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Cell Spreading (cell + spreading)
Selected AbstractsNucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cellsACTA PHYSIOLOGICA, Issue 2 2010I. Grimm Abstract Aim:, The adult subventricular zone (SVZ) contains neural stem cells that generate neuroblasts migrating to the olfactory bulb (OB) and differentiating into interneurones. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, functional integration and cell survival are poorly understood. We have previously shown that cultured adult neural stem cells express a considerable variety of nucleotide receptors and that nucleotides and epidermal growth factor (EGF) induce converging intracellular signalling pathways that carry potential for synergism in the control of neural stem cell proliferation and cell survival. Here we investigate the role of EGF and the nucleotides ATP, ADP,S and UTP in neural stem cell migration. Methods:, Neural stem cells were prepared from adult mice and subjected to adherent culture. Labelling of F-actin was performed with tetramethylrhodamine isothiocyanate-phalloidin. Images were processed for quantitative evaluation of fluorescence labelling. Agonist-induced phosphorylation of AKT and focal adhesion kinase was analysed by quantitative Western blotting. Agonist-dependent cell migration was assayed using 48-well microchemotaxis chambers. Results:, Nucleotides and EGF induce the formation of stress fibres, an increase in the cortical actin cytoskeleton and in cell spreading. This is associated with increased phosphorylation of AKT and focal adhesion kinase. Using microchemotaxis chambers we demonstrate a parallel increase in cell migration. Conclusion:, Our results suggest that nucleotides and EGF acting as paracrine or autocrine signalling substances can be of relevance for structuring and maintaining the cytoarchitecture of the SVZ and the stream of neuroblasts migrating to the OB. [source] Novel interactors and a role for supervillin in early cytokinesis,CYTOSKELETON, Issue 6 2010Tara C. Smith Abstract Supervillin, the largest member of the villin/gelsolin/flightless family, is a peripheral membrane protein that regulates each step of cell motility, including cell spreading. Most known interactors bind within its amino (N)-terminus. We show here that the supervillin carboxy (C)-terminus can be modeled as supervillin-specific loops extending from gelsolin-like repeats plus a villin-like headpiece. We have identified 27 new candidate interactors from yeast two-hybrid screens. The interacting sequences from 12 of these proteins (BUB1, EPLIN/LIMA1, FLNA, HAX1, KIF14, KIFC3, MIF4GD/SLIP1, ODF2/Cenexin, RHAMM, STARD9/KIF16A, Tks5/SH3PXD2A, TNFAIP1) co-localize with and mis-localize EGFP-supervillin in mammalian cells, suggesting associations in vivo. Supervillin-interacting sequences within BUB1, FLNA, HAX1, and MIF4GD also mimic supervillin over-expression by inhibiting cell spreading. Most new interactors have known roles in supervillin-associated processes, e.g. cell motility, membrane trafficking, ERK signaling, and matrix invasion; three (KIF14, KIFC3, STARD9/KIF16A) have kinesin motor domains; and five (EPLIN, KIF14, BUB1, ODF2/cenexin, RHAMM) are important for cell division. GST fusions of the supervillin G2-G3 or G4-G6 repeats co-sediment KIF14 and EPLIN, respectively, consistent with a direct association. Supervillin depletion leads to increased numbers of bi- and multi-nucleated cells. Cytokinesis failure occurs predominately during early cytokinesis. Supervillin localizes with endogenous myosin II and EPLIN in the cleavage furrow, and overlaps with the oncogenic kinesin, KIF14, at the midbody. We conclude that supervillin, like its interactors, is important for efficient cytokinesis. Our results also suggest that supervillin and its interaction partners coordinate actin and microtubule motor functions throughout the cell cycle. © 2010 Wiley-Liss, Inc. [source] The effect of combined hypergravity and microgrooved surface topography on the behaviour of fibroblastsCYTOSKELETON, Issue 7 2006W. A. Loesberg Abstract This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 1 ,m, width: 1, 2, 5, 10 ,m), which undergo artificial hypergravity by centrifugation (10, 24 and 50 g; or 1 g control). The aim of the study was to clarify which of these parameters was more important to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell spreading and alignment. Confocal laser scanning microscopy visualised distribution of actin filaments and vinculin anchoring points through immunostaining. Finally, expression of collagen type I, fibronectin, and ,1 - and ,1 -integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata (control), cells spread out in a random fashion. The alignment of cells cultured on grooved surfaces increased with higher g-forces until a peak value at 25 g. An ANOVA was performed on the data, for all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, most gene levels were reduced by hypergravity. Still, collagen type 1 and fibronectin are seemingly unaffected by time or force. