RESUMEN
Microscale slender swimmers are frequently encountered in nature and are now used in microrobotic applications. The swimming mechanism examined in this paper is based on small transverse axisymmetric traveling wave deformations of a cylindrical long shell. The thin-shelled device is assumed to be inextensible at the middle surface and extensible at the surface wetted by the fluid. Assuming low-Reynolds-number hydrodynamics, an analytical solution is derived for waves of small amplitudes compared with the cylinder diameter. We show that swimming velocity increases with ß(1) (the ratio of cylinder radius to wavelength) and that swimming velocity is linearly dependent on wave propagation velocity, increasing to leading order with the square of the ratio of wave amplitude to wavelength ß(2) and decreasing with the wall thickness. A fourth-order correction in ß(2) was also calculated and was found to have a negative effect on the swimming velocity. The results for a shell of negligible-wall thickness were compared with Taylor's solution for an inextensible two-dimensional flat membrane undergoing a waving motion and Felderhof's results [Phys. Fluids 22, 113604 (2010)] for an unbounded flow field and negligible-wall thickness. We show that Taylor's analytic solution is a particular limiting case of the present solution, assuming zero wall thickness and infinite values of ß(1). The present mechanism was also compared with Taylor's well known solutions of waving planar and helical circular tails. We show that the present approach yields higher velocities as ß(1) increases, whereas, the opposite occurs for waving tails. Indeed, in the region where ß(1)>15, the present approach yields velocities nearly as fast as Taylor's helical waving tail while consuming less power and with a design that is considerably more compact. In this regime, the axisymmetric swimmer is twice as fast as Taylor's planar-tail swimmer for an additional investment of only one-third of the power. Experiments were conducted using a macroscale autonomous model immersed in highly viscous silicone fluid. We outlined how the proposed mechanism was realized to propel an elongated, yet finite, swimmer. Measured data demonstrate the effects of wave velocity and wavelength on swimming speed, showing good agreement with analytical results.
Asunto(s)
Biomimética/métodos , Modelos Teóricos , Reología/métodos , Robótica/métodos , Natación , Simulación por Computador , Movimiento (Física)RESUMEN
The introduction of retroviral vector producer cells (VPC) into tumors as a means of increasing transduction efficiency has recently been employed in human gene therapy trials. However, the fate of these xenogeneic cells in humans is not well understood. In the present study, we used an in vitro model to examine the survival of commonly used VPC lines in serum from humans and various other species. VPC derived from the murine NIH-3T3 cell line, including PA317, Psi CRIP, and GP + E-86, were effectively killed in sera from Old World primates, including human and baboon. Conversely, the same murine cell lines survived exposure to sera from dog, rabbit, rat, and mouse. This pattern of serum killing parallels the occurrence of the anti-alpha-galactosyl natural antibody (Ab) found exclusively in Old World primates. The anti-alpha-galactosyl Ab targets the terminal glycosidic structure Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-galactosyl epitope) found on the surface of mammalian cells, excluding Old World primates. All murine-derived VPC tested expressed high levels of the alpha-galactosyl epitope as determined by FACS analysis. VPC killing was complement-mediated, because preincubation of human serum with a functionally blocking anti-C5 mAb completely abolished cell lysis. Furthermore, addition of soluble galactose(alpha 1-3)galactose (Gal alpha 1-3Gal) to human serum or down-regulation of the alpha-galactosyl epitope on the surface of VPC effectively reduced VPC killing, indicating that complement activation by these cells is primarily initiated by natural antibody recognition of the alpha-galactosyl epitope. Finally, VPC incubated with human serum for 8 hr in the presence of complement inhibition continued to produce viable retroviral particles, thus demonstrating a correlation between VPC and particle survival. Taken together, these data suggest that elimination of the alpha-galactosyl epitope or complement blockade may provide a strategy to prolong the survival of VPC and the particles that they produce in vivo.
