人蛋白酶体亚基PSMB1的重组表达、纯化及在蛋白酶体抑制剂体外筛选中的应用

蛋白酶体是真核细胞中的一类多亚基蛋白酶复合物,它在胞内蛋白质降解的泛素-蛋白酶体通路中起关键作用。重组表达蛋白酶体的活性亚基可以用于在体外筛选、寻找具有蛋白酶体抑制剂作用的化合物。将人蛋白酶体催化亚基(PSMB1)cDNA的编码区(全长726 bp)克隆至原核表达载体pET28a(+),构建重组质粒pET28a-PSMB1,转化大肠杆菌BL21(DE3),通过1 mmol/L IPTG,20℃过夜诱导,获得相对分子量约为27 kDa的重组蛋白,采用IMAC亲和层析柱纯化重组蛋白,纯化后的重组蛋白纯度超过95%。重组蛋白酶解后经NanoLC-MS/MS鉴定表明所表达的融合蛋白氨基酸序列完全正确。在体外BIAcore分析中,重组蛋白表现出对不同化合物的选择性结合能力,其中与蛋白酶体抑制剂雷公藤红素的结合较强,10μmol/L的雷公藤红素与重组蛋白的结合达到27 RU,并且具有良好的浓度依赖型。本研究建立了表达、纯化人蛋白酶体催化亚基PSMB1的方法,并应用于具有蛋白酶体抑制活性化合物的体外筛选。     

A Founder Mutation in the GK1 Gene Is Responsible for Galactokinase Deficiency in Roma (Gypsies)

Galactokinase deficiency is an inborn error in the first step of galactose metabolism. Its major clinical manifestation is the development of cataracts in the first weeks of life. It has also been suggested that carriers of the deficiency are predisposed to presenile cataracts developing at age 20-50 years. Newborn screening data suggest that the gene frequency is very low worldwide but is higher among the Roma in Europe. Since the cloning of the galactokinase gene (GK1) in 1995, only two disease-causing mutations, both confined to single families, have been identified. Here we present the results of a study of six affected Romani families from Bulgaria, where index patients with galactokinase deficiency have been detected by the mass screening. Genetic linkage mapping placed the disease locus on 17q, and haplotype analysis revealed a small conserved region of homozygosity. Using radiation hybrid mapping, we have shown that GK1 is located in this region. The founder Romani mutation identified in this study is a single nucleotide substitution in GK1 resulting in the replacement of the conserved proline residue at amino acid position 28 with threonine (P28T). The P28T carrier rate in this endogamous population is approximately 5%, suggesting that the mutation may be an important cause of early childhood blindness in countries with a sizeable Roma minority.     

Biological rhythm of saliva ghrelin in humans

Background: We previously reported that ghrelin in saliva, orexigenic hormone that induces NPY release, was produced and released by salivary glands in humans. The purpose of this study was to investigate a possible circadian rhythm in saliva ghrelin concentration in human subjects as a function of time and meal. Saliva samples were collected at three-hour intervals throughout a 24-h period in 12 healthy volunteer males and ten healthy volunteer females who were provided with meals on a fixed schedule, and saliva collections were made within 15 minutes after each meal. Saliva ghrelin levels were measured by using a commercial radioimmunoassay (RIA) kit that uses ¹²⁵I-labeled bioactive ghrelin as a tracer and a rabbit polyclonal antibody raised against full-length octanoylated human ghrelin. Immunohistochemical analysis of salivary glands was also performed. The results of this investigation indicated the following. (1) The saliva ghrelin level was slightly higher in female subjects in comparison with male subjects. (2) Saliva ghrelin levels were elevated before each meal and fell to trough levels after eating. (3) Saliva ghrelin levels showed a circadian rhythm that rose throughout the day to a zenith at 0300, then dropped at 0600 - 0900. (4) Saliva ghrelin also weakly correlated with BMI. (5) Immunohistochemical analysis showed that ghrelin was localized in the striated and excretory ducts of salivary glands of human. The present work is the first report of the circadian rhythm of saliva ghrelin level in human subjects as a function of time and meal. Meal plays an important role in lowering saliva ghrelin concentration in humans. However, present data did not exclude whether the circadian changes in saliva ghrelin expression were regulated by the biological clock or by food intake.     

Angiotensin II Inhibits Iron Uptake and Release in Cultured Neurons

Based on the well-confirmed roles of angiotensin II (ANGII) in iron transport of peripheral organs and cells, the causative link of excess brain iron with and the involvement of ANGII in neurodegenerative disorders, we speculated that ANGII might also have an effect on expression of iron transport proteins in the brain. In the present study, we investigated effects of ANGII on iron uptake and release using the radio-isotope methods as well as expression of cell iron transport proteins by Western blot analysis in cultured neurons. Our findings demonstrated for the first time that ANGII significantly reduced transferrin-bound iron and non-transferrin bound iron uptake and iron release as well as expression of two major iron uptake proteins transferrin receptor 1 and divalent metal transporter 1 and the key iron exporter ferroportin 1 in cultured neurons. The findings suggested that endogenous ANGII might have a physiological significance in brain iron metabolism.