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by hypergravity forces. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] CSF-1 and PI 3-kinase regulate podosome distribution and assembly in macrophagesCYTOSKELETON, Issue 3 2006Ann P. Wheeler Abstract Podosomes are actin-rich adhesive foci found in several cell types, including macrophages. They have a core containing actin and actin-binding proteins and a peripheral ring of integrins and associated proteins. We show that podosomes are abundant in polarized mouse bone marrow-derived macrophages (BMM) and are found primarily in lamellae. We investigated the effects of CSF-1, which induces membrane ruffling, cell spreading, and subsequent polarization and migration, on podosome formation. CSF-1 induces a transient increase in podosome number and enhances the formation of circular arrays of podosomes. Conversely, CSF-1 withdrawal leads to a reduction in podosomes and a decrease in polarized cells. The PI 3-kinase inhibitor LY294002 induces loss of podosomes together with rapid retraction of lamellae and loss of polarity. Our results indicate that CSF-1 acts via PI 3-kinase to enhance podosome assembly and that this is linked to macrophage polarization. Cell Motil. Cytoskeleton, 2006. © 2006 Wiley-Liss, Inc. [source] RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisationCYTOSKELETON, Issue 3 2005Elen Griffith Abstract TES was originally identified as a candidate tumour suppressor gene and has subsequently been found to encode a novel focal adhesion protein. As well as localising to cell-matrix adhesions, TES localises to cell-cell contacts and to actin stress fibres. TES interacts with a variety of cytoskeletal proteins including zyxin, mena, VASP, talin and actin. There is evidence that TES may function in actin-dependent processes as overexpression of TES results in increased cell spreading and decreased cell motility. Together with TES's interacting partners, these data suggest that TES might be involved in regulation of the actin cytoskeleton. Here, for the first time, we have used RNAi to successfully knockdown TES in HeLa cells and we demonstrate that loss of TES from focal adhesions results in loss of actin stress fibres. Similarly, and as previously reported, RNAi-mediated knockdown of zyxin results in loss of actin stress fibres. TES siRNA treated cells show reduced RhoA activity, suggesting that the Rho GTPase pathway may be involved in the TES RNAi-induced loss of stress fibres. We have also used RNAi to examine the requirement of TES and zyxin for each other's localisation at focal adhesions, and we propose a hierarchy of recruitment, with zyxin being first, followed by VASP and then TES. Cell Motil. Cytoskeleton 60:140,152, 2005. © 2005 Wiley-Liss, Inc. [source] Early molecular events in the assembly of the focal adhesion-stress fiber complex during fibroblast spreadingCYTOSKELETON, Issue 3 2004Baruch Zimerman Cell adhesion to the extracellular matrix triggers the formation of integrin-mediated contact and reorganization of the actin cytoskeleton. Examination of nascent adhesions, formed during early stages of fibroblast spreading, reveals a variety of forms of actin-associated matrix adhesions. These include: (1) small (,1 ,m), dot-like, integrin-, vinculin-, paxillin-, and phosphotyrosine-rich structures, with an F-actin core, broadly distributed over the ventral surfaces of the cells; (2) integrin-, vinculin-, and paxillin-containing "doublets" interconnected by short actin bundles; (3) arrays of actin-vinculin complexes. Such structures were formed by freshly plated cells, as well as by cells recovering from latrunculin treatment. Time-lapse video microscopy of such cells, expressing GFP-actin, indicated that long actin cables are formed by an end-to-end lining-up and apparent fusion of short actin bundles. All these structures were prominent during cell spreading, and persisted for up to 30,60 min after plating. Upon longer incubation, they were gradually replaced by stress fibers, associated with focal adhesions at the cell periphery. Direct examination of paxillin and actin reorganization in live cells revealed alignment of paxillin doublets, forming long and highly dynamic actin bundles, undergoing translocation, shortening, splitting, and convergence. The mechanisms underlying the assembly and reorganization of actin-associated focal adhesions and the involvement