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PubMed2005

Fibronectin adsorption and arrangement on copolymer surfaces and their significance in cell adhesion.

The adsorption of fibronectin (FN) to (styrene/methyl methacrylate) copolymer surfaces, both sulfonated (hydrophilic) and nonsulfonated (hydrophobic), was studied by means of the radioisotope (125I-FN) and ELISA assays; the latter employed monoclonal antibodies. It was found that the radioiodination-derived isotherms did not follow the Langmuir-type adsorption law within the FN concentration range studied; rather, a quasi-linear FN surface density versus bulk concentration dependence was observed. These isotherms, and our recent ELISA measurements with polyclonal antibodies, allowed us to estimate saturative FN surface densities, which were, within the experimental error, similar on both types of surfaces. This suggested the amount of adsorbed FN to be not responsible for observed differences in leukaemia L1210 cell adhesion (FN-coated sulfonated surfaces are far more pro-adhesive than their nonsulfonated analogues). The presumption that these differences are induced by changes in the FN arrangement was confirmed by the use of monoclonal antibodies directed against distinct FN domains, and by the blocking of alpha5beta1 integrin receptor with the synthetic Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide. The RGD sequence located within the FN cell-binding domain seems to be masked in the structure adopted on nonsulfonated surfaces, which hinders the integrin-ligand interaction.

Adsorption↗
PubMed2007

Integrin alpha(5) controls osteoblastic proliferation and differentiation responses to titanium substrates presenting different roughness characteristics in a roughness independent manner.

Integrin alpha(5)beta(1) regulates osteoblast proliferation and differentiation on smooth synthetic surfaces presenting different chemistries, but it is not known whether this integrin controls osteoblast behavior on surfaces that have micron-scale rough topographies. We cultured MG63 human osteoblast-like cells on titanium substrates with three different roughness characteristics: chemically polished (PT), grit blasted and acid etched with a complex topography consisting of 20-100 mum craters and 0.5-2 mum micropits (SLA), and plasma-sprayed Ti with irregular projections (TPS). Cells spread well on PT but displayed a smaller footprint on SLA or TPS. Nuclei were larger on PT as well. alpha(5)beta(1) binding and FAK phosphorylation were greater on the rougher surfaces (TPS > SLA > PT). Antibodies against the alpha(5)beta(1) binding site on fibronectin had no effect on cell number at 3 days, but [(3)H]-thymidine incorporation was increased, suggesting that binding to fibronectin was necessary for cell cycle regulation. Antibodies to the alpha(5) subunit reduced cell number at 3 days on PT and TPS and reduced DNA synthesis on all substrates in a surface microstructure-independent manner. At 7 days, cell numbers were reduced on PT, and DNA synthesis was reduced by 50% on all surfaces. At 7 days, anti-alpha(5) antibodies caused a partial reduction in alkaline phosphatase enzyme activity on all surfaces, but this effect was independent of surface microstructure. These results indicate that surface micron-scale topography modulates alpha(5)beta(1) integrin binding and FAK activation. Signaling via alpha(5)-dependent mechanisms is required for DNA synthesis and regulation of alkaline phosphatase, but this effect is independent of surface microstructure.

Alkaline Phosphatase↗
PubMed2006

Biological activity of rainbow trout Ea4-peptide of the pro-insulin-like growth factor (pro-IGF)-I on promoting attachment of breast cancer cells (MDA-MB-231) via alpha2- and beta1-integrin.

