 (DSS)。PG相对于以下各项的效力等级顺序 转录后效应是HP-PGs&gt；&gt；HS-PGs&gt；&gt；&gt；DS-PGs&gt；&gt；chondroitin 硫酸盐-PG(CS-PG)和活性GAG中的HPS&>DSS。 我们建议完成正在进行的结构-功能分析，以定义 活性PG和GAG的化学，使它们能够调节基因 表情。我们完成的研究使我们能够专注于HPS， PG中活性最强的菌种HP-PGs的生物活性成分 能够调节肝细胞中的基因表达。HSS是密切相关的 在化学上，是不活跃的，并将被用作阴性对照。这个 生物检测，所有记录的幽门螺杆菌敏感，将是： 检测核形状变化的形态分析；]2)分子 胰岛素反应基因合成和丰度的杂交检测 基因；以及3)缝隙连接的电生理检测。单元格将是 在无血清培养液中用测试HP或HP组分治疗 仅补充完全定义和纯化的激素或生长 因子、成分和浓度为每个基因量身定做。这个 分析格式为：1)完成HPS和OF的屏幕 具有类似幽门螺杆菌活性的聚阴离子(例如苏拉明)使我们能够识别 特别是活性物种；2)对最活跃的物种进行剖析 分子量(链长)、电荷密度、度数的贡献 和硫酸盐化的形式，以及抗凝血活性的程度； 并从结构上分析最活跃的HPS中最活跃的部分 和聚阴离子；4)从化学分析中推断出 馏分有共同点，并相应地制备修饰版；5) 评估修饰版本的生物活性，6)重复3-5次 直到鉴定出活性糖序列(S)。] 确定的最有活性的HP糖(S)将用于引发 幽门螺杆菌/胰岛素信号转导机制的研究(S) 基因表达的调控。我们首先将重点放在研究 钙和细胞内pH，因为我们之前的研究表明 膜-已知的第二信使cAMP的永久类似物 在转录速率的调节中无法与HPS协同作用 检测了任何肝脏特异的基因。[我们还将评估HPS是否修改 胰岛素受体水平或胰岛素自身的结合亲和力 受体或IGF I或II受体。]

Expression of liver-specific genes, especially their mRNA synthesis, was found dependent upon cooperative effects of peptide hormones and heparins (HPs). Comparison of more than 40+ species of intact, native glycosaminoglycans (GAGs) and PGs indicates that only HPs or PGS with HP- like GAG chains, can cooperate with peptide hormones to regulate mRNA synthesis of tissue-specific genes. By contrast, heparin sulfates (HSs), at all concentrations and from all sources, proved inactive or even inhibitory. Posttranscriptional effects were observed with all tested PGs, and of the GAGs, with all species of HPs and weakly with dermantan sulfates (DSs). The rank order of potency of the PGs with respect to posttranscriptional effects was HP-PGs>>HS-PGs>>>DS-PGs>>chondroitin sulfate-PG (CS-PG) and of the active GAGs, HPs>>>>DSs. We propose to complete a structure-function analysis, ongoing, to define the chemistry of active PGs and GAGs enabling them to regulate gene expression. Our completed studies allow us to focus on HPs, the biologically active components of HP-PGs, the most active species of PG able to regulate gene expression in liver cells. HSs are closely related chemically, are inactive, and will be used as negative controls. The biological assays, all documented to be HP sensitive, will be: [1) morphological assays to detect nuclear shape changes;] 2) molecular hybridization assays for mRNA synthesis and abundance of insulin-responsive genes; and 3) electrophysiological assays for gap junctions. Cells will be treated with a test HP or HP fraction when in serum-free medium supplemented only with completely defined and purified hormones or growth factors, the composition and concentrations tailored for each gene. The format of the analysis is to: 1) complete the screens of HPs and of polyanions with HP-like activity (e.g. suramin) allowing us to identify especially active species; 2) dissect the most active ones for contributions of molecular weight (chain length), charge density, degree and form of sulfation, and extent of anticoagulant activity; [3) chemically and structurally analyze the most active fractions of the most active HPs and polyanions; 4) deduce from the chemical analysis what the active fractions have in common and prepare modified versions accordingly; 5) assess the biological activity of the modified versions and 6) repeat 3-5 until the active saccharide sequences (s) are identified.] The most active HP saccharide(s) identified will be used to initiate studies on the signal transduction mechanism(s) involved in HP/insulin's regulation of gene expression. We will focus initially on studies of calcium and intracellular pH, since our prior studies indicated that membrane-permanent analogues of one known second messenger, cAMP, were unable to synergize with HPs in the regulation of transcription rates for any liver-specific gene assayed. [We will also assess whether HPs modify insulin receptor levels or the binding affinity of insulin for its own receptor or for the receptors for IGF I or II.]