非病毒载体介导的siRNA细胞内导入机理研究

比较了两类非病毒载体,包括阳离子聚合物载体(HPAMAM-PHE45,PEI)和阳离子脂质载体(DOTAP)运载siRNA进入细胞的动态过程.DOTAP/siRNA能够很快地被内吞进入内涵体途径,6 h内就能将绝大多数的siRNA从内涵体中释放出来.而HPAMAMPHE45,PEI/siRNA复合物在与细胞接触0.5 h后首先附着在细胞膜表面,逐渐地部分复合物也经由内涵体途径将siRNA从内涵体中释放出来进入细胞质.但也有一部分复合物可能从窖蛋白介导的内吞途径进入细胞.实验结果显示,细胞摄取阳离子脂质/siRNA主要通过网格蛋白介导的内吞实现,而阳离子聚合物/siRNA主要通过网格蛋白介导的内吞和窖蛋白介导的内吞共同完成.这两种不同的内吞方式可能对非病毒载体的不同转染机理及优化理论有较大影响.深入研究其中的关键因素可以为非病毒载体运载siRNA内吞和胞内途径的机理提供线索.     

中空介孔二氧化硅纳米材料(HMSNs)的合成及其生物相容性

以CaCO₃为模板合成中空介孔二氧化硅纳米材料（HMSNs）, 并通过透射电镜（TEM）和小角X射线粉末衍射（SAXRD）对样品进行表征, 通过流式
细胞仪检测HMSNs样品对A549的细胞内吞能力及生物相容性. 结果表明: CaCO₃ HMSNs是以CaCO₃为核, 介孔二氧化硅为壳的纳米粒子; 当材料的质量浓度为62.5 μg/mL时, 细胞的存活率为100%; 有92.80%的细胞吸收了样品HMSNs, 即HMSNs具有较高的细胞内吞能力及生物相容性.     

CD23 (the low-affinity IgE receptor) as a C-type lectin: a multidomain and multifunctional molecule

This review, regards the low-affinity receptor CD23 as a C-type lectin and compares it with other C-type lectins and C-type lectin-like receptors. C-type lectins such as the asialoglycoprotein receptor, as well as the dendritic cell immunoreceptor and the dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin on dendritic cell lectin, possess amino acid sequences which interact with Ca⁺⁺ and sugar, and many of them possess an endocytosis signal sequence that includes tyrosine or serine in the cytoplasmic region. In contrast, natural killer receptors lack the Ca⁺⁺ and sugar-binding amino acids but conserve homologous cysteines in the form of C-type lectin, and possess an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic region which inhibits killer activity when they recognize the self major histocompatibility (MHC) class I molecule. Since human CD23a form has a similar amino acid sequence, the possibility that this sequence is an endocytosis signal or an ITIM is discussed. The function of the reverse RGD and RGD-binding inhibitory peptide in human CD23 from the point of view of the relation between a C-type lectin and MHC class II molecules is also considered. Received 21 May 2001; received after revision 28 November 2001; accepted 29 November 2001     

A role for the hinge/ear domain of the β chains in the incorporation of AP complexes into clathrin-coated pits and coated vesicles

The clathrin-associated adaptor protein (AP) complexes drive the polymerization of clathrin in coated pits to form coated vesicles. It has previously been shown that the carboxyl-terminal hinge/ear domain of the β2 chain contains a binding site for clathrin and that removal of this domain from APs or from isolated β2 chains abrogates their ability to form clathrin coats in vitro. We show here that the hinge/ear domain is necessary for efficient incorporation of AP complexes into coated pits and coated vesicles in cells, a result that is consistent with the view that the β chains indeed provide an important interaction between the AP complexes and clathrin. Received 7 April 1997; received after revision 22 May 1997; accepted 28 May 1997     

Agonist-induced trafficking of the low-affinity formyl peptide receptor FPRL1

The formyl peptide-like receptor FPRL1 is a member of the chemoattractant subfamily of G protein- coupled receptors involved in regulating leukocyte migration in inflammation. To elucidate mechanisms underlying the internalization of ligand-bound FPRL1 and possible receptor recycling, we characterized the endocytic itinerary of FPRL1. We show that agonist-triggered internalization from the plasma membrane into intracellular compartments is prevented by perturbation of clathrin-mediated endocytosis, such as expression of the dominant-negative clathrin Hub mutant, siRNA-mediated depletion of cellular clathrin and expression of a dominant-negative mutant of the large GTPase dynamin. Internalized FPRL1 co-localized with endocytosed transferrin and the small GTPases Rab4 and Rab11 in vesicular structures most resembling recycling endosomes. Recycling of FPRL1 was significantly reduced by pretreatment with PI3-kinase inhibitors. Thus, ligand-bound FPRL1 undergoes primarily clathrin-mediated and dynamin-dependent endocytosis and the receptor recycles via a rapid PI3-kinase-sensitive route as well as pathways involving perinuclear recycling endosomes.Received 19 March 2004; received after revision 26 April 2004; accepted 12 May 2004     

