Brain Ligatin: A Membrane Lectin That Binds Acetylcholinesterase

Ligatin, a lectin that recognizes phosphorylated sugars, has been demonstrated in mammalian tissues to bind specific hydrolases to cell surfaces. Ligatin exists as a filament that can be released from membranes still complexed with its bound hydrolases by treatment of membrane preparations with CaCl₂ and/or pH 8.0. The ligatin-hydrolase complexes subsequently can be dissociated with ethyleneglycol-bis(β-amino-ethyl ether) N, N′-tetraacetic acid, resulting in a concurrent depolymerization of the ligatin filament. From membrane preparations of cerebrum, this procedure solubilized ligatin and a membrane-bound acetylcholinesterase (EC 3.1.1.7). Binding of the cosolubilized acetylcholinesterase to ligatin could be demonstrated in vitro by affinity chromatography using the immobilized lectin. Ligatin-hydrolase complexes have been shown to be dissociated by specific phosphorylated sugars (mannose 6-phosphate and glucose 1-phosphate). These sugars were also effective in eluting bound brain acetylcholinesterase from ligatin affinity columns. Analysis of labeled glycitols produced by tritiated borohydride reduction confirmed the presence of phosphorylated sugars on the ligatin-cosolubilized material from brain.     

Calcium-Dependent and Calcium-Independent Adhesive Mechanisms Are Present During Initial Binding Events of Neural Retina Cells

The hypothesis that intercellular adhesion can be subdivided into two separable phenomena, an initial recognition event and a subsequent stabilization, is supported by the use of a new cell binding assay that provides a quantitative measure of intercellular binding strengths. Radioactive single cells are brought into contact with cell monolayers at 4°C in sealed compartments. The compartments are inverted and a centrifugal force is then applied tending to dislodge the probe cells from the monolayers. By varying the speed of centrifugation, the force maintaining associations between embryonic chick neural retina cells was determined to be on the order of 10^?5 dynes after incubation at 4°C. Brief incubations at 37°C resulted in significant strengthening of the intercellular bond. Using this cell binding assay, neural retina cells were shown to exhibit both a Ca⁺⁺-independent and a Ca⁺⁺-dependent mechanism in their initial binding to one another.     

细丝蛋白A在细胞黏附和迁移中的作用

细胞迁移在发育、伤口愈合、炎症反应和肿瘤转移等多种病理生理过程中发挥重要作用。细丝蛋白A（filamin A,FlnA）是一种在各组织细胞中广泛表达的微丝结合蛋白,其表达异常导致细胞迁移功能障碍。该文回顾了相关的文献,首先介绍生理情况下细丝蛋白A的功能,接着介绍细丝蛋白A基因突变和表达异常导致的多种遗传性疾病及其与肿瘤转移的关系,突出细丝蛋白A对迁移的影响在这些疾病发病中的作用,最后深入探讨了细丝蛋白A影响细胞迁移和黏附的可能机制。     

神经细胞粘附分子与记忆

记忆的形成阶段包含着神经元突触的可塑性变化过程.近年来的研究表明,神经细胞粘附分子可同时增进突触的可塑性和维持突触结构的稳定性.许多研究证实神经细胞粘附分子对与学习和记忆相关的过程起着一定的调节作用.     

P-选择素及其细胞黏附与血栓形成

P-选择素是选择素家族的重要黏附分子,作为血小板/内皮细胞活化标志和细胞黏附受体,其可通过介导血小板、内皮细胞黏附及与白细胞的相互作用,启动参与了包括炎症和血栓形成等多种病理生理起始过程,是炎症/血栓的重要介质和靶分子。抑制P-选择素及其与配体的结合和作用,可使病理状态下血栓局部白细胞聚集减少、细胞因子及组织因子表达降低、纤维蛋白生成减少,从而有助于抑制血栓的形成。因此,随着P-选择素及其细胞黏附与血栓形成研究的不断深入和阐明,以P-选择素为靶标的血栓性疾病的诊断和抗黏附治疗,也已引起人们关注并具有良好的临床应用价值和前景。     

Melatonin is Involved in Regulation of the Expression of Neural Cell Adhesion Molecules in the Rat Brain

