The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles

The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP^C). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20–50, 100–200, and 300–400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP^C. STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP^C-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1–PrP^C signaling.     

Lymphokine dependence of in vivo expression of MHC class II antigens by endothelium

Changes in the expression of class II antigens of the major histocompatibility complex (MHC) have an integral role in the regulation of immune responses, and are brought about in vitro by soluble mediators. However, the mechanism that underlies in vivo expression of MHC class II antigens in, for example, endothelial cells in the absence of immunological stimulation has not been studied. We demonstrate here that expression of MHC class II antigens is not a constitutive property of endothelial cells, for MHC class II antigen-positive endothelial cells do not express these antigens during treatment with the immunosuppressive agent cyclosporin A. In vivo MHC class II antigen expression by canine endothelial cells is therefore dependent on factors, probably the lymphokine gamma-interferon produced by the immune system, whose secretion is inhibited by cyclosporin A.     

Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD117)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.     

Phagocytosis of apoptotic cells: a matter of balance

Efficient clearance of apoptotic cells is required to control homeostasis in normal and pathological circumstances, and inappropriate clearance of cell corpses may lead to autoimmune diseases and inflammation. The multiplicity of phagocytotic mechanisms points to the relevance of removing apoptotic cells. A variety of surface molecules present in either the apoptotic bodies or phagocytes help in attachment and initiation of engulfment. Nonetheless, uncontrolled phagocytosis of apoptotic cells and other particles may lead to tissue injury; therefore, negative signals are important in balancing phagocytotic activity. This review aims at a systematic examination of positive and negative signals that modulate the uptake of apoptotic bodies and the signaling mechanisms involved in the clearance of apoptotic cells.Received 13 November 2004; received after revision 5 March 2005; accepted 8 March 2005     

Targeted mutation of Cyln2 in the Williams syndrome critical region links CLIP-115 haploinsufficiency to neurodevelopmental abnormalities in mice

Williams syndrome is a neurodevelopmental disorder caused by the hemizygous deletion of 1.6 Mb on human chromosome 7q11.23. This region comprises the gene CYLN2, encoding CLIP-115, a microtubule-binding protein of 115 kD. Using a gene-targeting approach, we provide evidence that mice with haploinsufficiency for Cyln2 have features reminiscent of Williams syndrome, including mild growth deficiency, brain abnormalities, hippocampal dysfunction and particular deficits in motor coordination. Absence of CLIP-115 also leads to increased levels of CLIP-170 (a closely related cytoplasmic linker protein) and dynactin at the tips of growing microtubules. This protein redistribution may affect dynein motor regulation and, together with the loss of CLIP-115-specific functions, underlie neurological alterations in Williams syndrome.     

两种质粒编码的Borrelia burgdorfei寡肽结合蛋白(OppA)的表达和纯化

为了弄清Borrelia burgdorferi中OppA4和OppA5蛋白的生物学功能,oppAIV和oppAV基因分别被克隆并在大肠杆菌中表达．大肠杆菌BL21CodonPlus（DE3）-RIL被用来克服由A＋T丰富引起的密码偏差,十二烷基-β—D-麦芽糖苷和甘油分别用于提高脂蛋白的分离效果和维护蛋白的稳定性．经过金属离子亲和层析,OppA4和OppA5纯蛋白产量可达到1mg／L细胞以上．