FOLATE DEFICIENCY AND ONE-CARBON METABOLISM

这项提案的广泛目标是确定是否存在缺陷 甲基团从头合成所需的叶酸， 导致与缺乏预制甲基类似的代谢后果 组。不含蛋氨酸和胆碱的饮食会导致自发 肝癌。乙硫氨酸，蛋氨酸的类似物，干扰 甲基化，是一种致癌物质，但当注射到胆碱缺乏时 小鼠，乙硫氨酸会迅速引起急性胰腺炎。一种解释是 甲基缺乏的影响是S的比例下降- 腺苷蛋氨酸(SAM)以S-腺苷-同型半胱氨酸(SAH)为结果。 当多种细胞甲基化反应被抑制时， SAM/SAH的比率降低，包括DNA的甲基化 控制真核生物中的基因表达。私人侦探已经证明了这一点。 叶酸缺乏会降低肝脏中SAM/SAH的比率 和胰腺，这与甲基引起的胰腺一样大 缺乏症。这种SAM/SAH的比例是由甘氨酸N- 甲基转移酶(GNMT)，含有紧密结合的叶酸。 免疫组织化学研究表明，GNMT集中在 胰腺外分泌。高浓度的GNMT意味着甲基化 和叶酸对某些类型的细胞特别重要。这些 在具体目标方面进行了调查，其中包括： 1.研究叶酸缺乏是否影响胰腺分泌 用AR42J细胞系进行体内、原位和组织培养。 2.研究叶酸缺乏是否影响脑组织的甲基化 小分子(肌酸、磷脂)和大分子(DNA)。 3.将GNMT的免疫组织化学定位扩展到多种 纸巾。 4.建立检测体内叶酸的免疫组织化学方法 纸巾。 叶酸的免疫组织化学定位将仿照 神经化学家用于脑定位的方法 神经递质。通过以下途径可使大鼠迅速产生叶酸缺乏 喂食Walzem和Clifford定义的氨基酸饮食。甲基化不足 的能力将通过酶结合的能力进行评估 通过改变聚A RNA的表达而增加甲基，以及 通过改变DNA对限制性内切酶的反应。 叶酸缺乏时DNA甲基化不足会影响基因表达。 评估组织内特定细胞的叶酸水平的能力 可以提供一种寻找局部叶酸缺乏作为原因的方法 吸烟者和口服避孕药使用者的肿瘤前病变。

The broad objective of this proposal is to determine whether a deficiency of folate, which is needed for the de novo synthesis of methyl groups, causes similar metabolic consequences as a deficiency of preformed methyl groups. Diets that contain no methionine and choline cause spontaneous liver cancer. Ethionine, an analogue of methionine that interferes with methylation, is a carcinogen, but when injected to choline deficient mice, ethionine rapidly causes acute pancreatitis. One explanation for the effects of methyl deficiency has been the decreased ratio of S- adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) that results. A wide variety of cellular methylation reactions are inhibited when the ratio of SAM/SAH is decreased, including the methylation of DNA which controls gene expression in eukaryotes. It has been shown by the P.I. that folate deficiency produces a decrease in the SAM/SAH ratio, in liver and pancreas, which is as great as that caused by methyl group deficiency. This SAM/SAH ratio is regulated by the enzyme, glycine N- methyltransferase (GNMT), which contains tightly bound folate. Immunohistochemical studies have shown that GNMT is concentrated in the exocrine pancreas. A high concentration of GNMT implies that methylation and folate are particularly important for certain cell types. These aspects are investigated in the specific aims which are: 1. To investigate whether folate deficiency affects pancreatic secretion in vivo, in situ and in tissue culture using the AR42J cell line. 2. To investigate whether folate deficiency affects the methylation of small molecules (creatine, phospholipids) and macromolecules (DNA). 3. To extend the immunohistochemical localization of GNMT to a variety of tissues. 4. To develop an immunohistochemical method for detecting folate within tissues. Immunohistochemical localization of folate will be modeled after the method used by the neurochemists for the localization of brain neurotransmitters. Rapid folate deficiency will be produced in rats by feeding the amino defined diet of Walzem and Clifford. Undermethylation of DNA will be evaluated by the ability to enzymatically incorporate additional methyl groups, by an altered expression of poly A+ RNA, and by an altered response of DNA to restriction endonucleases. Undermethylation of DNA in folate deficiency can affect gene expression. The ability to estimate folate levels of specific cells within tissues may provide a means of looking for localized folate deficiency as a cause of pre-neoplastic lesions in smokers and oral-contraceptive users.