Identification of a factor that links apoptotic cells to phagocytes

Apoptotic cells are rapidly engulfed by phagocytes to prevent the release of potentially noxious or immunogenic intracellular materials from the dying cells, thereby preserving the integrity and function of the surrounding tissue. Phagocytes engulf apoptotic but not healthy cells, indicating that the apoptotic cells present a signal to the phagocytes, and the phagocytes recognize the signal using a specific receptor. Here, we report a factor that links apoptotic cells to phagocytes. We found that milk fat globule-EGF-factor 8 (MFG-E8), a secreted glycoprotein, was produced by thioglycollate-elicited macrophages. MFG-E8 specifically bound to apoptotic cells by recognizing aminophospholipids such as phosphatidylserine. MFG-E8, when engaged by phospholipids, bound to cells via its RGD (arginine-glycine-aspartate) motif--it bound particularly strongly to cells expressing alpha(v)beta(3) integrin. The NIH3T3 cell transformants that expressed a high level of alpha(v)beta(3) integrin were found to engulf apoptotic cells when MFG-E8 was added. MFG-E8 carrying a point mutation in the RGD motif behaved as a dominant-negative form, and inhibited the phagocytosis of apoptotic cells by peritoneal macrophages in vitro and in vivo. These results indicate that MFG-E8 secreted from activated macrophages binds to apoptotic cells, and brings them to phagocytes for engulfment.     

Angiogenesis selectively requires the p110alpha isoform of PI3K to control endothelial cell migration

Phosphoinositide 3-kinases (PI3Ks) signal downstream of multiple cell-surface receptor types. Class IA PI3K isoforms couple to tyrosine kinases and consist of a p110 catalytic subunit (p110alpha, p110beta or p110delta), constitutively bound to one of five distinct p85 regulatory subunits. PI3Ks have been implicated in angiogenesis, but little is known about potential selectivity among the PI3K isoforms and their mechanism of action in endothelial cells during angiogenesis in vivo. Here we show that only p110alpha activity is essential for vascular development. Ubiquitous or endothelial cell-specific inactivation of p110alpha led to embryonic lethality at mid-gestation because of severe defects in angiogenic sprouting and vascular remodelling. p110alpha exerts this critical endothelial cell-autonomous function by regulating endothelial cell migration through the small GTPase RhoA. p110alpha activity is particularly high in endothelial cells and preferentially induced by tyrosine kinase ligands (such as vascular endothelial growth factor (VEGF)-A). In contrast, p110beta in endothelial cells signals downstream of G-protein-coupled receptor (GPCR) ligands such as SDF-1alpha, whereas p110delta is expressed at low level and contributes only minimally to PI3K activity in endothelial cells. These results provide the first in vivo evidence for p110-isoform selectivity in endothelial PI3K signalling during angiogenesis.     

Interaction between integrin αIIbβ3 and synthesized cyclic hexapeptide containing RGD

The RGD sequence generally exists in the extracellular matrix proteins and can be recognized by many integrin proteins. The binding ability of immobilized biotinylated cyclic hexapeptide [cyclo(-Arg-Gly-Asp-D-Phe-Lys-Gly-)] containing RGD to integrin α_αbβ₃ was tested by the methods of ELISA and SPR. Results showed that a spacer of 1.48–2.2 nm between the peptide and the biotin residue was long enough to send the RGD sequence into the binding center embccedded within α_IIbβ₃, and the equilibrium dissociation constant was 1.1 μm. The work provides an ideal model system for the research of cell adhesion on solid surfaces.     

ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM

Antigen-specific cell contacts in the immune system are strengthened by antigen-nonspecific interactions, mediated in part by lymphocyte-function associated (LFA) antigens. The LFA-1 antigen is widely expressed on cells of haematopoietic origin and is a major receptor of T cells, B cells and granulocytes. LFA-1 mediates the leukocyte adhesion reactions underlying cytolytic conjugate formation, helper T-cell interactions, and antibody-dependent killing by natural killer cells and granulocytes. Recently, ICAM-1 (intercellular adhesion molecule-1) has been defined as a ligand for LFA-1. Monoclonal antibodies to ICAM-1 block T lymphocyte adhesion to fibroblasts and endothelial cells and disrupt the interaction between cytotoxic T cells and target cells. In addition, purified ICAM-1 reconstituted into artificial membranes binds LFA-1+ cells. ICAM-1 is found on leukocytes, fibroblasts, epithelial cells and endothelial cells and its expression is regulated by inflammatory cytokines. LFA-1 has been placed in the integrin family of cell surface receptors by virtue of the high sequence similarity between the LFA-1 and integrin beta chains. The adhesion ligands of the integrin family are glycoproteins bearing the Arg-Gly-Asp (RGD) sequence motif, for example, fibronectin, fibrinogen, vitronectin and von Willebrand factor. Here we show that a complementary DNA clone ICAM-1 contains no RGD motifs, but instead is homologous to the neural cell adhesion molecule NCAM.     