Asunto(s)
Anticuerpos/inmunología , Supervivencia Celular/genética , Proteínas del Sistema Complemento/inmunología , Galactosa/inmunología , Vectores Genéticos , Retroviridae/genética , Animales , Secuencia de Carbohidratos , Células Cultivadas , Perros , Epítopos/inmunología , Citometría de Flujo , Humanos , Ratones , Datos de Secuencia Molecular , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Papio/metabolismo , Conejos , Ratas , Retroviridae/metabolismoRESUMEN
The major obstacle to successful discordant xenotransplantation is the phenomenon of hyperacute rejection (HAR). In the pig-to-primate discordant transplant setting, HAR results from the deposition of high-titre anti-alpha-galactosyl antibodies and complement activation leading to endothelial cell destruction and rapid graft failure. To overcome HAR, we developed an enzymatic carbohydrate remodelling strategy designed to replace expression of the Gal alpha-1,3-Gal xenoepitope on the surface of porcine cells with the non-antigenic universal donor human blood group O antigen, the alpha-1,2-fucosyl lactosamine moiety (H-epitope). Xenogenic cells expressing the human alpha-1,2-fucosyltransferase expressed high levels of the H-epitope and significantly reduced Gal alpha-1,3-Gal expression. As a result, these cells were shown to be resistant to human natural antibody binding and complement-mediated cytolysis.
Asunto(s)
Disacáridos/metabolismo , Fucosiltransferasas/metabolismo , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Unión Competitiva , Línea Celular , Activación de Complemento , Cartilla de ADN , Fucosiltransferasas/genética , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Rechazo de Injerto/inmunología , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Transfección , Trasplante Heterólogo/inmunología , Células Tumorales Cultivadas , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
The hyperacute rejection (HAR) of xenotransplanted organs is initiated by the deposition of natural antibodies on donor endothelium followed by the activation of the recipient complement system, which rapidly destroys the graft. Studies of the role of activated complement in HAR have suggested that natural antibody as well as early (C3a, C3b) and late (C5a, C5b-9) activated complement components may contribute to cell activation and damage. Attenuation of HAR has been achieved by blockade of C3 activation with soluble CR1 or consumptive depletion of complement with cobra venom factor; however, similar studies using specific inhibitors of terminal complement components have not been described. To address the contribution of C5a and the membrane attack complex (C5b-9, MAC) to complement-mediated xenogeneic cell and organ damage, we utilized functionally blocking monoclonal antibodies directed against the human terminal complement components C5 and C8. Our data show that both anti-C5 and anti-C8 mAbs protect porcine aortic endothelial cells from membrane damage mediated by human C5b-9. Additionally, both the anti-C5 and anti-C8 mAbs blocked complement-mediated generation of membrane prothrombinase activity on porcine aortic endothelial cells challenged with human serum. To test the ability of these antibodies to attenuate antibody and complement-mediated damage of xenogeneic organs, an ex vivo model was developed wherein isolated rat hearts were perfused with human serum in the presence or absence of the anti-C5 and anti-C8 mAbs. Our data demonstrate that mAbs directed against human C5 and C8 prevented organ damage by human serum complement and suggest that these molecules may serve as potent inhibitors of HAR.
Asunto(s)
Complemento C5/inmunología , Complemento C8/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Rechazo de Injerto , Animales , Anticuerpos Monoclonales , Activación de Complemento , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Endotelio Vascular/inmunología , Humanos , Miocardio/inmunología , Perfusión , Ratas , Porcinos , Tromboplastina/metabolismoRESUMEN
Human cells express cell surface complement regulatory molecules that inhibit the activity of the C3/C5 convertases (DAF, MCP, CR1) or inhibit the membrane attack complex (CD59). A single molecule that inhibits both the convertase activity and formation of the membrane attack complex has never been characterized. To this end, we have developed two reciprocal chimeric complement inhibitors (CD, NH2-CD59-DAF-GPI; and DC, NH2-DAF-CD59-GPI) that contain the functional domains of decay accelerating factor (DAF; CD55) and CD59. Cell surface expression of the CD and DC chimeric proteins was detected with DAF- and CD59-specific antisera. Cell surface C3d deposition was inhibited on cells expressing the chimeric molecules, thereby indicating that the DAF moiety was functional in both molecules. Conversely, Ab-blocking experiments demonstrated that only the DC molecule retained CD59 function. Therefore, the DC molecule represents a novel potent chimeric bifunctional complement inhibitor that retains the functional domains of two distinct complement regulatory molecules.