Cell adhesion molecules play a diverse role in neural development, signal transduction, structural linkage to extracellular and intracellular proteins, synaptic stabilization, neurogenesis, and learning. Neural cell adhesion molecules (NCAM) are members of the immunoglobulin superfamily and are involved in synaptic rearrangements in the mature brain. There are three major NCAM isoforms: NCAM 180, NCAM 140, and NCAM 120. Several studies reported that NCAM play a central role in memory formation. We investigated the effects of melatonin on the expression of NCAM in the hippocampus, cortex, and cerebellum of rats. The levels of NCAM isoforms were determined by Western blotting. After administration of melatonin for 7 days, the expression of NCAM 180 increased both in the hippocampus and in the cortex, as compared with the control. In contrast, in rats exposed to constant illumination for 7 days (a procedure that inhibits endogenous production of melatonin), levels of NCAM 180 dropped in the hippocampus and became undetectable in the cortex and cerebellum. Levels of NCAM 140 in the hippocampus of light-exposed rats also decreased. There was no change in the expression of NCAM 120 in any brain region. This is the first report indicating that melatonin exerts a modulatory effect on the expression of NCAM in brain areas related to realization of cognitive functions. Melatonin may be involved in structural remodeling of synaptic connections during memory and learning processes.     

The Apical Lamina and its Role in Cell Adhesion in Sea Urchin Embryos

The hyaline layer (HL) is an extracellular matrix surrounding sea urchin embryos which has been implicated in a cell adhesion and morphogenesis. The apical lamina (AL) is a fibrous meshwork that remains after removal of hyalin from the HL and the fibropellins (FP) are glycoproteins thought to be the principal components of the AL. Using anti-FP antibodies (AL-1 and AL-2) we report immunoprecipitations and affinity purifications yield a high molecular weight complex comprised of the FP glycoproteins. The three components form a complex, stabilized by disulphide cross-linking and have stochiometric ratios of 2 FPIa molecules to 1 each of FPIb and FPIII. Pulse chase experiments indicate all 3 FP's are synthesized throughout development with peaks in synthesis during cleavage and a sustained peak beginning at hatching. Using immunogold and immunoperoxidase localization, the FP localize to a fibrillar complex forming the innermost layer of the HL. In cell adhesion experiments, cells adhere to affinity purified FP in a temperature, time and concentration dependent manner. Cell adhesion to Fp is about 70% of that seen when hyalin is used as a substrate. Pretreating with AG1 and AG2 reduces in vitro cell adhesion by about 65%. We conclude FP's form a fibrillar complex, which is synthesized throughout early development and functions, with other components of the HL, as a substrate for cell adhesion.     

神经细胞粘连分子和多聚唾液酸在神经发育和再生中的作用

神经系统的形成依赖于细胞间的互相粘连。本文综述了神经细胞粘连分子（ＮＣＡＭ）及其多聚唾液酸（ＰＳＡ）组份对神经发育和再生的作用。ＮＣＡＭ的基本功能是介导细胞粘连,ＰＳＡ则由于其特殊的分子结构而降低细胞间的粘连。研究表明,鸡胚的发育过程中,ＰＳＡ含量在三个关键时期表达的高低决定了运动神经元能否准确地识别和支配肌肉。成年大鼠周围神经损伤后,肌肉内ＮＣＡＭ含量的高低决定于该肌肉的神经支配状况。成年大鼠脑内,切断内嗅皮层与海马的神经联系,发现齿回外分子层ＰＳＡ含量显著增加,并至少可持续６０天。已有的研究资料提示在去神经靶区域ＰＳＡ的重新表达可能有利于移植神经元轴突的生长并与宿主重建突触联系。     

Cytokines and Cell Adhesion Molecules in Tumor-Endothelial Cell Interaction and Metastasis

Metastasis is a multistep process in which a metastatic tumor cell detaches from the primary tumor, invades the surrounding tissues, passes through supporting structures such as interstitial stroma and extracellular matrix, and enters the lymphatic or blood circulation (Poste and Fidler, 1980). Only a few of the neoplastic cells released into the circulation, that survive hemodynamic pressure and host defense mechanisms, will form metastases. The arrest of tumor cells in the capillary bed of secondary organs through an interaction with vascular or lymphatic endothelium and subendothelial basement membrane is followed by their extravasation into the tissue parenchyma, and then micro-metastasis formation. Therefore cell-cell and cell-substrate adhesions occur at different moments in this process. With the recent identification and characterization of cell surface molecules, it has become of particular interest to clarify their role in tumor progression and metastasis (Albelda, 1993).     