蒙脱土/聚丙烯成核机理的α→β转变

蒙脱土(MMT)填充等规聚丙烯(i PP)仅形成α-晶。该文利用MMT的Ca2+与庚二酸(PA)反应形成庚二酸钙(Ca PA)制备了表面负载β-成核剂的MMT(β-MMT),FTIR和TGA证实了β-MMT表面Ca PA的形成。采用DSC和XRD研究了β-MMT填充i PP纳米复合材料的β-成核作用和β-晶含量,观察到随着MMT/PA质量比减小(即PA用量增加),i PP复合材料的结晶峰温提高,α-晶熔融峰强逐渐减弱,β-晶熔融峰强逐渐增强,最后仅出现β-晶熔融单峰,表明β-成核作用增强。XRD结果表明随着MMT/PA质量比减小,β-晶含量逐步提高,当MMT/PA质量比为100制备的β-MMT填充i PP纳米复合材料全部形成β-晶。以上结果表明,MMT粒子表面负载β-成核剂可实现MMT填充i PP复合材料结晶从α-晶转变为β-晶。     

Xanomeline新型衍生物SBG"PK-014促进APPsw的cr剪切简

研究了xanomeline新型衍生物SBG-PK-014对毒蕈碱型M1乙酰胆碱受体的激活能力以及对APP基因瑞典型突变体(APPsw)的α-剪切的作用.利用M1激动剂筛选细胞模型检测了SBG-PK-014的EC₅₀和最大响应倍数(FA_max),并在小鼠神经母细胞瘤N2a细胞中同时过表达APPsw和M1受体,分析了该化合物和xanomeline对sAPPα分泌的影响.结果显示,SBG-PK-014的EC₅₀(40.2 nmol/L)与xanomeline (28.4 nmol/L)接近,但FA_max是xanomeline的3.5倍.SBG-PK-014通过激活M1受体促进APPsw的α-剪切,且在0.1 μmol/L和1 μmol/L的浓度下,其效果显著强于同剂量的xanomeline.可见,SBG-PK-014比xanomeline更能有效地激活M1受体,还能促进APPsw的α-剪切和神经保护性sAPPα的生成,在调节阿尔茨海默病的Aβ病理途径上可能有一定潜力,值得进一步研究.     

Molecular simulation study of the binding mechanism of [α-PTi<Subscript>2</Subscript>W<Subscript>10</Subscript>O<Subscript>40</Subscript>]<Superscript>7−</Superscript> for its promising broad-spectrum inhibitory activity to FluV-A neuraminidase

Polyoxometalate (POM) has promising antiviral activities. It shows broad-spectrum inhibiting ability, high efficiency, and low tox-icity. Experimental assays show that titanium containing polyoxotungstates have anti-influenza-virus activity. In this paper, the bind-ing mechanisms of five isomers of di-Ti-substituted polyoxotungstate, [α-1,2-PTi₂W₁₀O₄₀]^7– (α-1,2), [α-1,6-PTi₂W₁₀O₄₀]^7– (α-1,6), [α-1,5-PTi₂W₁₀O₄₀]^7– (α-1,5), [α-1,4-PTi₂W₁₀O₄₀]^7– (α-1,4) and [α-1,11-PTi₂W₁₀O₄₀]^7– (α-1,11), to five subtypes of influenza virus A neuraminidase (FluV-A NA) were investigated in the context of aqueous solution by using molecular docking and molecular dynamics studies. The results show that the isomer α-1,2 is superior to other isomers as a potential inhibitor to neuraminidase. The positively charged arginine residues around the active site of NA could be induced by negatively charged POM to adapt themselves and could form salt bridge interactions and hydrogen bond interactions with POM. The binding free energies of POM/NA complexes range from –5.36 to –8.31 kcal mol^–1. The electrostatic interactions are found to be the driving force during the binding process of POM to NA. The conformational analysis shows that POM tends to bind primarily with N1 and N8 at the edge of the active pocket, which causes the conformational change of the pincers structure comprising residue 347 and loop 150. Whereas, the active pockets of N2, N9 and N4 are found to be more spacious, which allows POM to enter into the active pockets directly and anchor there firmly. This study shows that negatively charged ligand as POM could induce the reorganization of the active site of NA and highlights POM as a promising inhibitor to NA despite the ever increasing mutants of NA.     

Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice

The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.     

Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics

Integrins are important adhesion receptors in all Metazoa that transmit conformational change bidirectionally across the membrane. Integrin alpha and beta subunits form a head and two long legs in the ectodomain and span the membrane. Here, we define with crystal structures the atomic basis for allosteric regulation of the conformation and affinity for ligand of the integrin ectodomain, and how fibrinogen-mimetic therapeutics bind to platelet integrin alpha(IIb)beta3. Allostery in the beta3 I domain alters three metal binding sites, associated loops and alpha1- and alpha7-helices. Piston-like displacement of the alpha7-helix causes a 62 degrees reorientation between the beta3 I and hybrid domains. Transmission through the rigidly connected plexin/semaphorin/integrin (PSI) domain in the upper beta3 leg causes a 70 A separation between the knees of the alpha and beta legs. Allostery in the head thus disrupts interaction between the legs in a previously described low-affinity bent integrin conformation, and leg extension positions the high-affinity head far above the cell surface.     

Alteration of cadherin isoform expression and inhibition of gap junctions in stomach carcinoma cells

To explore cell malignant phenotype correlated changes of cell surface adhesion molecules and cell-cell communication in carcinogenesis, human stomach transformed and cancer cell lines were investigated. Expressions of E-cadherin, N-cadherin,α-catenin, β-catenin as well as gap junction (GJ) protein Cx32 were studied by utilization of immunoblotting, immunocytochemical and fluorescent dye transfer methods. Mammalian normal stomach mucosal cells expressed E-cadherin but not N-cadherin. E-cadherin im-munofluorescence was detected at cell membranous adher-ens junctions (AJ) where colocalization with immunofluo-rescent staining of inner surface adhesion plaque proteins αnd β-catenins was observed. The existence of E-cadherin/ catenin (α-, β-) protein complexes as AJ was suggested. In transformed and stomach cancer cells E-cadherin was inhibited, instead, N-cadherin was expressed and localized at membranous AJ where co-staining with α- and β-catenin fluorescence was observed. Formation of N-cadherin/catenin (α-, β-) protein complex at AJs of transformed and cancer cells was suggested. The above observations were further supported by immunoblotting results. Normal stomach muscosal and transformed cells expressed Cx32 at membranous GJ and were competent of gap junction communication (GJIC). In stomach cancer cells, Cx32 was inhibited and GJIC was defective. The results suggested that changes of signal pathways mediated by both cell adhesion and cell communication systems are associated intracellular events of stomach carcinogenesis. The alteration of cadherin isoform from E- to N-cadherin in transformed and stomach cancer cells is the first report.     

Expression, distribution and function of the focal adhesion kinase (pp125FAK) during murine ectoplacental cone outgrowthin vitro

Mouse embryo implantation is a complex process that includes trophoblast cells derived from ectoplacental cone (EPC) adhesion to and migration through the extracellular matrix (ECM) of uterine endometrium and invasion into the decidua. At the time of implantation, fibronectin (FN) is abundant in the decidua and is distributed pericellularly around each individual stromal cell, and its receptor (integrin α-5β-1) expression on trophoblast populations is up-regulated. The focal adhesion kinase, a 125 ku protein tyrosine kinase (pp125 FAK), is tyrosine phosphorylated upon integrin engagement with its ECM ligand, and its tyrosine phosphorylation sites then serve as the binding sites which couple it with cellular proteins that contain Src SH2 or SH3 domains. Through these linkages, pp125 FAK may integrate multiple signals triggered by integrins. The model of EPC culture %in vitro% was used to study the expression, distribution and function of pp125 FAK during EPC outgrowth on FN. Results indicated that, pp125 FAK primarily expressed and distributed in cellular focal adhesions of the front edge of trophoblast outgrowth from EPC, and was localized in the peripheral region of the individual migrating trophblast cell; antibody or antisense oligodeoxynucleotide to pp125 FAK inhibited EPC attachment and outgrowth, as well as trophoblast cells spreading and migration. This experiment demonstrated that pp125 FAK as an integrin-mediated signaling molecule was involved in EPC outgrowth %in vitro%, and played an important role during trophoblast cells interaction with FN.     

Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function

The motility and morphogenesis of endothelial cells is controlled by spatio-temporally regulated activation of integrin adhesion receptors, and integrin activation is stimulated by major determinants of vascular remodelling. In order for endothelial cells to be responsive to changes in activator gradients, the adhesiveness of these cells to the extracellular matrix must be dynamic, and negative regulators of integrins could be required. Here we show that during vascular development and experimental angiogenesis, endothelial cells generate autocrine chemorepulsive signals of class 3 semaphorins (SEMA3 proteins) that localize at nascent adhesive sites in spreading endothelial cells. Disrupting endogenous SEMA3 function in endothelial cells stimulates integrin-mediated adhesion and migration to extracellular matrices, whereas exogenous SEMA3 proteins antagonize integrin activation. Misexpression of dominant negative SEMA3 receptors in chick embryo endothelial cells locks integrins in an active conformation, and severely impairs vascular remodelling. Sema3a null mice show vascular defects as well. Thus during angiogenesis endothelial SEMA3 proteins endow the vascular system with the plasticity required for its reshaping by controlling integrin function.     