Asunto(s)
Antígenos CD55/fisiología , Antígenos CD59/fisiología , Convertasas de Complemento C3-C5/antagonistas & inhibidores , Proteínas Inactivadoras de Complemento/fisiología , Complejo de Ataque a Membrana del Sistema Complemento/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Animales , Secuencia de Bases , Antígenos CD55/genética , Antígenos CD59/genética , Línea Celular , Convertasas de Complemento C3-C5/genética , Proteínas Inactivadoras de Complemento/biosíntesis , Proteínas Inactivadoras de Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/antagonistas & inhibidores , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
Type C retroviruses endogenous to various nonprimate species can infect human cells in vitro, yet the transmission of these viruses to humans is restricted. This has been attributed to direct binding of the complement component C1q to the viral envelope protein p15E, which leads to classical pathway-mediated virolysis in human serum. Here we report a novel mechanism of complement-mediated type C retrovirus inactivation that is initiated by the binding of "natural antibody" [Ab] (anti-alpha-galactosyl Ab) to the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R expressed on the retroviral envelope. Complement-mediated inactivation of amphotropic retroviral particles was found to be restricted to human and other Old World primate sera, which parallels the presence of anti-alpha-galactosyl natural Ab. Blockade or depletion of anti-alpha-galactosyl Ab in human serum prevented inactivation of both amphotropic and ecotropic murine retroviruses. Similarly, retrovirus was not killed by New World primate serum except in the presence of exogenous anti-alpha-galactosyl Ab. Enzyme-linked immunosorbent assays revealed that the alpha-galactosyl epitope was expressed on the surface of amphotropic and ecotropic retroviruses, and Western blot analysis further localized this epitope to the retroviral envelope glycoprotein gp70. Finally, down-regulation of this epitope on the surface of murine retroviral particle producer cells rendered them, as well as the particles liberated from these cells, resistant to inactivation by human serum complement. Our data suggest that anti-alpha-galactosyl Ab may provide a barrier for the horizontal transmission of retrovirus from species that express the alpha-galactosyl epitope to humans and to other Old World primates. Further, these data provide a mechanism for the generation of complement-resistant retroviral vectors for in vivo gene therapy applications where exposure to human complement is unavoidable.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Sangre/virología , Cebidae/inmunología , Cercopithecidae/inmunología , Epítopos/inmunología , Galactosa/inmunología , Virus de la Leucemia Murina/fisiología , Proteínas Oncogénicas de Retroviridae/inmunología , Proteínas del Envoltorio Viral/inmunología , Células 3T3 , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/biosíntesis , Sangre/inmunología , Secuencia de Carbohidratos , Cebidae/sangre , Cercopithecidae/sangre , Proteínas del Sistema Complemento/inmunología , Humanos , Inmunidad Innata , Mamíferos/sangre , Mamíferos/inmunología , Ratones , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/inmunología , Proteínas Oncogénicas de Retroviridae/biosíntesis , Especificidad de la Especie , Proteínas del Envoltorio Viral/biosíntesisRESUMEN
Herpesvirus saimiri (HVS) is a lymphotropic herpesvirus that induces T-cell transformation in vitro and causes lymphomas and leukemias in New World primates other than its natural host, the squirrel monkey. Nucleotide sequence analysis of the HVS genome revealed two open reading frames with significant homology to genes for human complement regulatory molecules. One of these genes encodes a predicted protein (designated HVSCD59) with 48% amino acid sequence identity to the human terminal complement regulatory protein CD59 (HuCD59). The CD59 homolog from squirrel monkey (SMCD59) was cloned, and the corresponding amino acid sequence showed 69% identity with HVSCD59. BALB/3T3 cells stably expressing HVSCD59, SMCD59, or HuCD59 were equally protected from complement-mediated lysis by human serum. However, only HVSCD59-expressing cells were effectively protected from complement-mediated lysis when challenged with rat serum, suggesting that HVSCD59 was less species restrictive. The complement regulatory activity of HVSCD59 and SMCD59 occurred after C3b deposition, indicating terminal complement inhibition. Treatment of BALB/3T3 stable transfectants with phosphatidylinositol-specific phospholipase C prior to complement attack decreased the complement regulatory function of HVSCD59, suggesting cell surface attachment via a glycosyl-phosphatidylinositol anchor. Cells expressing HVSCD59 effectively inhibited complement-mediated lysis by squirrel monkey serum in comparison with SMCD59-expressing cells. Finally HVSCD59-specific transcripts were detected in owl monkey cells permissive for lytic HVS replication but not in T cells transformed by HVS, which failed to produce virions. These data are the first to demonstrate a functional, virally encoded terminal complement inhibitor and suggest that HVSCD59 represents a humoral immune evasion mechanism supporting the lytic life cycle of HVS.