of mechanical forces in regulating their dynamic properties are discussed. Cell Motil. Cytoskeleton 58:143,159, 2004. © 2004 Wiley-Liss, Inc. [source] Neural tube defects and impaired neural progenitor cell proliferation in G,1 -deficient miceDEVELOPMENTAL DYNAMICS, Issue 4 2010Hiroaki Okae Abstract Heterotrimeric G proteins are well known for their roles in signal transduction downstream of G protein,coupled receptors (GPCRs), and both G, subunits and tightly associated G,, subunits regulate downstream effector molecules. Compared to G, subunits, the physiological roles of individual G, and G, subunits are poorly understood. In this study, we generated mice deficient in the G,1 gene and found that G,1 is required for neural tube closure, neural progenitor cell proliferation, and neonatal development. About 40% G,1,/, embryos developed neural tube defects (NTDs) and abnormal actin organization was observed in the basal side of neuroepithelium. In addition, G,1,/, embryos without NTDs showed microencephaly and died within 2 days after birth. GPCR agonist-induced ERK phosphorylation, cell proliferation, and cell spreading, which were all found to be regulated by G,i and G,, signaling, were abnormal in G,1,/, neural progenitor cells. These data indicate that G,1 is required for normal embryonic neurogenesis. Developmental Dynamics 239:1089,1101, 2010. © 2010 Wiley-Liss, Inc. [source] Cathepsin X cleaves the ,2 cytoplasmic tail of LFA-1 inducing the intermediate affinity form of LFA-1 and ,-actinin-1 bindingEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2009Zala Jevnikar Abstract The motility of T cells depends on the dynamic spatial regulation of integrin-mediated adhesion and de-adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T-cell migration by interaction with lymphocyte function associated antigen-1 (LFA-1). LFA-1 adhesion to the ICAM-1 is controlled by the association of actin-binding proteins with the cytoplasmic tail of the ,2 chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S769, E768 and A767 from the C-terminal of the ,2 cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein ,-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with ,-actinin-1. Increased levels of intermediate affinity LFA-1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM-1-coated surface. Gradual cleavage of LFA-1 by cathepsin X enables the transition between intermediate and high affinity LFA-1, an event that is crucial for effective T-cell migration. [source] Expression of PI(4,5)P2 -binding proteins lowers the PI(4,5)P2 level and inhibits Fc,RIIA-mediated cell spreading and phagocytosisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2008Ewelina Szyma Abstract We found that Fc,RII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P2 and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase I, (PIP5-kinase I,) that colocalized with PI(4,5)P2 and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C374,440 fragment of PIP5-kinase I, or the pleckstrin homology domain of phospholipase C,1 (PLC,1 -PH), two probes binding PI(4,5)P2. These probes reduced the amount of PI(4,5)P2 in the cells, evoked reorganization of the actin cytoskeleton and abolished PI(4,5)P2 elevation during phagocytosis. Simultaneously, PLC,1 -PH-GFP reduced the amount of PIP5-kinase I, associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase I,-GST bound PI(4,5)P2, phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLC,1 -PH domain, and possibly also the C374,440 fragment, when expressed in cells, can compete with endogenous PIP5-kinase I, for PI(4,5)P2 binding in the plasma membrane leading eventually to PI(4,5)P2 depletion. [source] Death-associated protein kinase (DAPK) and signal transduction: blebbing in programmed cell deathFEBS JOURNAL, Issue 1 2010Miia Bovellan Death-associated protein kinase (DAPK) regulates many distinct signalling events, including apoptosis, autophagy and membrane blebbing. The role of DAPK in the blebbing process is only beginning to be understood and, in this review, we will first summarize what is known about the cytoskeletal proteins and signalling cascades that participate in bleb growth and retraction and then highlight how DAPK integrates with these processes. Membrane blebs are quasispherical cellular protrusions that have a lifetime of approximately 2 min. During expansion, blebs are initially devoid of actin, although actomyosin contractions provide the motive force for growth. Once growth slows, an actin cortex reforms and actin-bundling and contractile proteins are recruited. Finally, myosin contraction powers bleb retraction into the cell body. Blebbing occurs in a variety of cell types, from cancerous cells to embryonic cells, and can be seen in cellular