RESUMO
J waves appear on an electrocardiogram as an elevation of the J point in the terminal portion of the QRS complex. J waves are often benign, but may be associated with malignant ventricular arrhythmias. In some cases, such problems appear to have been precipitated by propofol infusions. We observed a sudden increase in J waves and profound hypotension following a single intravenous dose of propofol in an 84-year-old woman with early repolarisation in the inferior ventricular wall. When early repolarisation (as shown by electrocardiographic J waves) is observed in the inferior ventricular wall pre-operatively, patients should be carefully monitored. Myocardial ischaemia and the use of drugs that might worsen J waves should be avoided.
Assuntos
Anestésicos Intravenosos/efeitos adversos , Complicações Intraoperatórias/induzido quimicamente , Propofol/efeitos adversos , Fibrilação Ventricular/induzido quimicamente , Idoso de 80 Anos ou mais , Eletrocardiografia/efeitos dos fármacos , Feminino , Humanos , Hipotensão/induzido quimicamente , Monitorização Intraoperatória/métodosRESUMO
We report on singlet-singlet annihilation and exciton diffusion in as-prepared p-type and annealed n-type thin films of the low-bandgap quinoidal quaterthiophene [QQT(CN)4] using ultrafast transient absorption measurements. The decay dynamics of exciton populations are well described by a one-dimensional diffusion-limited bimolecular recombination, indicating that the singlet excitons migrate preferentially along the stacking direction. Our results show that the exciton diffusion constants in QQT(CN)4 films do not vary significantly upon thermal annealing. Exciton diffusion lengths are measured to be as high as 4 and 5 nm in as-prepared and annealed QQT(CN)4 films, respectively. We also observe an influence of the excitation densities on the singlet exciton diffusion, which is attributed to phonon scattering. Because of the possibility of patterning p-n regions in QQT(CN)4 films by thermal nanolithography techniques, this study provides important insight not only into the photophysical properties of quinoidal oligothiophene derivatives but also for their future integration into high-performance p-n nanostructured near infrared light-sensing devices.
RESUMO
The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell-cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell-cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and beta-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell-cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell- cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell-cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell-cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell- cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell- cell adhesion.
Assuntos
Adesão Celular , Proteínas de Ligação ao GTP/fisiologia , Transativadores , Actinas/fisiologia , Animais , Caderinas/metabolismo , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Tamanho Celular , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Fosfoproteínas/metabolismo , Solubilidade , Transfecção , Proteína da Zônula de Oclusão-1 , beta Catenina , Proteína cdc42 de Ligação ao GTP , Proteínas rac de Ligação ao GTP , Proteínas rho de Ligação ao GTPRESUMO
Evidence is accumulating that rho p21, a ras p21-related small GTP-binding protein (G protein), regulates the actomyosin system. The actomyosin system is known to be essential for cell motility. In the present study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein (named rho GDI), its stimulatory GDP/GTP exchange protein (named smg GDS), and Clostridium botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in cell motility (chemokinesis) of Swiss 3T3 cells. We quantitated the capacity of cell motility by measuring cell tracks by phagokinesis. Microinjection of the GTP gamma S-bound active form of rhoA p21 or smg GDS into Swiss 3T3 cells did not affect cell motility, but microinjection of rho GDI into the cells did inhibit cell motility. This rho GDI action was prevented by comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 but not with the same form of rhoA p21 lacking the C-terminal three amino acids which was not posttranslationally modified with lipids. The rho GDI action was not prevented by Ki-rasVal-12 p21 or any of the GTP gamma S-bound form of other small GTP-binding proteins including rac1 p21, G25K, and smg p21B. Among these small G proteins, rhoA p21, rac1 p21, and G25K are known to be substrates for rho GDI. The rho GDI action was not prevented by comicroinjection of rho GDI with smg GDS. Microinjection of C3 into Swiss 3T3 cells also inhibited cell motility. These results indicate that the rho GDI-rho p21 system regulates cell motility, presumably through the actomyosin system.