Asunto(s)
Antígenos CD/inmunología , Proteínas Inactivadoras de Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Herpesvirus Saimiriino 2/inmunología , Inmunidad Celular/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas Virales/inmunología , Células 3T3 , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Secuencia de Bases , Antígenos CD59 , Muerte Celular , Complemento C3/metabolismo , Proteínas Inactivadoras de Complemento/biosíntesis , Proteínas Inactivadoras de Complemento/efectos de los fármacos , Proteínas Inactivadoras de Complemento/genética , Herpesvirus Saimiriino 2/genética , Humanos , Glicoproteínas de Membrana/genética , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Hidrolasas Diéster Fosfóricas/farmacología , Ratas , Saimiri/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transcripción Genética , Proteínas Virales/biosíntesis , Proteínas Virales/efectos de los fármacos , Proteínas Virales/genéticaRESUMEN
A blood vessel's response to denudation injury will determine its final luminal diameter as well as its function. The synthesis, deposition, and remodeling of extracellular matrix components and migration by vascular endothelial cells are major factors in determining luminal diameter, cellular proliferative and migratory responses, and mononuclear cell adhesion at sites of injury. Previously, we have shown that after in vivo and in vitro denudation injury, endothelial cell migration is dramatically influenced by the amount of fibronectin synthesized and deposited by the responding endothelial cell population. The aim of this study was to elucidate the roles of fibronectin in modulating mononuclear cell adhesion to the endothelial cell population during in vitro migration. In this report we demonstrate that U937 cell binding to the migrating fronts of endothelial cell monolayers is modulated by the amount of fibronectin synthesized and deposited by the endothelial cells. Agents which increase fibronectin deposition, such as transforming growth factor-beta 1, elicit greater U937 cell adhesion. Manipulations that decrease fibronectin deposition, such as transfection and overexpression of pp60c-src proto-oncogene in endothelial cells, reduce U937 cell adhesion. These results suggest that changes in endothelial cell extracellular matrix synthesis and deposition modulate, in part, the adhesive properties of the vessel wall after injury. In turn, the intensity and duration of mononuclear cell adhesion at sites of vessel wall injury determines, in part, the vessel wall response.
Asunto(s)
Endotelio Vascular/citología , Fibronectinas/fisiología , Células Tumorales Cultivadas/citología , Animales , Aorta/citología , Bovinos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas/química , Resistencia a Medicamentos/genética , Endotelio Vascular/química , Técnica del Anticuerpo Fluorescente , Humanos , Integrinas/análisis , Leucemia Promielocítica Aguda , Neomicina/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas pp60(c-src)/análisis , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales Cultivadas/ultraestructuraRESUMEN
Scatter factor (SF) was first identified as a fibroblast-derived protein which disperses (i.e., "scatters") cohesive colonies of epithelium. SF-like proteins were found in human smooth muscle cell conditioned medium, amniotic fluid, and placental tissue. SFs markedly stimulate migration of epithelial, carcinoma, and vascular endothelial cell types at picomolar concentrations. Hepatocyte growth factors (HGFs) were originally described as platelet- and serum-derived proteins which stimulate hepatocyte DNA synthesis. Partial amino acid sequence data for mouse and human SFs indicate significant homology with HGFs. We used biological, biochemical, and immunological assays to evaluate and compare the activities, properties, and mechanisms of action of mouse SF, human SF (fibroblast or placenta derived), and recombinant human HGF (hrHGF). We report the following findings: (a) mouse SF exhibits species-related differences in biological activities relative to the human factors; (b) human SF and hrHGF show significant overlap in biological activities (i.e., hrHGF stimulates motility of multiple normal and carcinoma cell types, whereas human SF stimulates DNA synthesis in several normal cell types); (c) the three factors contain common antigenic determinants; and (d) all three proteins stimulate rapid phosphorylation of tyrosine residues on the c-met protooncogene protein product (the putative receptor for HGF) and on another protein with Mr 110,000. A few biological and immunological differences between human SFs and hrHGF were observed. These may reflect minor variations in amino acid sequence or posttranslational modification related to the sources of the factors. Taken as a whole, our findings suggest that by structural, functional, immunological, and mechanistic criteria, human SF and human HGF are essentially identical.
