Analysis of the Tetrahydrobiopterin Biosynthesizing System in Cultured Neuron-Derived Cells

培养的神经元来源细胞中四氢生物蝶呤生物合成系统的分析

• 批准号: 10670050
• 负责人: OTA Akira
• 金额: $ 2.11万
• 依托单位: Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670050/
• 关键词: NIE-115 cell line; 6R-tetrahydrobjopterin; lipopolysaccharide; GTP cyclohydrolase "I"; CD14; CD18; Toll-like receptor; tumor necrotizing factorα; リポポリサッカライド受容体; N1E-115細胞; IκB; IκBキナーゼ; 一酸化窒素; 一酸化窒素合成酵素

6R-Tetrahydrobiopterin (BH4) is a naturally occurring essential cofactor for aromatic amino acid hydroxylases and nitric oxide (NO) synthase (NOS). Lipopolysaccharide(LPS), a component of the outer membrane of gram-negative bacilli, has been shown to enhance the BH4 content of the murine neuroblastoma cell line N1E-115 as well as the release of BH4 into culture medium. Such alterations in BH4 production were at first caused by the enhancement of the expression level of RNA encoding GTP cyclohydrolase "I"(GTPCH), a rate-limiting enzyme in the BH4 de novo biosynthetic pathway. Moreover, the expression levels of mRNAs encoding 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase which place at the 2nd and the final step in BH4 biosynthesic pathway, respectively. However, we found no evidence in N1E-115 cells showing the expression of mRNA encoding CD14. CD14 is an membrane-anchoring protein and it is already recognized as the LPS receptor on macrophages. Therefore we searched the candidates for LPS receptor except for CD14 in N1E-115 cells. In a series of experiments, we found the expressions of mRNA encoding CD18, another subtype of LPS receptor on macrophages, and type 4 Toll-like receptor (T1r-4) in this cell line by way of reverse transcriptase-polymerase chain reaction. This should be the first evidence that neuron-derived cell line expresses T"I"r-4..Tumor necrotizing factor α(TNF-α) was also capable of enhancing BH4 production in N1E-115 cells. It is currently under investigation in my laboratory whether the increase in BH4 production induced by TNF-α was caused by the activation of the pathway common to the LPS stimulation.In spite of the increase in BH4 production by LPS stimulation. we could detect neither the increase in NO production nor the increase in the expression levels of mRNAs encoding neuronal and inducible NOS in N1E-115 cells. NOS enzymes in these cell lines were speculated to be saturated by BH4.

6R四氢无菌蛋白酶（BH4）是芳族氨基酸羟化酶和一氧化氮（NO）合酶（NOS）的天然必需辅助因子。脂多糖（LPS）是革兰氏阴性杆菌外膜的一个成分，已被证明可以增强鼠神经母细胞瘤细胞系N1E-1115的BH4含量，以及将BH4释放到培养基中。 BH4产生的这种改变起初是由编码GTP环氢酶“ I”（GTPCH）的RNA表达水平的提高引起的，这是BH4 DE NOVO生物合成途径中的速率限制酶。此外，编码6-吡喃四氢蛋白酶合酶和Sepipterin降低的mRNA的表达水平分别位于2nd和BH4 Biosynthesic途径中的最后一步。但是，我们在N1E-115细胞中没有发现表达编码CD14的MRNA的表达的证据。 CD14是一种膜锚定蛋白，已经被认为是巨噬细胞上的LPS受体。因此，我们在N1E-115细胞中搜索了候选者的LPS受体。在一系列实验中，我们通过逆转录酶 - 聚合酶链反应在该细胞系中发现了编码CD18的mRNA编码CD18的表达，LPS受体的另一种亚型，以及巨噬细胞上的另一种亚型和4型Toll样受体（T1R-4）。应该是第一个证据表明，神经衍生的细胞系表达t“ i” R-4 ..肿瘤坏死因子α（TNF-α）也能够增强N1E-1115细胞中的BH4产生。目前，我的实验室正在研究TNF-α引起的BH4产生的增加是由LPS刺激的激活引起的。在LPS刺激中增加了BH4产生的增加，我们既不能检测到NON产生的增加，也不能检测到编码神经元和nos nos nos中的MRNAS表达水平的增加，而NOS nos nos nos nos nos in n os n oscobil Nos in n os nos nos in n os-115。推测这些细胞系中的NOS酶被BH4饱和。

项目成果

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Akira Nakashima 等人：“多巴胺对 1 型人酪氨酸羟化酶的抑制是由该酶 N 末端的特定部分控制的”《神经化学杂志》。

Nakashima A et al.: "Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme"Journal of Neurochemistry. 72・5. 2145-215* (1999)

Nakashima A 等：“人酪氨酸羟化酶 1 型的多巴胺抑制是由酶 N 末端的特定部分控制的”Journal of Neurochemistry 72·5* (1999)。

Nakashima A et al.: "Expression of human tyrosine hydroxylase type I in Escherichia coli as a protease-cleavable fusion protein. Short communication"Journal of Neural Transmission. 106・9/10. 819-824 (1999)

Nakashima A等人：“人酪氨酸羟化酶I型在大肠杆菌中作为蛋白酶可裂解的融合蛋白的表达。短通讯”106·9/10（1999）。