phenomena as diverse as cell spreading, movement, cytokinesis and cell death. Although the machinery that executes this is still undefined in detail, the conservation of blebbing phenomenon suggests a fundamental role in metazoans and DAPK offers a door to further dissect this fascinating process. [source] A novel function of WAVE in lamellipodia: WAVE1 is required for stabilization of lamellipodial protrusions during cell spreadingGENES TO CELLS, Issue 5 2005Daisuke Yamazaki When a cell spreads and moves, reorganization of the actin cytoskeleton pushes the cell membrane, and the resulting membrane protrusions create new points of contact with the substrate and generate the locomotive force. Membrane extension and adhesion to a substrate must be tightly coordinated for effective cell movement, but little is known about the mechanisms underlying these processes. WAVEs are critical regulators of Rac-induced actin reorganization. WAVE2 is essential for formation of lamellipodial structures at the cell periphery stimulated by growth factors, but it is thought that WAVE1 is dispensable for such processes in mouse embryonic fibroblasts (MEFs). Here we show a novel function of WAVE in lamellipodial protrusions during cell spreading. During spreading on fibronectin (FN), MEFs with knockouts (KOs) of WAVE1 and WAVE2 showed different membrane dynamics, suggesting that these molecules have distinct roles in lamellipodium formation. Formation of lamellipodial structures on FN was inhibited in WAVE2 KO MEFs. In contrast, WAVE1 is not essential for extension of lamellipodial protrusions but is required for stabilization of such structures. WAVE1-deficiency decreased the density of actin filaments and increased the speed of membrane extension, causing deformation of focal complex at the tip of spreading edges. Thus, at the tip of the lamellipodial protrusion, WAVE2 generates the membrane protrusive structures containing actin filaments, and modification by WAVE1 stabilizes these structures through cell-substrate adhesion. Coordination of WAVE1 and WAVE2 activities appears to be necessary for formation of proper actin structures in stable lamellipodia. [source] Colocalization of Intracellular Osteopontin With CD44 Is Associated With Migration, Cell Fusion, and Resorption in Osteoclasts,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2002K. Suzuki Abstract Although osteopontin (OPN) is recognized generally as a secreted protein, an intracellular form of osteopontin (iOPN), associated with the CD44 complex, has been identified in migrating fibroblastic cells. Because both OPN and CD44 are expressed at high levels in osteoclasts, we have used double immunofluorescence analysis and confocal microscopy to determine whether colocalization of these proteins has functional significance in the formation and activity of osteoclasts. Analysis of rat bone marrow-derived osteoclasts revealed strong surface staining for CD44 and ,1- and ,3-integrins, whereas little or no staining for OPN or bone sialoprotein (BSP) was observed in nonpermeabilized cells. In permeabilized perfusion osteoclasts and multinucleated osteoclasts, staining for OPN and CD44 was prominent in cell processes, including filopodia and pseudopodia. Confocal microscopy revealed a high degree of colocalization of OPN with CD44 in motile osteoclasts. In cells treated with cycloheximide (CHX), perinuclear staining for OPN and BSP was lost, but iOPN staining was retained within cell processes. In osteoclasts generated from the OPN-null and CD44-null mice, cell spreading and protrusion of pseudopodia were reduced and cell fusion was impaired. Moreover, osteoclast motility and resorptive activity were significantly compromised. Although the area resorbed by OPN-null osteoclasts could be rescued partially by exogenous OPN, the resorption depth was not affected. These studies have identified an intracellular form of OPN, colocalizing with CD44 in cell processes, that appears to function in the formation and activity of osteoclasts. [source] Src and FAK mediate cell,matrix adhesion-dependent activation of met during transformation of breast epithelial cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Angela Y. Hui Abstract Cell,matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand-independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non-neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto-activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with ,1 integrins and is co-localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src-dependent phosphorylation of Met requires cell,matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src-induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF-dependent and Src/integrin-dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells. J. Cell. Biochem. 