Asunto(s)
Proteínas Sanguíneas/fisiología , Citocinas/fisiología , Sustancias de Crecimiento/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/química , Cápsulas , Línea Celular , Movimiento Celular/fisiología , Citocinas/química , ADN/biosíntesis , Fibroblastos , Sustancias de Crecimiento/química , Factor de Crecimiento de Hepatocito , Humanos , Procesamiento de Imagen Asistido por Computador , Hígado/citología , Hígado/metabolismo , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
Cellular motility is a critical function in embryonic development, tissue repair, and tumor invasion. We used assays of scattering (epithelial colony dispersion), cell migration, and cell invasion to study cytokine-regulated motility in epithelial and carcinoma cell lines. Tumor necrosis factor (TNF) stimulated motility in 12 of 14 cell lines in one or more assay systems. The motility-stimulating activity of TNF did not correlate with its antiproliferative activity. In lines whose migration was stimulated by both TNF and scatter factor (SF), a fibroblast-derived cytokine which stimulates epithelial cell motility, saturating concentrations of TNF plus SF induced greater migration than either agent alone. Anti-TNF monoclonal antibody blocked TNF- but not SF-stimulated motility. While various other factors (basic fibroblast growth factor, interleukin 6, interleukin 2, colony-stimulating factor 1) had little or not motility-stimulating activity, phorbol-12-myristate-13-acetate (PMA), a tumor-promoting phorbol ester, scattered and/or stimulated migration in all cell lines studied. Combinations of saturating concentrations of TNF plus PMA or of SF plus PMA induced greater migration than did any agent alone. These findings suggest that (a) carcinoma cell motility may be mediated by multiple biochemical pathways and (b) TNF stimulates epithelial motility by a mechanism different from that of SF and PMA. In vivo, TNF might enhance invasiveness of some carcinomas or stimulate epithelial wound healing.
Asunto(s)
Neoplasias/fisiopatología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiotaxis , Citocinas/farmacología , Perros , Relación Dosis-Respuesta a Droga , Epitelio/fisiología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor de Crecimiento de Hepatocito , Humanos , Técnicas In Vitro , Interleucina-2/farmacología , Interleucina-6/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Invasividad Neoplásica , Neoplasias/tratamiento farmacológico , Ratas , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
Scatter factor (SF) is a fibroblast-derived cytokine which stimulates motility of epithelial and vascular endothelial cells. We used a quantitative assay based on migration of cells from microcarrier beads to flat surfaces to study the regulation of motility in bovine brain endothelial cells (BBEC). Peptide growth factors (EGF, ECGF, basic FGF) did not stimulate migration. Tumor promoting phorbol esters (PMA, PDD) markedly stimulated migration, while inactive phorbol esters (4a-PDD, phorbol-13,20-diacetate) did not affect migration. Both SF- and PMA-stimulated migration were inhibited by 1) TGF-beta; 2) protein kinase inhibitors (e.g., staurosporine, K-252a); 3) activators of the adenylate cyclase signaling pathway (e.g., dibutyryl cyclic AMP, theophylline); 4) cycloheximide; and 5) anti-cytoskeleton agents (e.g., cytochalasin B, colcemid). However, PMA and SF pathways were distinguishable: 1) PMA induced additional migration at saturating SF concentrations; 2) the onset of migration-stimulation was immediate for PMA and delayed for SF; and 3) down-modulation of protein kinase C (PKC) ablated PMA but not SF responsiveness. Assessment of PKC by (3H)-phorbol ester (PDBu) binding and by immunoblot showed 1) scatter factor does not cause significant redistribution or down-modulation of PDBu binding or alpha-PKC; and 2) PDBu mediates redistribution and down-modulation of both binding and alpha-PKC. These findings suggest two pathways for BBEC motility: a PKC-dependent pathway and an SF-stimulated/PKC-independent pathway.