Cytokines and Cell Adhesion Molecules in Tumor-Endothelial Cell Interaction and Metastasis

Metastasis is a multistep process in which a metastatic tumor cell detaches from the primary tumor, invades the surrounding tissues, passes through supporting structures such as interstitial stroma and extracellular matrix, and enters the lymphatic or blood circulation (Poste and Fidler, 1980). Only a few of the neoplastic cells released into the circulation, that survive hemodynamic pressure and host defense mechanisms, will form metastases. The arrest of tumor cells in the capillary bed of secondary organs through an interaction with vascular or lymphatic endothelium and subendothelial basement membrane is followed by their extravasation into the tissue parenchyma, and then micro-metastasis formation. Therefore cell-cell and cell-substrate adhesions occur at different moments in this process. With the recent identification and characterization of cell surface molecules, it has become of particular interest to clarify their role in tumor progression and metastasis (Albelda, 1993).     

A Protein Kinase Activity Is Associated with and Specifically Phosphorylates the Neural Cell Adhesion Molecule LI

The neural cell adhesion molecule L1 is a phosphorylated integral membrane glycoprotein that is recovered from adult mouse brain by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, 80, and 50 kilodaltons (L1-200, L1-180, L1-140, L1-80, and L1-50, respectively). In the present study, we show that two kinase activities are associated with immunopurified L1: One specifically phosphorylates L1-200 and L1-80 but not L1-180, L1-140, or L1-50. This pattern of phosphorylation corresponds to the one described for L1 after metabolic phosphate incorporation into cultures of cerebellar cells. In both cases, serine is the main amino acid that is labeled by radioactive phosphate. The kinase activity is not activated by Ca2+, calmodulin, phosphatidylserine, diolein, cyclic AMP, or cyclic GMP, a result suggesting that the enzyme is distinct from Ca2+/calmodulin-dependent kinases, from protein kinase C, or from cyclic AMP/cyclic GMP-dependent kinases and may belong to the independent kinase group. The other kinase phosphorylates only casein but not L1, utilizes GTP as well as ATP, and is strongly inhibited by heparin. Because the primary structure of the L1 protein does not contain consensus sequences characteristic for known kinases, we believe that the catalytic activities detectable in immunopurified L1 are due to kinases that are strongly enough associated with L1 to withstand the stringent purification procedures.     

EphB/ephrinB Signaling in Cell Adhesion and Migration

Eph receptors and their ligands, ephrins, represent the largest group of the receptor tyrosine kinase (RTK) family, and they mediate numerous developmental processes in a variety of organisms. Ephrins are membrane-bound proteins that are mainly divided into two classes: A class ephrins, which are linked to the membrane by a glycosylphosphatidylinositol (GPI) linkage, and B class ephrins, which are transmembrane ligands. Based on their domain structures and affinities for ligand binding, the Eph receptors are also divided into two groups. Trans-dimerization of Eph receptors with their membrane-tethered ligands regulates cell-cell interactions and initiates bidirectional signaling pathways. These pathways are intimately involved in regulating cytoskeleton dynamics, cell migration, and alterations in cellular dynamics and shapes. The EphBs and ephrinBs are specifically localized and modified to promote higher-order clustering and initiate of bidirectional signaling. In this review, we present an in-depth overview of the structure, mechanisms, cell signaling, and functions of EphB/ephrinB in cell adhesion and migration.     

Effects of Nanoparticles on the Adhesion and Cell Viability on Astrocytes

In recent years, both pharmaceutical companies and manufacturing industries have expressed heightened interest in the potential applications of magnetic nanoparticles for therapeutic and technological purposes. Specifically, pharmaceutical companies seek to employ magnetic nanoparticles as carriers to facilitate effective drug delivery, especially in areas of the brain. Manufacturing industries desire to use these nanoparticles as ferrofluids and in magnetic resonance imaging. However, data concerning the effects of magnetic nanoparticles on the nervous system is limited. This study tested the hypotheses that nanoparticles can (1) inhibit adherence of astrocytes to culture plates and (2) cause cytotoxicity or termination of growth, both end points representing surrogate markers of neurotoxicity. Using light microscopy, changes in plating patterns were determined by visual assessment. Cell counting 4 days after plating revealed a significant decrease in the number of viable astrocytes in nanoparticle treated groups (p < 0.0001). To determine the cytotoxic effects of nanoparticles, astrocytes were allowed to adhere to culture plates and grow to maturity for 3 weeks before treatment. Membrane integrity and mitochondrial function were measured using colorimetric analysis lactate dehydrogenase (LDH) and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTS), respectively. Treatment with nanoparticles did not significantly alter astrocytic LDH release (p > 0.05) in the control group (100% ± 1.56) vs the group receiving treatment (97.18% ± 2.03). However, a significant increase in MTS activity (p < 0.05) between the control (100% ± 3.65) and treated groups (112.8% ± 3.23) was observed, suggesting astrocytic mitochondrial uncoupling by nanoparticles. These data suggest that nanoparticles impede the attachment of astrocytes to the substratum. However, once astrocytes attach to the substratum and grow to confluence, nanoparticles may cause mitochondrial stress.     