Assuntos
Alquil e Aril Transferases , Movimento Celular , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina , Células 3T3 , Animais , Nucleotídeos de Guanina/metabolismo , Técnicas In Vitro , Camundongos , Microinjeções , Processamento Pós-Transcricional do RNA , Transferases/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico , Proteína rhoA de Ligação ao GTPRESUMO
Insulin and hepatocyte growth factor (HGF) induced morphologically different membrane rufflings in KB cells. Insulin-induced membrane ruffling was inhibited by microinjection of rho GDI, an inhibitory GDP/GTP exchange regulator for both rho p21 and rac p21 small GTP-binding proteins, but not inhibited by microinjection of botulinum exoenzyme C3, known to selectively ADP-ribosylate rho p21 and to impair its function. This rho GDI action was prevented by comicroinjection with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound rac1 p21. In contrast, HGF-induced membrane ruffling was inhibited by microinjection of rho GDI or C3. This rho GDI action was prevented by comicroinjection with GTP gamma S-bound rhoA p21, and this C3 action was prevented by comicroinjection with GTP gamma S-bound rhoAIle-41 p21, which is resistant to C3. Microinjection of either GTP gamma S-bound rac1 p21 or rhoA p21 alone induced membrane ruffling in the absence of the growth factors. The rac1 p21-induced membrane ruffling was morphologically similar to the insulin-induced kind, whereas rhoA p21-induced ruffling was apparently different from both the insulin- and HGF-induced kinds. Membrane ruffling was also induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore or microinjection of a dominant active Ki-ras p21 mutant (Ki-rasVal-12 p21). The phorbol ester-induced membrane ruffling was morphologically similar to the rhoA p21-induced kind and inhibited by microinjection of rho GDI or C3. These results indicate that rac p21 and rho GDI are involved in insulin-induced membrane ruffling and that rho p21 and rho GDI are involved in HGF- and phorbol ester-induced membrane rufflings.
Assuntos
Toxinas Botulínicas , Membrana Celular/ultraestrutura , Proteínas de Ligação ao GTP/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Insulina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , ADP Ribose Transferases/farmacologia , Membrana Celular/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Células KB , Microinjeções , Ligação Proteica , Proteínas rac de Ligação ao GTP , Proteínas rho de Ligação ao GTPRESUMO
The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.
Assuntos
Citoesqueleto/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina , Proteínas rab de Ligação ao GTP , Actinas/metabolismo , Animais , Adesão Celular , Linhagem Celular , Citoesqueleto/efeitos dos fármacos , Cães , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rab5 de Ligação ao GTP , Proteínas rac de Ligação ao GTP , Proteína rhoA de Ligação ao GTPRESUMO
Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. We investigated here the actions of ROCK and mDia, both of which have been identified to be putative downstream target molecules of Rho, in Madin-Darby canine kidney cells. The dominant active mutant of RhoA induced the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induced the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induced only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibited the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia were not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton.
Assuntos
Actinas/ultraestrutura , Proteínas de Transporte/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Rim/citologia , Proteínas Serina-Treonina Quinases/metabolismo , ADP Ribose Transferases/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Proteínas de Ligação ao GTP/genética , Genes Dominantes , Peptídeos e Proteínas de Sinalização Intracelular , Rim/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Acetato de Tetradecanoilforbol/farmacologia , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTPRESUMO
3-Hydroxy-3-methylglutaryl coenzyme A reductase activity and the rate of sterol biosynthesis are positively correlated with DNA synthesis and proliferation of mammalian cells. The total (active plus latent) activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and the activity of its active form in hepatocellular carcinoma (HCC) from seven patients were measured and compared with those in liver tissue from five control subjects. The activity of the active form in HCC was 61 +/- 21 (SD) pmol/min/mg microsomal protein, while it was only 17 +/- 9.8 pmol/min/mg protein in the liver tissue from the controls; the difference was significant (P less than 0.005). The total activity of the reductase was also higher in HCC although the difference was not significant. The microsomal contents of the enzyme protein also were not significantly different. The rate of cholesterol biosynthesis was 307 +/- 81 pmol/h/mg tissue in HCC and 79.6 +/- 52 in normal liver tissue, indicating a significant increase in the rate in HCC (P less than 0.001). Thus, enhanced synthesis of cholesterol in human HCC seems to result partly from an increase in the active form of the reductase.