Asunto(s)
Encéfalo/irrigación sanguínea , Endotelio Vascular/fisiología , Proteínas/fisiología , Animales , Bovinos , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Citoesqueleto/efectos de los fármacos , Regulación hacia Abajo , Endotelio Vascular/citología , Sustancias de Crecimiento/fisiología , Factor de Crecimiento de Hepatocito , Técnicas In Vitro , Nucleótidos Cíclicos/fisiología , Ésteres del Forbol/farmacología , Inhibidores de Proteínas Quinasas , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
Interleukin-6 (IL-6) is a cytokine which regulates host response to injury. Various preparations of recombinant human IL-6 stimulated migration of bovine brain and bovine aortic endothelial cells, with maximal responses at 100-600 ng/ml. The migration response was inhibited by anti-IL-6 monoclonal antibody. IL-6 also inhibited endothelial cell proliferation in a dose-dependent fashion. Combinations of IL-6 and tumor necrosis factor induced additive stimulation of migration. Studies with inhibitors and stimulators of various metabolic processes suggest that IL-6-induced motility: 1) does not require a pertussis toxin-sensitive G-protein, protein kinase C, or DNA synthesis; and 2) is regulated differently from the motility induced by scatter factor. A possible role for IL-6 in the regulation of physiologic angiogenesis is discussed.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Endotelio Vascular/fisiología , Interleucina-6/farmacología , Animales , Aorta , Células CHO , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Circulación Cerebrovascular , Cricetinae , Citocinas/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Interleucina-6/genética , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
Scatter factors (SFs) are heat- and trypsin-sensitive cytokines secreted by fibroblastic and vascular smooth muscle cell lines which stimulate motility of normal epithelium, carcinoma cells, and vascular endothelium. Human and mouse SFs have been purified and identified as 90 kD heterodimeric proteins consisting of heavy (58 kD) and light (31 kD) disulfide-bonded subunits. Partial amino acid sequence data from SF-derived tryptic peptides indicate marked sequence homology with hepatocyte growth factors, suggesting a common multigene family. In this chapter we describe the regulation by SF of vascular endothelial cell chemotaxis and chemokinesis; migration from microcarrier beads to flat surfaces; invasion through porous filters coated with reconstituted basement membrane; secretion of plasminogen activator; and in vitro capillary-like tube formation on a basement membrane surface.
Asunto(s)
Capilares/fisiología , Movimiento Celular/fisiología , Quimiotaxis/fisiología , Citocinas/farmacología , Endotelio Vascular/fisiología , Animales , Movimiento Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Fibroblastos/fisiología , Factor de Crecimiento de Hepatocito , HumanosRESUMEN
Fibroblasts and smooth muscle cells release a protein activity which causes epithelial sheets to "scatter" into isolated cells. Purification of scatter factor (SF) activity from ras-transformed 3T3 cells was reported recently. We purified ras-3T3 SF by a slightly different method with essentially similar findings. Purified factor showed a single band at 77 +/- 3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Scatter activity was eluted from gel slices at this molecular size. Reduction with mercaptoethanol caused the loss of activity and the appearance of two bands (58 and 31 kDa). We report the amino acid composition of ras-3T3 SF and sequences of several tryptic peptides. These sequences were not similar to the known proteins in the Protein Database. We have shown previously that partially purified ras-3T3 scatter activity stimulates migration of epithelial and vascular endothelial cells in a new migration assay utilizing microcarrier beads. We now demonstrate that the same purified ras-3T3 protein scatters epithelial cells and stimulates epithelial and endothelial migration in microcarrier bead and Boyden chamber assays. Partially purified human smooth muscle scatter activity shares these activities, but the protein(s) responsible has not been isolated. Migration-stimulating activity was maximal at ras-3T3 protein concentrations less than 10 ng/ml (0.13 nM). ras-3T3 SF had no collagenolytic activity and did not stimulate DNA synthesis in fibroblast growth factor-responsive human melanocytes. ras-3T3 SF appears to be a new protein which regulates endothelial and epithelial mobility; and, therefore, it may be involved in vascular repair and wound healing.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Epitelio/efectos de los fármacos , Proteínas/farmacología , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , División Celular/efectos de los fármacos , Línea Celular , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , Células Epiteliales , Fibroblastos , Factor de Crecimiento de Hepatocito , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas/aislamiento & purificaciónRESUMEN