E-peptide of pro-IGF-I was considered as biologically inactive. We have demonstrated that rainbow trout (rt) Ea4-peptide exerted biological activities in several established tumor cell lines [Chen et al., 2002; Kuo and Chen, 2002]. Here we report the activity of rtEa4-peptide in promoting attachment of human breast cancer cells (MDA-MB-231). While rtEa2-, rtEa3-, and rtEa4-peptides enhanced the attachment of MDA-MB-231 cells in a dose dependent manner, rtEa4-peptide possessed the highest activity. Antibodies specific to alpha2 and beta1 integrins significantly inhibited the attachment of cells to rtEa4-peptide coated-plates by 40%. In addition, rtEa4-peptide induced the expression of fibronectin 1 and laminin receptor genes in MDA-MB-231 cells. Blocking new protein synthesis by cycloheximide significantly reduced the attachment of MDA-MB-231 cells to rtEa4-peptide coated wells by 50%. These results suggest that rtEa4-peptide may promote cell attachment by interacting with alpha2/beta1 integrin receptors at the cell surface and by inducing the expression of fibronectin 1 and laminin receptor genes. Expression of fibronectin 1 gene induced by rtEa4-peptide in MDA-MB-231 cells was abolished by inhibitors of PI3K, PKC, Mek1/2, JNK1/2, and p38 MAPK signaling transduction molecules. These results suggested that induction of fibronectin 1 gene expression in MDA-MB-231 cells by rtEa4-peptide may be mediated via PI3K, PKC, Mek1/2, JNK1/2, and p38 MAPK signal transduction molecules.

Animals↗
PubMed2001

Tenascin-R is expressed by Schwann cells in the peripheral nervous system.

The extracellular matrix glycoprotein tenascin-R (TN-R) has been implicated in a variety of cell-matrix interactions involved in the molecular control of axon guidance and neural cell migration during development and regeneration of the central nervous system (CNS). Whereas TN-R is amply expressed in the early postnatal and adult mammalian CNS, the protein has so far not been detected in different compartments of the peripheral nervous system (PNS). Here we provide first evidence that TN-R (predominantly TN-R 160 isoform) is transiently expressed in the sciatic nerve of late embryonic (E14-18) and neonatal mice, while at later developmental stages, both protein and mRNA are downregulated. In vitro, TN-R protein was found to be expressed by both undifferentiated and neuronally differentiated PC12 cells and by L1-positive Schwann cells (SC), but not by other neural and non-neural cell types in cell cultures derived from embryonic (E17/18) hindlimbs and neonatal sciatic nerves. In the developing PNS, TN-R expression correlated with axon growth and SC migration during the period of skeletal muscle innervation. Based on different in vitro approaches, we found that the substrate-bound glycoprotein selectively inhibits the fibronectin-dependent: (1) neurite outgrowth from dorsal root ganglion neurons (strongly expressing alpha5beta1 integrin and the disialoganglioside GD3) by a ganglioside-sensitive signaling mechanism; and (2) migration of primary myoblasts and other non-neuronal cells in a ganglioside-independent manner. Our findings suggest the functional role of TN-R in PNS pattern formation during distinct stages of axon pathfinding and skeletal muscle innervation.

Aging↗
PubMed1996

Guidance and activation of murine macrophages by nanometric scale topography.

We studied the guidance and activation of macrophages from the P388D1 cell line and rat peritoneum by topographic features on a nanometric scale. Cells were plated on plain fused silica substrata or substrata with microfabricated grooves and steps, 30-282 nm deep. The contact of cells with the patterned surface activated cell spreading and adhesion and increased the number of protrusions of the cell membrane. These changes were accompanied by an increase in the amount of F-actin in cells. The accumulation of F-actin and vinculin in cells was observed along the edges of single steps or grooves. Formation of focal contacts along discontinuities in the substratum was accompanied by the phosphorylation of tyrosine colocalized with F-actin and vinculin. A similar pattern of staining was seen in cells stained for vitronectin receptor, alphaV integrin, but not for integrins alpha5beta1 or alpha3beta1. Cells cultured on nanogrooves showed a higher phagocytotic activity than cells cultured on plain substrata. We show that macrophages can react to ultrafine features of topography of a size comparable to that of a single collagen fiber and become activated by the contact with topographic features.

Animals↗
PubMed1997

Cell-cell adhesion molecules and the development of an epithelial phenotype in cultured human retinal pigment epithelial cells.