107: 1168,1181, 2009. © 2009 Wiley-Liss, Inc. [source] NG2 proteoglycan mediates ,1 integrin-independent cell adhesion and spreading on collagen VIJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2002Emmanuelle Tillet Abstract Collagens V and VI have been previously identified as specific extracellular matrix (ECM) ligands for the NG2 proteoglycan. In order to study the functional consequences of NG2/collagen interactions, we have utilized the GD25 cell line, which does not express the major collagen-binding ,1 integrin heterodimers. Use of these cells has allowed us to study ,1 integrin-independent phenomena that are mediated by binding of NG2 to collagens V and VI. Heterologous expression of NG2 in the GD25 line endows these cells with the capability of attaching to surfaces coated with collagens V and VI. The specificity of this effect is emphasized by the failure of NG2-positive GD25 cells to attach to other collagens or to laminin-1. More importantly, NG2-positive GD25 cells spread extensively on collagen VI. ,1 integrin-independent extension of ruffling lamellipodia demonstrates that engagement of NG2 by the collagen VI substratum triggers signaling events that lead to rearrangement of the actin cytoskeleton. In contrast, even though collagens V and VI each bind to the central segment of the NG2 ectodomain, collagen V engagement of NG2 does not trigger cell spreading. The distinct morphological consequences of NG2/collagen VI and NG2/collagen V interaction indicate that closely-related ECM ligands for NG2 differ in their ability to initiate transmembrane signaling via engagement of the proteoglycan. J. Cell. Biochem. 86: 726,736, 2002. © 2002 Wiley-Liss, Inc. [source] A short history of blebbingJOURNAL OF MICROSCOPY, Issue 3 2008G.T. CHARRAS Summary Blebs are protrusions of the cell membrane. They are the result of actomyosin contractions of the cortex, which cause either transient detachment of the cell membrane from the actin cortex or a rupture in the actin cortex. Then, cytosol streams out of the cell body and inflates the newly formed bleb. During expansion, which lasts ,30 s, the bleb is devoid of actin and the surface area increases through further tearing of membrane from the cortex and convective flows of lipids in the plane of the membrane through the bleb neck. Once expansion slows, an actin cortex is reconstituted. First actin-membrane linker proteins, such as ezrin, are recruited to the bleb, then actin, actin-bundling proteins and finally myosin motor proteins. Retraction lasts ,2 min and is powered by myosin motor proteins. Though it has been less studied than other actin-based membrane protrusions such as lamellipodia or filopodia, blebbing is a common feature of cell physiology during cell movement, cytokinesis, cell spreading and apoptosis. This review will succinctly attempt to summarize what we know about the mechanisms involved in blebbing, when it appears in cell physiology and what open questions remain. [source] Microtopography of metal surfaces influence fibroblast growth by modifying cell shape, cytoskeleton, and adhesionJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 11 2007David O. Meredith Abstract Stainless Steel (SS), titanium (cpTi), and Ti-6Al-7Nb (TAN) are frequently used metals in fracture fixation, which contact not only bone, but also soft tissue. In previous soft tissue cytocompatibility studies, TAN was demonstrated to inhibit cell growth in its "standard" micro-roughened state. To elucidate a possible mechanism for this inhibition, cell area, shape, adhesion, and cytoskeletal integrity was studied. Only minor changes in spreading were observed for cells on electropolished SS, cpTi, and TAN. Cells on "standard" cpTi were similarly spread in comparison with electropolished cpTi and TAN, although the topography influenced the cell periphery and also resulted in lower numbers and shorter length of focal adhesions. On "standard" microrough TAN, cell spreading was significantly lower than all other surfaces, and cell morphology differed by being more elongated. In addition, focal adhesion numbers and mean length were significantly lower on standard TAN than on all other surfaces, with 80% of the measured adhesions below a 2-µm threshold. Focal adhesion site location and maturation and microtubule integrity were compromised by the presence of protruding ,-phase microspikes found solely on the surface of standard TAN. This led us to propose that the impairment of focal adhesion numbers, maturation (length), and cell spreading to a possibly sufficient threshold observed on standard TAN blocks cell cycle progress and eventually cell growth on the surface. We believe, as demonstrated with standard cpTi and TAN, that a difference in surface morphology is influential for controlling cell behavior on implant surfaces. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1523,1533, 2007 [source] Ethanol Treatment Reduces Bovine Bronchial Epithelial Cell MigrationALCOHOLISM, Issue 4 2005John R. Spurzem Background: Chronic ethanol abuse is associated with significant lung disease. Excessive alcohol intake increases risk for a variety of respiratory tract diseases, including pneumonia and bronchitis. Damage to airway epithelium is critical to the pathogenesis of airway disorders such as chronic bronchitis and chronic obstructive pulmonary disease. The ability of the airway epithelium to repair itself is an important step in the resolution of airway inflammation and disease. Ethanol exposure is known to modulate signaling systems in bronchial epithelial cells. We hypothesize that chronic ethanol exposure down-regulates the adenosine 3,:5,-cyclic monophosphate signaling cascade in airway epithelial cells, resulting in decreased epithelial cell migration and repair. Methods: We evaluated the effect of ethanol on primary cultures of bovine bronchial epithelial cells in in vitro models of cell migration, wound repair, cell attachment, and cell spreading. Results: Ethanol causes a concentration-dependent effect on closure of mechanical wounds in cell monolayers. Pretreatment of cells with 100 mm ethanol for 24 hr further slows wound closure. Ethanol pretreatment also reduced the protein kinase A response to wounding and made the cells unresponsive to stimuli of protein kinase A that accelerate wound closure. The effects of ethanol on cell migration in wound closure were confirmed in another assay of migration, the Boyden chamber cell migration assay. Prolonged treatment with ethanol also reduced other cell functions, such as spreading and attachment, which are necessary for epithelial repair. Conclusions: Ethanol modulates signaling systems that are relevant to airway injury and repair, suggesting that chronic, heavy ethanol ingestion has a detrimental impact on airway repair. Impaired response to inflammation and injury may contribute to chronic airway disease. [source] Effect of a novel botanical agent Drynol Cibotin on human osteoblast cells and implications for osteoporosis: promotion of cell growth, calcium uptake and collagen productionPHYTOTHERAPY RESEARCH, Issue S2 2010Barbara Wegiel Abstract Osteoporosis is a widespread problem afflicting millions of people. Drynol Cibotinis is a newly developed proprietary botanical combination of eight botanicals including Angelica sinensis, Glycine max, Wild yam, Ligustrum lucidum, Astragalus membranaceus, Cuscuta chinensis, Psoraleae corylifoliae, and Drynaria fortune. Each of the botanicals has been used in traditional Chinese medicine to treat osteoporosis. The effect of Drynol Cibotinis, with the specific combination of these anti-osteoporosis botanicals for promoting bone growth, was examined in this study. The effects of Drynol Cibotin on cell growth, apoptosis, cell spreading, calcium uptake and production of bone matrix proteins Collagen I and Laminin B2 on human osteoblast cells were assessed by BrdU incorporation, TUNEL assay, cell staining, intracellular Ca2+ measurement and Western blot analysis. The results showed that Drynol Cibotin significantly increased cell proliferation and inhibited apoptosis in osteoblasts (P < 0.01). In addition, Drynol Cibotin was found to promote cell spreading and greatly increase calcium uptake both instantaneously and in the long term (P < 0.01). Furthermore, Drynol Cibotin significantly increased production of two key extracellular matrix proteins in bone cells: Collagen I and Laminin B2. These results indicate that Drynol Cibotin alone or in combination with amino acids and vitamins may have prophylactic potentials in osteoporosis. Copyright © 2009 John Wiley & Sons, Ltd. [source] Characterization and effect of modified surface on the morphology, structure and function of rabbit bone marrow-derived mesenchymal stem cells 1ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING, Issue 5 2009K. D. Song Abstract The surface morphologies modified with chitosan and gelatin respectively, the effects on cell spreading, adhesion and distribution of rabbit bone marrow-derived mesenchymal stem cells (rMSCs) were observed under atomic force microscope (AFM). The structures and morphologies of rMSCs on coverslips were qualitatively and quantitatively assayed under AFM. Thereafter, the growth status and viability of rMSCs on the different coated surfaces were examined under inverted microscope and by Hoechst/PI double staining. The results showed that the surfaces modified with