Effect of HDL1 infusion on biliary secretion in perfused rat liver

The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in thein vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria plus lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant.     

GD1b-Derived Gangliosides Modulate Fc��RI Endocytosis in Mast Cells

The role of the mast cell–specific gangliosides in the modulation of the endocytic pathway of FcεRI was investigated in RBL-2H3 cells and in the ganglioside-deficient cell lines, E5 and D1. MAb BC4, which binds to the α subunit of FcεRI, was used in the analysis of receptor internalization. After incubation with BC4-FITC for 30 min, endocytic vesicles in RBL-2H3 and E5 cells were dispersed in the cytoplasm. After 1 hr, the endocytic vesicles of the RBL-2H3 cells had fused and formed clusters, whereas in the E5 cells, the fusion was slower. In contrast, in D1 cells, the endocytic vesicles were smaller and remained close to the plasma membrane even after 3 hr of incubation. When incubated with BC4-FITC and subsequently imunolabeled for markers of various endocytic compartments, a defect in the endocytic pathway in the E5 and D1 cells became evident. In the D1 cells, this defect was observed at the initial steps of endocytosis. Therefore, the ganglioside derivatives from GD1b are important in the endocytosis of FcεRI in mast cells. Because gangliosides may play a role in mast cell–related disease processes, they provide an attractive target for drug therapy and diagnosis.     

LDL受体介导的血浆低密度脂蛋白胆固醇的内吞

胆固醇是动物细胞细胞膜的重要组成成分,其做为细胞和环境之间的屏障调节细胞膜的流动性。胆固醇是体内所有的类固醇激素和胆酸合成的前体物质,参与体内代谢。同时胆固醇在神经系统的发育中也起着重要的作用。在血浆中胆固醇以低密度脂蛋白和高密度脂蛋白这两种胆固醇运载血脂蛋白的形式运输。动物细胞通过细胞表面的低密度脂蛋白受体(LDL receptor,LDLR)介导的内吞可以从血液中摄取富含胆固醇的低密度脂蛋白,当细胞表面的LDLR的功能缺陷时,可以导致高胆固醇血症,继而引起动脉粥样硬化、冠心病和中风等严重疾病。本文综述了LDL受体的概述及其通过内吞调节血液中低密度脂蛋白胆固醇水平的作用,并对LDL受体的调节进行了阐述。     

Routes to and from the plasma membrane: bulk flow versus signal mediated endocytosis

Transport of proteins via the secretory pathway is controlled by a combination of signal dependent cargo selection as well as unspecific bulk flow of membranes and aqueous lumen. Using the plant vacuolar sorting receptor as model for membrane spanning proteins, we have distinguished bulk flow from signal mediated protein targeting in biosynthetic and endocytic transport routes and investigated the influence of transmembrane domain length. More specifically, long transmembrane domains seem to prevent ER retention, either by stimulating export or preventing recycling from post ER compartments. Long transmembrane domains also seem to prevent endocytic bulk flow from the plasma membrane, but the presence of specific endocytosis signals overrules this in a dominant manner.     

Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles

Cholesterol is an abundant lipid of the trans-Golgi network (TGN) and of certain endosomal membranes where cholesterol-rich microdomains are important in the organization and compartmentalization of vesicular trafficking. Here we describe the development of a rapid method to isolate a cholesterol-rich endomembrane fraction. We show that widely used subcellular fractionation techniques incompletely separate cholesterol-rich membranes, such as the TGN, from organelles, such as late endosomes and lysosomes. To address this issue, we devised a new subcellular fractionation scheme involving two rounds of velocity centrifugation, membrane sonication, and discontinuous sucrose density gradient centrifugation. This strategy resulted in the isolation of a cholesterol and GM1 glycosphingolipid-enriched membrane fraction that was completely cleared of plasma membrane, endoplasmic reticulum, and mitochondria. This buoyant fraction was enriched for the TGN and recycling endosome proteins Rab11 and syntaxin-6, and it was well resolved from cis-Golgi and early and late endosomal membranes. We demonstrate that this technique can give useful insights into the compartmentation of phosphoinositide synthesis, and it facilitates the isolation of cholesterol-rich membranes from a population of TGN-trafficking vesicles.     

Overexpression of a novel sorting nexin, SNX15, affects endosome morphology and protein trafficking

Sorting nexin (SNX) 15 is a novel member of the SNX family of proteins. Although the functions of most SNXs have not yet been determined, several family members (e.g., SNX1, SNX2, SNX3, and SNX8) are orthologs of yeast proteins involved in protein trafficking. Overexpression of myc-tagged SNX15 in COS-7 cells altered the morphology of several endosomal compartments. In transient transfection experiments, myc-SNX15 was first seen in small punctate spots and small ring structures. Later, myc-SNX15 was found in larger rings. Finally, myc-SNX15 was observed in large, amorphous membrane-limited structures. These structures contained proteins from lysosomes, late endosomes, early endosomes, and the trans-Golgi network. However, the morphology of the endoplasmic reticulum and Golgi was not affected by overexpression of myc-SNX15. In myc-SNX15-overexpressing cells, the endocytosis of transferrin was severely inhibited and endocytosis of tac-trans-Golgi network (TGN) 38 and tac-furin was slowed. In addition, the recycling of internalized tac-TGN38 and tac-furin was also inhibited. Both the morphological and biochemical data indicate that SNX15 plays a crucial role in trafficking through the endocytic pathway. This is the first demonstration that a mammalian SNX protein is involved in protein trafficking.     

Electrostatically induced growth of spiral lipid domains in the presence of cholesterol

The formation of crystalline domains of the phospholipid l--dimyristoyl-phosphatidic acid containing 1 mol% cholesterol, was studied as a function of head group charge by fluorescence microscopy with monolayers at the air/water interface. It is shown that the usual dendritic growth occurs at low pH (8), whereas spiral domains are formed at high pH (11), where the head group contains two negative charges. The findings are ascribed to an electrostatically induced chain tilt that, in conjunction with an in-plane dipole moment, causes a ferroelectric state. This allows for domain aggregation and orientation originating in elongated domains that, additionally, are bent because of the chirality of the molecules. The structure is stabilized and further elongated due to the anisotropic edge activity of cholesterol.     

高通量筛选系统在定向改造中的新进展

酶和细胞工厂是工业生物技术的核心,在医药、化工、食品、农业、能源等诸多领域发挥重要作用.一般天然酶和细胞均需通过分子改造提高其催化效率、稳定性及立体选择性等.定向改造为快速改善酶和细胞工厂的性能提供了可能性,其中灵敏可靠的高通量筛选方法是决定酶和细胞工厂成功高效定向改造的关键.文中阐述并分析讨论了各种筛选方法的优缺点、...