For most epithelial cells, the adherens junction protein E-cadherin is an epithelial morphogen, inducing the development of an epithelial phenotype in vitro after cell contact at confluency. Here retinal pigment epithelial cells (RPE), which lack E-cadherin but express a cadherin that is also found in many non-epithelial cells (N-cadherin), were examined for the ability to produce an epithelial phenotype in vitro. Subpopulations of grossly epithelioid or fusiform cells were selected for analysis from RPE cultures derived from adult human donors. After confluency, epithelioid RPE cells were observed to undergo time-dependent changes that were similar to those previously found in epithelial cells expressing E-cadherin: the cadherin gradually developed a zonular distribution of detergent-resistant protein that co-localized with forming circumferential actin bundles; Na/K ATPase accumulated at cell contact sites, then polarized to its tissue-specific domain (the apical membrane for RPE); the cells formed elevated domes on the impermeant culture substrate. In contrast to cells expressing E-cadherin, these events in RPE required weeks rater than days at confluency. Additional proteins were examined in epithelioid RPE cells revealing that cytokeratins reorganized after confluency producing a zonular array, and several other adhesion proteins (alpha5beta1 integrin, ICAM-1, PECAM-1, NCAM) became enriched at cell-cell contact sites, each developing a distinct pattern at a distinct postconfluency interval. In contrast to epithelioid RPE, in fusiform RPE the adhesion molecules did not develop discrete distribution patterns after confluency, although the same complement of adhesion proteins was expressed. In cells expressing E-cadherin, the absence of epithelial properties is often due to underexpression of the cadherin or of the catenins, adherens junction proteins that link the cadherin to actin. Fusiform RPE, however, were not deficient in these proteins, expressing amounts of N-cadherin, alpha-catenin, beta-catenin, plakoglobin, p120, alpha-actinin and vinculin that were equivalent to epithelioid cells. It appears, therefore, that a subset of epithelial cells that express N-cadherin can produce a highly-developed epithelial phenotype in vitro through a slow morphogenetic process. However, the expression alone of adhesion molecules, including those with a morphoregulatory function in other cells, is insufficient to produce an epithelial phenotype in all cells derived from the pigment epithelium.

Adult↗
PubMed2002

Developmental regulation of beta1 integrins during angiogenesis in the central nervous system.

We examined beta1 integrin expression during angiogenesis in the developing mouse CNS. Maturation of blood vessels was accompanied by three main events: (1) marked upregulation of beta1 integrin expression; (2) a switch in beta1 integrin expression, from alpha4 and alpha5 at postnatal day 1 to alpha1 and alpha6 in the adult; and (3) downregulation of fibronectin and upregulation of laminin expression. Thus, blood vessel maturation was associated with a developmental switch, from fibronectin-mediated signaling early in angiogenesis, to laminin-mediated signaling at later stages. To investigate the potential role of alpha4 and alpha5 integrins in angiogenesis, we developed a novel approach to purify endothelial cells from the CNS by selective adhesion to fibronectin. BrdU incorporation showed that fibronectin induced more endothelial cell proliferation than laminin, and antibody-blocking studies revealed that fibronectin mediated its effect via both alpha4 and alpha5 integrins. This work provides the first evidence that there is a developmental switch in the use of beta1 integrins during angiogenesis. Furthermore, it suggests that regulation of beta1 integrin and ECM expression by endothelial cells are important factors influencing vascular development in the CNS.

Aging↗
PubMed2001

Reconstruction of basement membrane in skin equivalent; role of laminin-1.

To reconstruct the basement membrane in a skin equivalent, the epidermodermal interface was coated with porcine type IV collagen and mouse laminin-1 at various ratios before keratinocyte seeding. Laminin-1, a component of the basement membrane, induced massive infiltration of keratinocytes into the dermal equivalent, while type IV collagen induced discrete demarcation between dermal and epidermal compartments without any infiltrating cells. Immunohistochemical staining indicated that the laminin-induced infiltrating cells expressed endogenous type IV collagens at the cell periphery, which were not incorporated into the basement membrane structure. The infiltrating cells did not express fibronectin receptor alpha5beta1 integrin but showed MMP-9 secretion and cell surface associated MMP-2. However, when laminin-1 was preincubated with type IV collagen, laminin-1-induced keratinocyte infiltration as well as MMP-9 induction were almost completely suppressed to basal levels. Therefore, replenishment of the type IV collagen lattice seemed to cause laminin-stimulated cells to anchor to the lattice, in a similar manner to the basal cells on the basement membrane of normal skin. Our study suggests that the molar ratio of basement membrane components may determine the behavior of basal cells within the wound healing microenvironment, which is probably regulated either by extracellular matrix deposition or degradation.