different materials presented different morphologies. The rMSCs adhered to the surfaces modified with 2 mg/ml chitosan and 0.5 mg/ml gelatin solution showed better viabilities than those of other groups, and the latter also presented the optimal cell adhesion, spreading and distribution with clear cellular structure such as cell nucleus, villus and interaction among various rMSCs. It is indicated that the surface modification played an important role on the development of cellular structure and function of rMSCs, and thus provided theoretical guidance to the fabrication and surface modification of embedded biomaterials in further clinical applications. Copyright © 2009 Curtin University of Technology and John Wiley & Sons, Ltd. [source] Effect of spatial architecture on cellular colonizationBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Yan Huang Abstract The spatial cell-material interaction remains vital issue in forming biodegradable scaffolds in Tissue Engineering. In this study, to understand the influence of spatial architecture on cellular behavior, 2D and 3D chitosan scaffolds of 50,190 kD and >310 kD MW were synthesized through air drying and controlled rate freezing/lypohilization technique, respectively. In addition, chitosan was emulsified with 19, 76, and 160 kD 50:50 poly lactide-co-glycolide (PLGA) using 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) as stabilizer. 2D and 3D scaffolds were formed by air drying and lyophilization as before. Tensile and compressive properties of films and scaffolds were analyzed in wet conditions at 37°C. Alterations in the cell spreading, proliferation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) were studied. These results showed that the formed 3D chitosan scaffolds had interconnected open pore architecture (50,200 µm size). HUVECs and MEFs had reduced spreading areas and circular morphology on 2D chitosan membranes compared with 3D chitosan scaffolds. The fluorescence photomicrographs for actin (using Alexa Fluor 488 phalloidin) and cytoplasm staining (using carboxyfluorescein diacetate-succinimidyl ester) demonstrated that the cells spread within 3D chitosan matrix. 2D and 3D emulsified chitosan and chitosan/PLGA scaffolds reduced the spreading of HUVECs and MEFs even further. Proliferation results, analyzed via MTT-Formazan assay and BrdU uptake assay, correlated with the spreading characteristics. The reductions in cell spreading area on emulsified surfaces were not detrimental to the viability and endocytic activity but to proliferation. The observed alterations in cellular colonization are in part due to the substrate stiffness and surface topography. In summary, these results suggest a significant influence of spatial architecture on cellular colonization. © 2005 Wiley Periodicals, Inc. [source] R-Ras promotes tumor growth of cervical epithelial cellsCANCER, Issue 3 2003Héctor Rincón-Arano B.S. Abstract BACKGROUND R-Ras is 55% identical to H-Ras. However, these two oncogenes seem to have different tumor-transforming potential. R-Ras induced cell transformation in fibroblasts but not in other cell types. R-Ras also reportedly induces a more invasive phenotype in breast epithelial cells through integrin activation. The authors studied the mechanisms whereby R-Ras induces a malignant phenotype. METHODS Dominant negative (R-Ras43N) and constitutively active (R-Ras87L) mutants of R-Ras were stably transfected into human cervical epithelium C33A cells. Transfected cells were analyzed for adhesion, cell spreading, migration, and growth in culture and in nude mice. The activity of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K) also was determined by Western blot analysis and by in vitro kinase assays. RESULTS R-Ras87L-transfected cells, but not R-Ras43 N-transfected cells, had a higher growth rate in nude mice and in culture compared with control cells. None of the transfected C33A cells showed an increase in cell adhesion to fibronectin or collagen I, nor did they show an increment of ,1 integrin affinity. However, cells that expressed R-Ras87L, but not cells that expressed R-Ras 43N, presented a marked increase in cell spreading and migration through collagen-coated membranes. Increases in cell proliferation, spreading, and migration induced by R-Ras87L were inhibited by the PI 3-K inhibitor LY294002. In addition, PI 3-K activity, but not ERK activity, was increased only in cells that expressed R-Ras87L. CONCLUSIONS These data suggest that the oncogene R-Ras promotes tumor growth of cervical epithelial cells and increases their migration potential over collagen through a pathway that involves PI 3-K. Cancer 2003;97:575,85. © 2003 American Cancer Society. DOI 10.1002/cncr.11093 [source] | ||||||||||||||||