Detection and quantification of 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) in rat retina,optic nerve,and brain

Methanol poisoning is characterized by the accumulation of formic acid, a metabolite of methanol, which can lead to metabolic acidosis and ocular toxicity. Formate metabolism to CO₂ is governed by tissue H₄folate and 10-FTHFDH levels. Presumably, rats are not normally susceptible to formate toxicity because they possess high hepatic H₄folate and 10-FTHFDH levels. However, the ability of target tissues to metabolize formate is not known. Therefore, studies were performed to determine whether 10-FTHFDH was present in rat retina, optic nerve, and brain. 10-FTHFDH levels were determined using Western blot analysis of mitochondiral and postmitochondrial preparations from these tissues. Hepatic mitochandrial and postmitochondrial levels of 10-FTHFDH were 13 and 12 ng/μg protein, respectively. Postmitochondrial levels of 10-FTHFDH in rat retina, optic nerve and whole brain were 0.2, 1.3, and 2.1 ng/μg protein; mitochondrial values in retina and brain were 0.2 and 1.5 ng/μg protein, respectively. Postmitochondrial values obtained for rat brain regions were similar to those found for whole brain. These results suggest that, in rats, target tissues possess the capacity to metabolize formate to CO₂ and may be protected from formate toxicity through this folate-dependent system.     

一个在肝癌中表达下调的基因syap1的克隆和鉴定

肿瘤细胞区别于正常细胞的最基本特征是细胞生长失控和分化受阻。这种细胞生长失控和分化受阻是由于多种遗传性缺陷积累的结果,这主要表现在两个方面:一是癌基因的激活或过度表达,阻止细胞分化,促进细胞生长;另一方面是肿瘤抑制基因的失活。在肝癌研究中人们发现有多处染色体DNA发生缺失如染色体1p、4q、5q、6q、8p、10p、11p、13q、16q、17p和22q区域,提示这些区域可能存在肿瘤抑制基因。其中13q、17p和8p区域的肿瘤抑制     

Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43

On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.     

细胞内物质转运调节分子ARFGAP3的克隆表达及生化活性

ADP核糖基化因子-GTP酶活化蛋白(ARF GAP)是重要的细胞内物质转运调节分子.在22周孕龄人胎肝cDNA文库中发现一种新基因,其编码的氨基酸序列与大鼠ARF1 GAP有32%同源性.将这种新基因命名为“ARFGAP3”,对其进行功能研究,利用逆转录-聚合酶链式反应(RT-PCR),从人胎盘总RNA中扩增ARFGAP3全长cDNA序列,并将其亚克隆到pGEM-T载体;采用RNA印迹法和斑点杂交法,检测其组织表达谱,发现在多种腺体和睾丸中有很高水平ARFGAP3基因转录,并且只有一种约2.7 kb的转录本.利用基因重组技术,构建表达质粒pBAD/Thio-ARFGAP3,在大肠杆菌中表达,采用亲和层析法纯化表达产物,利用肠激酶切除重组融合蛋白N端引导序列.检测重组ARFGAP3的生化活性,证实ARFGAP3对ARF1具有GAP活性,促进ARF1结合的GTP水解为GDP,磷脂酰肌醇二磷酸(PIP2)增强其GAP活性,而磷脂酰胆碱(PC)抑制其GAP活性.     

Cloning and expression of a novel retinoblastoma binding protein cDNA,RBBP10

A 2860-bp cDNA was isolated from a human fetal brain cDNA library by high throughput cDNA sequencing, which encodes a putative protein with 186 amino acids. The putative protein shares 90.7% identity with rat pBOG (3403163) and shares 93.4% identity with human RBBP9 (NPA conserved RB binding domain, L × C × E, located between residue 63 and 68 was recognized. Therefore, it was named RBBP10. Mapviewer analysis locates it on human chromosome 20q11.22. RBBP10 spans about 9.6 kb of the genome and consists of six exons and five introns. RT-PCR revealed that the gene was expressed widely in various human tissues, and the expression level is somewhat higher in tumor tissues than in normal tissues. But subsequent sequencing analysis did not found any mutation of this in tumor tissues. The COS 7 cell transfected with the ORF of RBBP10 showed that the protein was distributed both in the cytoplasm and in the nucleus. Our results suggest that RBBP10 is the orthologue of the rat BOG gene (AF025819) and a paralogue of human RBBP9 (AF039564).