3T3 Cells↗
PubMed1998

The internal and external protein scaffold of the T-tubular system in cardiomyocytes.

The transverse tubule system of the cardiomyocyte remains undeformed despite the extreme forces it undergoes during the contraction-relaxation cycle, but the morphological basis for its stability remains unclear. Therefore, we have investigated the architecture and subcellular protein scaffold of the cardiac T-tubules and compared it with that of the costameres and of the free sarcolemma. Tissue samples from normal rat and monkey hearts, and left ventricular tissue from normal and cardiomyopathic human hearts obtained at transplantation surgery were investigated using immunocytochemistry and confocal microscopy and by electron microscopy. In addition, we used a re-differentiation model of isolated, cultured adult rat cardiomyocytes. The cell membrane of the cardiac T-tubules was found to contain the cell-matrix focal adhesion molecules (FAMs) vinculin, talin, the alpha5beta1 integrin and the membrane-associated proteins (MAPs) dystrophin and spectrin. FAMs and MAPs were localized in the T-tubular membrane in a similar pattern: in longitudinally oriented myocytes as transverse punctate lines at the Z-level; in transversally cut myocytes a radial tubular network was found to extend throughout the interior of the cell. Immunolabeling for basement membrane components including collagen IV, fibronectin and laminin showed a colocalization with FAMs and MAPs parallel to the transverse T-tubules. The costameres of the sarcolemma showed a protein composition resembling that of the T-tubules but the intervening segments of free sarcolemma showed absence of FAMs and presence of MAPs. For the first time, we demonstrate the existence and protein composition of the T-tubular scaffold in the human heart. Furthermore, we show that cardiomyocytes from human failing hearts have less abundant but more dilated T-tubules than do experimental animals. These results indicate that the cardiac T-tubular system contains a subcellular scaffold closely resembling that of the costameres. It consists of FAMs, MAPs and basal lamina proteins that confer structural integrity to the cardiac T-tubular membrane during contraction/relaxation cycles.

Animals↗
PubMed2006

Characterization of the type 3 fimbriae with different MrkD adhesins: possible role of the MrkD containing an RGD motif.

Four novel mrkD alleles namely mrkD(V1), mrkD(V2), mrkD(V3), and mrkD(V4) were identified in seventeen Klebsiella pneumoniae meningitis strains using PCR-RFLP and sequence determination. Comparative analysis revealed a most variable region containing an RGD motif in the receptor domain of MrkD(V3). In order to determine if the sequence confers the K. pneumoniae mrkD(V3) the highest level of the fimbrial activity, a type 3 fimbriae display system was constructed in Escherichia coli. The E. coli JM109[pmrkABCD(V3)F] displaying meshwork-like fimbriae also had the most fimbrial activity, supporting a possible role of the varied sequences. In a dose-dependent manner, the GRGDSP hexapeptide appeared to inhibit the adhesion of the E. coli JM109[pmrkABCD(V3)F] to HCT-8, an ileocecal epithelial cell line. In addition, the adhesion activity was reduced by the addition of anti-alpha5beta1 integrin monoclonal antibody, indicating that the RGD containing region in MrkD(V3) is responsible for the binding of type 3 fimbriae to integrin.

Adhesins, Bacterial↗
PubMed2005

Effects of calcium ion implantation on human bone cell interaction with titanium.