Internalization of Trichosanthin via Different Endocytic Mechanisms

Trichosanthin （TCS） is a plant toxin with ribosome-inactivating activity. TCS can be internalized by the host cells and then attacks the ribosomes resulting in cell death. However, the manner for endocytic uptake of TCS is not well understood. The present work investigates the endocytosis pathway of TCS in human choriocarcinoma cells. The different endocytic mechanisms are interfered by potassium depletion, cholesterol-extraction/addition, or treatments of various drugs. The experiments detect their effects on the TCS-uptake. The results show that a large portion of the TCS can be internalized by clathrin-dependent, as well as by clathrin-independent but cholesterol-dependent endocytosis in human choriocarcinoma cells.     

Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration in collecting duct epithelial cells

Mutations in CLCN5, which encodes the voltage-dependent Cl⁻/H⁺antiporter, CLC-5, cause Dent’s disease. This disorder is characterized by low molecularweight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. Using a collecting duct cell model (mIMCD-3) in which endogenous clc-5 is disrupted by antisense clc-5 or overexpression of truncated clc-5, we demonstrate altered expression of the crystal adhesion molecule, annexin A2. Endogenously expressed annexin A2 is intracellular with limited plasma membrane localization. Following clc-5 disruption, there is both a marked increase in plasma membrane annexin A2 and an increase in cell surface crystal retention and agglomeration, which may be attenuated using pretreatment with anti-annexin A2 antibodies or wheat germ agglutinin lectin but not by concanavalin A. We hypothesize that in Dent’s disease, endocytic failure leads to an accumulation at the plasma membrane of crystal-binding molecules that include annexin A2 leading to retention of calcium crystals and ultimately nephrocalcinosis and nephrolithiasis. Received 22 October 2005; received after revision 26 November 2005; accepted 2 December 2005     

The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies

In recent years the interest in antimicrobial proteins and peptides and their mode of action has been rapidly increasing due to their potential to prevent and combat microbial infections in all areas of life. A detailed knowledge about the function of such proteins is the most important requirement to consider them for future application. Our research in recent years has been focused on the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum, which inhibits the growth of opportunistic zoo-pathogens including Aspergillus fumigatus, numerous plant-pathogenic fungi and the model organism Aspergillus nidulans. So far, the experimental results indicate that PAF elicits hyperpolarization of the plasma membrane and the activation of ion channels, followed by an increase in reactive oxygen species in the cell and the induction of an apoptosis-like phenotype. Detailed knowledge about the molecular mechanism of action of antifungal proteins such as PAF contributes to the development of new antimicrobial strategies that are urgently needed. Received 09 August 2007; received after revision 17 September 2007; accepted 19 September 2007     

Co-localization of galectin-1 with GM1 ganglioside in the course of its clathrin- and raft-dependent endocytosis

Mammalian galectin-1 (Gal-1), a beta-galactoside-binding lectin has a prominent role in regulating cell adhesion, cell growth and immune responses. Downregulation of these biological functions may occur via internalization of Gal-1. In the present study we have investigated the mechanism and possible mediator(s) of Gal-1 endocytosis. We show that internalization occurs at a temperature higher than 22 degrees C in an energy dependent fashion. After one hour incubation Gal-1 localizes in the Golgi system within the cells, and then disappears without accumulation in degradation compartments, such as lysosomes. Based on their strong intracellular co-localization, two glycoconjugates, GM1 ganglioside and CD7 are implicated in the sorting of internalized Gal-1 into Golgi. Other known Gal-1 binding glycoproteins on T cells (CD2, CD3, CD43 and CD45) do not cointernalize with the lectin. Internalization of Gal-1 depends on its lectin activity and follows dual pathways involving clathrin-coated vesicles and raft-dependent endocytosis.     

Low-density lipoprotein receptor-mediated endocytosis of PEGylated nanoparticles in rat brain endothelial cells

Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to diffuse through the blood-brain barrier after intravenous administration. However, the mechanism of transport of these nanoparticles into brain has not yet been clearly elucidated. The development of a model of rat brain endothelial cells (RBEC) in culture has allowed investigations into this mechanism. A study of the intracellular trafficking of nanoparticles by cell fractionation and confocal microscopy showed that nanoparticles are internalized by the endocytic pathway. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of the nanoparticles. In contrast, chlorpromazine and NaN₃ pretreatment, which interferes with clathrin and energy-dependent endocytosis, caused a significant decrease of nanoparticle internalization. Furthermore, cellular uptake experiments with nanoparticles preincubated with apolipoprotein E and blocking of low-density lipoprotein receptors (LDLR) clearly suggested that the LDLR-mediated pathway was involved in the endocytosis of PEGPHDCA nanoparticles by RBEC. Received 1 September 2006; received after revision 4 December 2006; accepted 18 December 2006