New evidence for microstructural evolution model of acid-activated montmorillonite

Ca-montmorillonite samples from Choushan treated at various acid concentrations were studied using chemical analysis, XRD, ²⁹Si and ²⁷Al MAS NMR, to investigate the microstructure of the activated montmorillonites. With the increase of acid concentration and dissolution of cations of montmorillonite, the intensity of d(001) decreased obviously and significant changes of the microstructure of Si and Al occurred. There were two new types of structural units of Si atoms formed: (SiO)₃SiOH units and Q⁴(0Al) units. For Al atoms, in the course of activation, the removal of one of a pair of octahedral aluminium atoms from montmorillonite removed two hydroxyl groups and left the other aluminum of the pair in the four-fold coordination. The ²⁷Al signal at δ 54.0 corresponding to Al^Ⅳ arose from the four- coordinated Al in the octahedral sheet. This study first con-firmed, by ²⁹Si and ²⁷Al MAS NMR spectra, that the model of microstructural evolution for activated montmorillonite postulated by Thomas et al. (1950) is correct.     

Different patterns of agonist-induced modulation of α1B-adrenoceptor density at different initial expression levels

We compared the norepinephrine (NE) induced α_1B-adrenoceptor (α_1B-AR) expression modulation between two transfected human embryonic kidney (MEK) 293 cell lines in which α_1B-AR densities were (6 336 ± 913) and (773 ± 164) fmol ▪ mg¹, respectively. Treatment of cells with NE (10 μmol ▪ L₁) for 48 h decreased high-level expressed α_1B-AR density, but increased low-level expressed α_1B-AR density. The protein kinase C inhibitor Calphostin C or Ro-31-8220 reversed, and its activator PMA mimicked the NE-induced down-regulation of high-level expressed α_1B-AR. Moreover, PMA induced a down-regulation of low-level expressed α_1B-AR. The endoplasmic reticulum Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) and the calcium chelator BAPTA/AM did not affect the down-regulation of high-level expressed α_1B-AR, but inhibited the up-regulation of low-level expression α_1B-AR induced by NE. These results suggest that α_1B-adrenoceptor densities at different initial expression levels are differentially regulated by NE and their signal transduction pathways are different.     

软骨血管生成抑制因子抑制血管生成及其抗肿瘤作用的研究

测定了小牛软骨血管生成抑制因子对血管内皮细胞细胞毒作用,对内皮细胞骨架系统及其运动迁移的抑制效应,对小鼠肿瘤生长的对抑制效应。     

A peptide fusion protein inhibits angiogenesis and tumor growth by blocking VEGF binding to KDR

Vascular endothelial growth factor (VEGF) binding to its tyrosine kinase receptors (KDR/FLK1, Flt-1) induces angiogenesis. In search of the peptides blocking VEGF binding to its receptor KDR/FLK1 to inhibit tumorangiogenesis and growth, we screened a phage display peptide library with KDR as target protein, and some candidate peptides were isolated. In this study, we cloned the DNA fragment coding the peptide K237 from the library, into a vector pQE42 to express fusion protein DHFR-K237 in E. coli M15. The affection of fusion protein DHFR-K237 on endothelial cell proliferation and angiogenesis was investigated. In vitro, DHFR-K237 could completely block VEGF binding to KDR and significantly inhibit the VEGF-mediated proliferation of the human vascular endothelial cells. In vivo, DHFR-K237 inhibited angiogenesis in chick embryo chorioallantoric membrane and tumor growth in nude mice. These results suggest that K237 is an effective antagonist of VEGF binding to KDR, and could be a potential agent for cancer biotherapy.     

Synthesis and crystal structure of hydroquinone-β-cyclodextrin complex

The title inclusion complex with the formula [(C₄₂H₇₀O₃₅)₂·(C₆O₂H₆)₃·(H₂O)_21.2(CH₃OH)₂] has been crystallized and characterized by single crystal X-ray analysis. The complex crystallizes in the triclinic system, space group P1, with a = 1.5500 (1), b = 1.5536(1), c = 1.8246(1) nm, α = 113.17(2), β = 99.12(2), γ = 103.37(3)o, V = 3.7739 (4) nm³, Z = 1; D_c = 1.343 g/cm³, μ = 0.124 mm^-1, F(000) = 1594, R = 0.0795 and S = 0.9639. Two independent molecules of β -cyclodextrin (β -CD) form a “head-to-head” dimer. The fairly important factor of the position and stability of the guest in the host cavity is the solvation of the polar group of the guest.