The use of calcium ion (Ca) implantation of titanium (Ti), previously reported to encourage osseointegration in vivo, has been investigated using an in vitro model in order to understand the basic mechanisms involved in the response of target cells to such surfaces. Polished Ti discs were implanted with high, medium and low (1x10(17), 1x10(16), 1x10(15)ionscm-2) doses of Ca ions at 40 keV. The effects of different levels of Ca implantation on morphology, attachment and spreading of MG-63 cells seeded on the surface of control (non-implanted) Ti and Ca-Ti discs were assessed. Further, to understand cell-material interactions at a molecular level, the expression of beta1 and alpha5beta1 integrins and the formation of vinculin-positive focal adhesion plaques were examined. In addition, the effects of pre-immersion of the Ca (high)-Ti in tissue culture medium on cell attachment were measured and correlated with specific chemical changes at the Ti surface. Our findings suggest that Ca implantation can affect the adhesion of MG-63 cells both qualitatively and quantitatively. However, this effect appears to depend on the level at which Ca ions are implanted. Results showed that although cell adhesion on Ca (high)-Ti was initially reduced, it nevertheless was not only restored but substantially increased with progressing culture times. In addition, a significantly enhanced cell spreading, formation of focal adhesion plaques and expression of integrins were measured on this particular surface. In contrast, no marked differences were observed in cell behaviour on Ca-Ti (low and medium). Pre-immersion studies indicated that the decrease in cell attachment to Ca (high)-Ti at early time periods may be linked to the presence of Ca- and P-rich particles on the surface. The absence of these particles at 24 h was consistent with a significant increase in cell attachment.

Calcium↗
PubMed2006

Identification of novel short chain 4-substituted indoles as potent alphavbeta3 antagonist using structure-based drug design.

The vitronectin receptor alpha(v)beta(3) has been identified as a promising potential target for the treatment of osteoporosis, diabetic retinopathy and cancer. We have recently reported 5-substituted indoles 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3-pyridyl)propionic acid 3 and 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3,4-methylenedioxyphenyl)propionic acid 4, as an original series of potent alpha(v)beta(3) antagonists with subnanomolar activity. Ligand-protein docking analyses have been performed to generate binding models of three different chemical classes of known alpha(v)beta(3) antagonists with alpha(v)beta(3). Results of this docking study suggested that indoles bearing the basic tetrahydronaphthyridine group at position 4 can easily adopt the correct binding conformation and should be as potent as our current 5-substituted indole leads 3 and 4. This hypothesis was nicely demonstrated by the synthesis of a series of 1,4-disubstituted indoles through a tandem of reactions involving: (i) the N-alkylation of indoles 15 and 22 with propargyl esters and cesium fluoride, and (ii) a Heck coupling reaction between 4-bromoindole and 7-vinyl-3,4-dihydro-2H-[1,8]naphthyridine-1-carboxylic acid tert-butyl ester 12, or (iii) a reductive amination involving the N-substituted-4-aminoindole 23 and the BOC-protected tetrahydro[1,8]naphthyridine aldehyde 13. Among the compounds assayed, 3-(3-pyridyl)-3-[4-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethyl]indol-1-yl]propionic acid 21 showed the most promising activity on alpha(v)beta(3) (IC(50)=0.5 nM), and was found to have the same potency as our current leads 3 and 4, while maintaining selectivity over alpha(IIb)beta(IIIa). Moreover, based on the reasonable apparent permeability coefficient in an in vitro CACO-2 cell monolayer assay (P(app) apical/basolateral=2.2 x 10(-6)cm/s, P(app) basolateral/apical=2.5 x 10(-6)cm/s), compound 21 is expected to be absorbed through the intestine in human. Thus, 1,4-disubstituted indole 21 represents a new lead for this novel class of conformationally restricted alpha(v)beta(3) antagonists. Additionally, this study validates the pharmacophore model previously postulated and provides an improved basis for further structure-based drug design in the field of alpha(v)beta(3).

Drug Design↗
PubMed2005

Fibronectin fibrillogenesis, a cell-mediated matrix assembly process.

