GCK基因和HNF-1α基因突变的研究

目的:研究一典型的青少年发病的成人型糖尿病家系并研究其基因突变位点。方法:以1个典型MODY家系的7名成员为研究对象,同时以10名无糖尿病家族史的普通2型糖尿病患者和10名健康人员为2个对照组。抽取外周血,分离白细胞,用快速盐析法提取基因组DNA,以基因组DNA为模板对HNF-1α基因的4号、2号和6号外显子和GCK基因的1号外显子进行PCR扩增,扩增产物经纯化后直接进行序列测定,并分别和各自的正常序列进行比较。结果:7名MODY家系成员HNF-1α2号外显子上游的内含子均存在一碱基G→A置换,即IVS2nt-42 G-A;4例存在HNF-1α6号外显子P380fsinsG移码突变,其中1例合并P379S点突变和IVS6nt-4G-A突变;1例存在P379S点突变;2例未发现突变和多态性。GCK基因的1号外显子及其内含子均未发现有突变或多态性。20名对照组成员均未发现有GCK1号外显子和HNF-1α2、4、6号外显子及其内含子的突变。结论:本家系是HNF-1α基因的6号外显子及其上游内含子突变(移码突变和/或点突变)和2号外显子上游内含子的一个碱基突变,该家系MODY属于MODY3。     

Notch inhibition promotes fetal liver stem/progenitor cells differentiation into hepatocytes via the inhibition of HNF-1β

In a previous study, the Notch pathway inhibited with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (also called DAPT) was shown to promote the differentiation of fetal liver stem/progenitor cells (FLSPCs) into hepatocytes and to impair cholangiocyte differentiation. The precise mechanism for this, however, was not elucidated. Two mechanisms are possible: Notch inhibition might directly up-regulate hepatocyte differentiation via HGF (hepatocyte growth factor) and HNF (hepatocyte nuclear factor)-4α or might impair cholangiocyte differentiation thereby indirectly rendering hepatocyte differentiation as the dominant state. In this study, HGF and HNF expression was detected after the Notch pathway was inhibited. Although our initial investigation indicated that the inhibition of Notch induced hepatocyte differentiation with an efficiency similar to the induction via HGF, the results of this study demonstrate that Notch inhibition does not induce significant up-regulation of HGF or HNF-4α in FLSPCs. This suggests that Notch inhibition induces hepatocyte differentiation without the influence of HGF or HNF-4α. Moreover, significant down-regulation of HNF-1β was observed, presumably dependent on an impairment of cholangiocyte differentiation. To confirm this presumption, HNF-1β was blocked in FLSPCs and was followed by hepatocyte differentiation. The expression of markers of mature cholangiocyte was impaired and hepatocyte markers were elevated significantly. The data thus demonstrate that the inhibition of cholangiocyte differentiation spontaneously induces hepatocyte differentiation and further suggest that hepatocyte differentiation from FLSPCs occurs at the expense of the impairment of cholangiocyte differentiation, probably being enhanced partially via HNF-1β down-regulation or Notch inhibition.     

Transcription of the human microsomal epoxide hydrolase gene (EPHX1) is regulated by an HNF-4α/CAR/RXR/PSF complex

Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes where they are involved in cholesterol excretion and metabolism, lipid digestion and regulating numerous signaling pathways. Previous studies have demonstrated the critical role of GATA-4 and a C/EBPα–NF/Y complex in the regulation of the mEH gene (EPHX1). In this study we show that HNF-4α and CAR/RXR also bind to the proximal promoter region and regulate EPHX1 expression. Bile acids, which inhibit the expression of HNF-4α also decrease the expression of EPHX1. Studies also established that the binding of HNF-4α was essential for the activation of EPHX1 activity by CAR suggesting the formation of a complex between these adjacent factors. The nature of this regulatory complex was further explored using a biotinylated oligonucleotide of this region in conjunction with BioMag beads and mass spectrometric analysis which demonstrated the presence of an additional inhibitory factor (PSF), confirmed by co-immunoprecipitation and ChIP analyses, which interacted with DNA-bound CAR/RXR/HNF-4α forming a 4-component regulatory complex.     

微生物发酵生产十一烷1，1 1二羧酸的研究

从热带假丝酵母(Candiada tropicalis)T25—14经过紫外线和亚硝酸的多次诱变,获得4株产十一烷l,11二羧酸(DC₁₃)较多的突变株,其中最优的NP-159株以20％(V／V)正十三烷(nC₁₃)为碳源摇瓶发酵4天,DC₁₃达80g／L左右。在16L罐上,以30％(V／V)nC₁₃发酵6天,DC₁₃高达139g／L,回收残烃后,对nC₁₃的转化率为80％以上。后处理收率为78．9％,DC₁₃的纯度为95．3％。     

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)

Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H⁺) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.     

Niemann–Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter

Niemann–Pick C1 Like 1 (NPC1L1) has been identified and characterized as an essential protein in the intestinal cholesterol absorption process. NPC1L1 localizes to the brush border membrane of absorptive enterocytes in the small intestine. Intestinal expression of NPC1L1 is down regulated by diets containing high levels of cholesterol. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that cholesterol absorption inhibitor ezetimibe specifically binds to an extracellular loop of NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice are resistant to diet-induced hypercholesterolemia, and when crossed with apo E null mice, are completely resistant to the development of atherosclerosis. Intestinal gene expression studies in Npc1l1 null mice indicated that no exogenous cholesterol was entering enterocytes lacking NPC1L1, which resulted in an upregulation of intestinal and hepatic LDL receptor and cholesterol biosynthetic gene expression. Polymorphisms in the human NPC1L1 gene have been found to influence cholesterol absorption and plasma low density lipoprotein levels. Therefore, NPC1L1 is a critical intestinal sterol uptake transporter which influences whole body cholesterol homeostasis.