Scatter factor (SF) is a cytokine that causes cohesive epithelial colonies to 'scatter' into isolated cells and stimulates epithelial cell migration. To investigate SF's mechanism(s), we screened agents that modulate various intracellular processes for effects on scattering of Madin-Darby canine kidney (MDCK) cells. Selected agents were studied in quantitative migration assays using microcarrier beads. Agents that activate the adenylate cyclase (AC) pathway caused mild to moderate inhibition of scattering and migration, while modulators of Ca2+/calmodulin pathways had little effect on scattering. In contrast, phorbol esters (PMA, PDD) and protein kinase C (PKC) inhibitors (staurosporine, H-7, 7,8-dihydroxychlorpromazine) markedly enhanced and accelerated scattering; PMA and staurosporine also stimulated migration. Diacylglycerol analogues (e.g. diC8), naphthalenesulfonamide PKC activators (SC-9, SC-10) and inactive phorbol esters (e.g. 4a-PDD) did not potentiate scattering, while PKC depletion by 48 h pre-incubation with PMA markedly stimulated scattering. Thus, PMA-enhanced scattering may be related to down-modulation of PKC. Scattering was blocked by inhibitors of protein and RNA but not DNA synthesis; SF- and agent-stimulated migration were ablated by cycloheximide. Scattering and migration were inhibited by an anti-microfilament (cytochalasin B) but not anti-microtubule (e.g. colcemid) agents. These findings suggest that SF-induced epithelial mobility may be mediated, in part, by protein synthesis, alterations in protein phosphorylation (?inhibition of PKC), and actin filament reorganization. They indicate directions for further studies.
Asunto(s)
Citocinas/metabolismo , Citoesqueleto/efectos de los fármacos , Proteínas/farmacología , Transducción de Señal/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Calcio/metabolismo , Calmodulina/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Supervivencia Celular , Epitelio/efectos de los fármacos , Factor de Crecimiento de Hepatocito , Canales Iónicos/efectos de los fármacos , Sustancias Macromoleculares , Fotomicrografía , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Proteínas/toxicidadRESUMEN
Scatter factor (SF) is a protein produced by cultured fibroblasts which causes epithelia to "scatter" into isolated cells. We found significant scatter activity in vivo in second trimester (but not term) human amniotic fluid and in human placenta. Placental SF was purified 500,000-fold and identified as a protein with Mr 78 kd. Factor scattered Madin-Darby canine kidney and human squamous carcinoma cells at 15 pM. Amino acid sequences from tryptic peptides did not match any known protein. Human placental fibroblasts produced high titers of scatter activity. SF may be involved in development and may enhance carcinoma invasion.
Asunto(s)
Proteínas Gestacionales/análisis , Proteínas/análisis , Secuencia de Aminoácidos , Líquido Amniótico/análisis , Línea Celular , Movimiento Celular , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Femenino , Factor de Crecimiento de Hepatocito , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Embarazo , Proteínas/farmacología , Células Tumorales CultivadasRESUMEN
Recent studies have identified a group of cytokines which appear to be cell-specific regulators of mobility in nonleukocytic mammalian cells. One example is scatter factor (SF), a soluble protein(s) produced by cultured fibroblasts and vascular smooth muscle cells which causes spreading and separation ("scattering") of tight, cohesive colonies of epithelial cells. Studies of SF action have been limited because the degree of scattering is difficult to quantitate and because scattering assays cannot be used to study potential target cells that do not form tight, cohesive colonies. We developed a simple, quantitative assay of SF-stimulated mobility based on migration of target cells off microcarrier beads onto plastic culture surfaces in 24-well plates. We showed that crude and partially purified SF derived from ras-transformed 3T3 cells stimulates migration of both epithelial and vascular endothelial cells but not of producer or nonproducer fibroblasts. Scatter and migration-stimulating activities copurified on cation exchange chromatography; and the degree of stimulation was closely correlated with scattering titer regardless of SF purity. Migration of endothelial cells from beads, while extremely sensitive to SF, was not affected by serum concentration (1 to 10%), various purified growth factors, or fibronectin. Both scattering and migration from beads were blocked by cycloheximide (0.1 microgram/ml) during assay incubation, suggesting that these processes require protein synthesis. The microcarrier bead assay may be a useful quantitative tool to study the biochemical mechanisms of SF-stimulated cell migration.
