The nutritional attempt to investigate progressive muscular dystrophy with Duchenne type in human.

研究人类进行性杜氏型肌营养不良症的营养尝试。

• 批准号: 01570292
• 负责人: YAMAGUCHI Tadatoshi
• 金额: $ 1.41万
• 依托单位: Miyazaki Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570292/
• 关键词: TDP Kinase; TDP-binding protein; Protein-bound TDP; Duchenne type muscular Dystrophy; Freezing-thawing treatment; Thiamin triphosphate; Thiamin diphosphate; Human erythrocytes; TDP kinase; チアミン二燐酸; チアミン三燐酸; 筋ジストロフィ-症; 金属キレ-ト剤; SH阻害剤; チアミン

This research has been started for diagnosis of the nutritional state of the patient with DMD. Vitamin B, (thiamin)is presence in blood as four chemical forms : thiamin monophosphate(TMP), thiamin diphosphate(TDP), thiamin triphosphate(TTP), and free thiamin. The quantitative analyses of these are performed by high-performance liquid chromatography(HPLC)according to a post column fluorescence method. In the resuits, although there is no difference of the total thiamin content between the DMD patient and control, a certain difference is observed in the proportions of TDP and TTP. On the other hand, the quantitative values by HPLC changed under the storage of normal human blood at various temperature. These facts might be due to the existence of the enzyme : TDP Kinase which catalysis the synthesis of TTPfrom TDP. The purification of TDP Kinase was performed using protein-bound TDP as a marker by a preparative isoelectric focusing apparatus(Rotofor)and TDP-ligand affinity chromatography. … More The isotated protein was used as reconstitutive material for HoLoenzyme state of the TDP Kinase, but the action of the Kinase was not observed. The TDP-binding activity of the isotated protein was assessed by an equilibrium dialysis apparatus, but the attempt was unsuccessful too(Heisei 1 year). Therefore, the nature of TDP Kinase has been further investigated and has been revealed that the enzyme possessed a SH-group, existed in erythrocytes, and required a certain metal ion and a certain low-molecular component in plasma as a cofactor'(Heisei 2 year). Fractionation with ammonium sulfate between 40 and 60% saturation and DEAE -SephaceL column were newly added in purification procedure. The addition of dithiothreitol to the sample effectively introduced three proteins having molecular weight as 21OKD, 18OKD, and 125KD. For separation of the three proteins, attempts to use TDP-ligand affinity column, Dye -ligand affinity column and Rotoformn and Rotofor have been carried out. While, the relationship between TDP Kinase and DMD was investigated by using of the blood of DMD patients. The results showed that the activity of TDP Kinase was more high in DMD patients than controls, therefore, the TTP content resulted in an increase(Heisei 3 year). It is unknown that the state of the patients is due to the disease or is a result of it, this study shoud be continued. Less

这项研究的目的是诊断DMD患者的营养状况。维生素B（硫胺素）在血液中以四种化学形式存在：一磷酸硫胺素（TMP）、二磷酸硫胺素（TDP）、三磷酸硫胺素（TTP）和游离硫胺素。根据柱后荧光法，通过高效液相色谱法（HPLC）进行定量分析。结果表明，DMD患者与对照组之间虽然总硫胺素含量无差异，但TDP和TTP的比例有一定差异。另一方面，正常人血液在不同温度下储存，HPLC定量值发生变化。这可能是由于存在催化TDP合成TTP的酶：TDP激酶。使用蛋白质结合的TDP作为标记物，通过制备型等电聚焦装置（Rotofor）和TDP-配体亲和层析进行TDP激酶的纯化。 ...更多信息 分离的蛋白质用作TDP激酶HoLoenzyme状态的重建材料，但未观察到激酶的作用。通过平衡透析装置评估分离的蛋白质的TDP结合活性，但尝试也不成功（平成1年）。因此，对TDP激酶的性质进行了进一步研究，并揭示了该酶具有SH基团，存在于红细胞中，并需要血浆中的某种金属离子和某种低分子组分作为辅因子“（Heisei 2年）。在纯化过程中增加了40 ~ 60%饱和度硫酸铵分级和DEAE-SephaceL柱。向样品中加入二硫苏糖醇有效地引入了分子量为21 OKD、18 OKD和125 KD的三种蛋白质。为了分离这三种蛋白质，已经尝试使用TDP-配体亲和柱、染料-配体亲和柱和Rotoform和Rotofor。同时，利用DMD患者的血液，探讨TDP激酶与DMD的关系。结果表明，DMD患者的TDP激酶活性高于对照组，因此，TTP含量增加（平成3年）。尚不清楚患者的状态是由于疾病还是疾病的结果，应继续进行本研究。少