The extracellular matrix provides a framework for cell adhesion, supports cell movement, and serves to compartmentalize tissues into functional units. Fibronectin is a core component of many extracellular matrices where it regulates a variety of cell activities through direct interactions with cell surface integrin receptors. Fibronectin is synthesized by many adherent cells which then assemble it into a fibrillar network. The assembly process is integrin-dependent and fibronectin-integrin interactions initiate a step-wise process involving conformational activation of fibronectin outside and organization of the actin cytoskeleton inside. During assembly, fibronectin undergoes conformational changes that expose fibronectin-binding sites and promote intermolecular interactions needed for fibril formation. In this review, the main steps of fibronectin assembly are described and recent studies on fibronectin conformational changes are discussed.

Actins↗
PubMed2005

Bi-potential behaviour of cytotrophoblasts in first trimester chorionic villi.

Murine trophoblast stem (TS) cells express fibroblast growth factor receptor 2 (FGFR2) and are maintained in their proliferative state by fibroblast growth factor 4 (FGF4). We show in this report that in the first trimester human placenta FGFR2 expression is similarly found in a subset of villous cytotrophoblast and in proximal anchoring columns. Western analysis demonstrated declining FGFR2 protein expression as gestation advanced, suggesting a similar role for FGF in early human trophoblast proliferation. Mouse TS cell differentiation is known to occur along two distinct transcriptionally-regulated pathways; extravillous trophoblast (EVT) cells invade the uterine wall to promote maternal blood flow whilst syncytiotrophoblast lines chorionic villi in the labyrinth. Similar differentiation steps occur in the human placenta though the fate of human trophoblast stem cells is presently unknown. To investigate the mechanisms underlying human cytotrophoblast differentiation we have developed a novel cultured floating first trimester villous explant model in which denuded first trimester villi spontaneously regenerate syncytiotrophoblast following 48 h of culture. Addition of FGF4 and heparin inhibited syncytiotrophoblast regeneration in favor of forming clumps of cytotrophoblast. Proximal cells in these clumps were FGFR2 immuno-reactive and proliferative, intermediate parts expressed alpha5beta1-integrin, while the distal portion expressed HLA-G and the invasive integrin alpha1beta1 indicating differentiation to the EVT phenotype. In contrast, non-denuded villi exposed to FGF4 exhibited similar proliferation of the cytotrophoblast; however, these cells did not express any of the invasive EVT markers. We conclude that FGFR2-positive chorionic cytotrophoblasts exhibit bi-potential behaviour, being capable of forming either syncytiotrophoblast or EVT. We suggest bipotential trophoblast progenitor cells persist during first trimester human placental development.

Animals↗
PubMed1999

HIV-1-trans-activating (Tat) protein: both a target and a tool in therapeutic approaches.

Tat proteins (trans-activating proteins) are present in all known lentiviruses and are early RNA binding proteins that regulate transcription. Tat from the human immunodeficiency virus type-1 is a protein comprising 86 amino acids and encoded by 2 exons. The first 72 amino acids are encoded by exon 1 and exhibit full trans-activating activity. The second exon encodes a 14-amino-acid C-terminal sequence that is not required for trans-activation but does contain an RGD motif, which is important in binding to alphavbeta3 and alpha5beta1 integrins. Tat has an unusual property for a transcription factor; it can be released and enter cells freely, yet still retain its activity, enabling it to up-regulate a number of genes. Tat also has an angiogenic effect; it is a potent growth factor for Kaposi sarcoma-derived spindle cells, and, separately, it has been shown to bind to a specific receptor, Flk-1/KDR, on vascular smooth muscle cells, as well as to integrin-like receptors present on rat skeletal muscle cells and the lymphocyte cell line H9. It appears that the basic domain of tat is important, not only for translocation but also for nuclear localisation and trans-activation of cellular genes. As such, targeting of tat protein or, more simply, the basic domain provides great scope for therapeutic intervention in HIV-1 infection. There is also opportunity for tat to be used as a molecular tool; the protein can be manipulated to deliver non-permeable compounds into cells, an approach that already has been employed using ovalbumin, beta-galactosidase, horseradish peroxidase, and caspase-3.

Anti-HIV Agents↗
PubMed2003

Nischarin inhibits Rac induced migration and invasion of epithelial cells by affecting signaling cascades involving PAK.