Asunto(s)
Factores Biológicos/farmacología , Endotelio Vascular/citología , Fibroblastos/citología , Riñón/citología , Animales , Bovinos , Línea Celular , Línea Celular Transformada , Movimiento Celular , Cicloheximida/farmacología , Citocinas , Perros , Endotelio Vascular/efectos de los fármacos , Células Epiteliales , Epitelio/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Factor de Crecimiento de Hepatocito , Riñón/efectos de los fármacos , Métodos , Ratones , Proteínas/farmacologíaRESUMEN
Invasion of normal tissues is a complex process which requires active locomotion of malignant cells. Recent studies have identified a group of proteins which appear to be specific regulators of cell movement. Various strains and lines of fibroblast-like and vascular smooth muscle cells release into culture medium a unique protein activity which causes contiguous sheets of normal epithelial cells (e.g., Madin-Darby canine kidney, MDCK, cells) to spread and separate into individual cells (i.e., to scatter). Crude conditioned medium and partially purified MDCK scattering activity derived from human iliac artery smooth muscle cells (HIAS) scattered several lines of human squamous carcinoma cells (FaDu and A253) and markedly stimulated migration of carcinoma cells out of multicellular spheroids onto plastic culture surfaces. The scattering activities for MDCK and carcinoma cells showed similar sensitivities to temperature, trypsin treatment, and alteration of pH; both activities were blocked in the presence of cycloheximide. Unlike HIAS-derived factor, a similar MDCK scattering factor derived from ras-transformed NIH 3T3 cells did not scatter human carcinoma cells. These findings indicate that specific normal tissue-derived proteins may affect the mobility of tumor cells. Further studies of such proteins may yield insights into the mechanisms of tumor cell locomotion and tumor invasion.
Asunto(s)
Factores Biológicos/fisiología , Carcinoma de Células Escamosas/fisiopatología , Músculo Liso Vascular/fisiología , Animales , Arterias/fisiología , Movimiento Celular/fisiología , Células Clonales , Humanos , Ratones , Invasividad Neoplásica/fisiopatología , Células Tumorales CultivadasRESUMEN
Hemoglobin chains were separated and their interaction with membrane ghosts was studied using their ability to quench the fluorescence intensity of a membrane embedded probe. It was observed that alpha chains bind faster and with higher affinity to the membrane sites than do beta chains. The fast reversible interaction of both chains with the membrane was followed by a time-dependent partial loss of reversibility. Band 3 cytoplasmic fragments (B3F) were isolated and their reaction with separated Hb chains was studied using fluorescence quenching techniques as well. The data demonstrate that the relative affinity of the chains for B3F and loss of reversibility of the reaction followed patterns similar to the corresponding interaction of the chains with whole membranes. Band 3 cytoplasmic poles are therefore suggested as the high-affinity sites on the membrane for hemoglobin chains. When globin was reacted with B3F, it was observed that this protein binds strongly to the same membrane sites, but practically irreversibly. Exchange of the HbA content of normal cells by separated alpha or beta chains resulted in membrane distortions in both cases, but alpha chains caused greater morphological changes than did beta chains. The results of this study may provide one explanation for the differences in the thalassemia syndromes when excess of either alpha or beta chains is involved.
Asunto(s)
Membrana Eritrocítica/metabolismo , Hemoglobina A/metabolismo , Talasemia/sangre , Membrana Eritrocítica/ultraestructura , Humanos , Sustancias Macromoleculares , Microscopía Electrónica de Rastreo , Unión ProteicaRESUMEN
The properties of the cellulosome (the cellulose-binding, multicellulase-containing protein complex) in Clostridium thermocellum were examined by comparing the cellulase systems derived from the wild type and an adherence-defective mutant. The growth conditions--specifically, growth either on cellulose (Avicel) or on cellobiose as insoluble or soluble carbon sources, respectively--were found to be critical to the distribution of the cellulosome in the mutant system: the cellobiose-grown mutant (in contrast to the wild type) lacked the cellulosome on its surface and produced only minor quantities of the extracellular cellulosome accompanied by other relatively low-molecular-weight cellulases. The polypeptide composition of the respective purified cellulosome was dependent on the nature of the carbon source and was similar for both wild-type and mutant cells. Ultrastructural analysis revealed the presence of novel polycellulosomal protuberances on the cell surface of the cellobiose-grown wild type which were absent in the mutant.