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.     

Unique Intracellular Trafficking Processes Associated With Neural Cell Adhesion Molecule and Its Intracellular Signaling

Abstract

Homophilic binding of the neural cell adhesion molecule (NCAM) results in intracellular signaling, which also involves heterophilic engagement of coreceptors such as the fibroblast growth factor receptor (FGFR) and receptor protein tyrosine phosphatase-α (RPTPα). NCAM's own cellular dynamic itinerary includes endocytosis and recycling to the plasma membrane. Recent works suggest that NCAM could influence the trafficking of other receptor molecules that it associates with, particularly the FGFR. Furthermore, it was demonstrated that NCAM could undergo proteolytic processing upon activation. A processed fragment of NCAM, together with an N-terminal fragment of focal adhesion kinase (FAK), is translocated into the nucleus. Here, the authors discuss these rather unique (though not without precedence and analogues) receptor trafficking activities that are associated with NCAM and NCAM signaling.     

Increased Resistance to Apoptosis in Cells Overexpressing Thymosin Beta Four: A Role for Focal Adhesion Kinase pp125FAK

Loss of adherence to substrate can, by itself, induce apoptosis (anoikis) in epithelial cells, but does not do so in fibroblasts. To test the idea that adherence transmits signals that inhibit apoptosis even in fibroblasts, we took advantage of the greatly increased adherence to the substratum observed in NIH3T3 cell lines that overexpress thymosin beta four. We treated overexpressing (OE) and vector control lines with either ultraviolet light (UV) or tumor necrosis factor alpha (TNFα). When the cells were on a substratum, the more adherent OE cells were 2-fold more resistant to apoptosis induced by either treatment than vector controls. In contrast, when the cells were treated with either agent while in suspension, the difference in resistance between OE cells and vector controls was lost. Thus the increased resistance to apoptosis was dependent on adherence. There was no difference in the content of bcl-2 in the OE cells vs the controls. A connection between pp^l25 FAK and resistance to apoptosis has been previously shown in primary cultures of fibroblasts. The Tβ₄ overexpressing cells have ～ 1.4× more pp¹²⁵ FAK than the controls, and the kinase is ～2-fold more phosphorylated in adherent OE cells than in the vector controls. The phosphorylation of pp¹²⁵ FAK decreased strikingly when the cells were put into suspension. In addition, twice as much paxillin associated with pp¹²⁵ FAK in OE adherent cells as in vector controls, but this difference was also lost in suspended cells. Our results support the concept of an adherence dependent pp^l25 FAK-paxillin signalling pathway in fibroblasts that inhibits damage-induced apoptosis.     

The Role of Cytoplasmic Serine Residues of the Cell Adhesion Molecule L1 in Neurite Outgrowth,Endocytosis, and Cell Migration

Summary 1. The cell adhesion molecule L1 has been implicated in adhesion and migration of cells, in axon growth, guidance, and fasciculation, in myelination and synaptic plasticity. The cytoplasmic domain of neuronal L1 is highly conserved between species and has been shown to be phosphorylated at serine and tyrosine residues. 2. To investigate the significance of L1 serine phosphorylation, mutants of L1 were generated in which ser-1152, ser-1181, ser-1204, and ser-1248 were exchanged for leucine and rat B35 neuroblastoma cells were stably transfected with the L1-cDNA constructs. 3. Neurite outgrowth on poly-l-lysine (PLL) as substrate was determined either with or without differentiation into a neuronal phenotype with dbcAMP. In addition, antibody-induced endocytosis and cell migration were examined. 4. Our observations indicate that phosphorylation of single serine residues of the cytoplasmic domain of L1 contributes to neurite outgrowth through different mechanisms. Neurite growth is increased when ser-1152 or ser-1181 is replaced by a non-phosphorylatable leucine and decreased when ser-1204 or ser-1248 is mutated to leucine. Furthermore, mutation of ser-1181 to leucine results in strongly enhanced antibody-induced endocytosis of L1 and also in enhanced cell migration.