Nischarin, a cytosolic protein that binds the alpha5beta1 integrin, plays an important role in fibroblast migration, and in regulation of the actin cytoskeleton. The effect of Nischarin on Rac induced migration and invasion by breast and colon epithelial cell lines has been determined. In these cells, Rac potently induced migration, as well as invasion of matrix; both of these events were strongly inhibited by overexpression of Nischarin. To understand the mechanism of Nischarin's inhibitory role in Rac induced cell migration, several effector domain mutants of Rac1 were employed. Nischarin was able to inhibit migration induced by Rac effector mutants that can activate PAK and JNK, but not migration stimulated by other Rac mutants. Further, Nischarin inhibited PAK induced cell migration, while not affecting migration induced by MEKK1, a Rac effector in the JNK pathway. In addition, Nischarin failed to inhibit migration induced by MEK1, a downstream effector in the Ras-Raf-MEK-Erk signaling cascade. Furthermore, Nischarin does not affect Rac mediated JNK and PI3K activities. However, Rac induced migration and invasion were effectively blocked by pharmacological inhibitors of PI-3 kinase and MEK. These results suggest that several pathways contribute to cell migration, but that Nischarin selectively inhibits Rac driven signaling cascades that affect migration through PAK.

Animals↗
PubMedDate not supplied

Entamoeba histolytica: a beta 1 integrin-like fibronectin receptor assembles a signaling complex similar to those of mammalian cells.

During tissue invasion, Entamoeba histolytica trophozoites interact with endothelial cells and extracellular matrix (ECM) proteins such as fibronectin (FN), collagen, and laminin. It has been demonstrated that trophozoites interact with FN through a beta1 integrin-like FN receptor (beta 1EhFNR), activating tyrosine kinases. In order to characterize the signaling process triggered by the amoebic receptor, activation, and association of tyrosine kinases and structural proteins were determined. As a result of FN binding by the beta 1EhFNR, the receptor itself, FAK, and paxillin were phosphorylated in tyrosine. Co-immunoprecipitation experiments showed that a multimolecular signaling complex was formed by the amoebic FN receptor, FAK, paxillin, and vinculin. These results strongly suggest that a signaling pathway, similar to the one used in mammalian cells, is activated when E. histolytica trophozoites adhere to FN.

Animals↗
PubMed2000

Molecular mechanisms of improved adhesion and growth of an endothelial cell line cultured on polystyrene implanted with fluorine ions.

Endothelial cells derived from the bovine pulmonary artery (line CPAE, CCL 209, American Tissue Culture Collection, Rockville, MD, USA) were cultured on pristine or fluorine ion-irradiated polystyrene (5 x 10(12) or 5 x 10(14) F ions/cm2, 150 keV). At 24-h post-seeding interval, the number of cells which adhered to the ion-modified polystyrene was significantly higher than on the unmodified material (+20 and +58% in cultures with the polystyrene irradiated by lower and higher ion doses, respectively). On day 7, the populations cultured on the irradiated substrates grew to higher densities, exceeding the controls at the lower and higher ion doses by 69 and 180%, respectively. The cells on ion-implanted samples were also larger (+70-95% and +90-99% at the lower and higher ion doses, respectively) and contained more protein (+16% at both ion doses). As was shown by ELISA, the polystyrene irradiated by the higher ion dose enhanced the expression of a cytoskeletal protein, vimentin (+65%) and protein of focal adhesion plaques, talin (+15%). The content of integrin alpha5beta1 (VLA-5), receptor for fibronectin, was increased at both lower and higher ion doses (+22 and +57%). In contrast to this, the content of ICAM-1 and vinculin was similar in cells grown on both pristine and ion-irradiated growth substrates. Moreover, the expression of VCAM-1 and ELAM-1 was lower by 11-14% in both ion dose groups. The present study has shown that ion implantation of polymers improves the adhesion and growth of endothelial cells without elevating the expression of immunoglobulin and selectin types of adhesion molecules. This surface modification should promote colonization of an artificial vascular prosthesis by endothelial cells and make it less vulnerable by immune system cells of the recipient.

Animals↗