Assuntos
Carcinoma Hepatocelular/metabolismo , Colesterol/biossíntese , Hidroximetilglutaril-CoA Redutases/metabolismo , Neoplasias Hepáticas/metabolismo , Microssomos Hepáticos/metabolismo , Carcinoma Hepatocelular/enzimologia , Humanos , Neoplasias Hepáticas/enzimologia , Microssomos Hepáticos/enzimologia , FosforilaçãoRESUMO
Malignant cells in culture express elevated levels of transforming growth factor beta 1 (TGF-beta 1) mRNA and secrete an abundant amount of TGF-beta protein, but little is known about the production of TGF-beta in human malignant tissues in vivo. We estimated the levels of TGF-beta 1 mRNA expression by Northern hybridization and measured TGF-beta protein using a radioreceptor assay in tumor tissues surgically obtained from six patients with hepatocellular carcinoma (HCC). TGF-beta 1 mRNA was expressed at much higher levels in HCC tissues from all the cases compared with normal human liver, suggesting an association of the activated TGF-beta 1 gene transcription with hepatocarcinogenesis. The content of TGF-beta was 207 +/- 121 ng/g wet tissue in the HCC tissue, and it showed correlation with the level of TGF-beta 1 mRNA in the tissue (r = 0.69; P less than 0.05). An immunohistochemical study demonstrated that TGF-beta 1 staining could be observed in HCC cells. These observations suggest that human HCC strongly expresses TGF-beta 1 mRNA in vivo, leading to a high content of TGF-beta protein.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/genética , Idoso , Northern Blotting , Carcinoma Hepatocelular/genética , Feminino , Humanos , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Ensaio Radioligante , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/metabolismoRESUMO
Rho small G protein regulates various actin-dependent cell functions. As to the functioning sites of Rho, Rho regulates formation of stress fibers and focal adhesions in many types of cultured cells, whereas we have shown that the association sites of actin filaments with the plasma membrane controlled by the ERM (Ezrin, Radixin, Moesin) family are the functioning sites of Rho in MDCK cells stably expressing myc-RhoA. We have investigated here the effect of microinjection of Rho GDI, a negative regulator of Rho which inhibits activation of Rho, C3, an exoenzyme of Clostridium botulinum which ADP-ribosylates Rho and inhibits its functions, or guanosine 5'-(3-O-thio) triphosphate-bound active form of Rho on the intracellular localization of both the ERM family and vinculin, which is one of the structural proteins of focal adhesions, in wild type MDCK cells. The ERM family was preferentially localized at peripheral bundles of actin filaments which are localized at the outer edge of colonies of the cells, microvilli and low Ca2+-induced cortical bundles of actin filaments in wild type MDCK cells. Microinjection of Rho GDI or C3 inhibited the localization of the ERM family at both the peripheral bundles and the low Ca2+-induced cortical bundles. On the other hand, vinculin was localized at both focal adhesions and basal edges of the colonies of the cells, and microinjection of Rho GDI or C3 inhibited the localization of vinculin at both of these sites. These results indicate that activation of Rho is necessary for the association of both the ERM family and vinculin with the plasma membrane in wild type MDCK cells. Microinjection of the guanosine 5'-(3-O-thio) triphosphate-bound form of Rho induced an increase in the localization of vinculin at focal adhesions, but did not induce an increase in the localization of the ERM family at the plasma membrane, indicating that activation of Rho itself is sufficient only for the association of vinculin with the plasma membrane at focal adhesions.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Fatores de Transcrição/fisiologia , Vinculina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Adenosina Difosfato Ribose/metabolismo , Animais , Cálcio/metabolismo , Adesão Celular , Compartimento Celular , Linhagem Celular , Membrana Celular/metabolismo , Cães , Substâncias Macromoleculares , Microvilosidades/ultraestrutura , Proteínas rho de Ligação ao GTPRESUMO
Rho small GTP-binding protein regulates various cell functions, such as formation of stress fibers and focal adhesions, cell motility, membrane ruffling, cytokinesis and smooth muscle contraction in mammalian cells and bud formation in the yeast Saccharomyces cerevisiae. As to the functioning sites of Rho in Saccharomyces cerevisiae, we have recently shown that RHO1 protein, a homologue of mammalian RhoA, is concentrated to the growth region of the cells where cortical actin patches are clustered. However, in mammalian cells, the functioning sites of Rho have not yet been studied. In the present study, MDCK cell lines stably expressing myc-tagged RhoA (myc-RhoA) were prepared and localization of myc-RhoA was first immunohistochemically examined using an anti-myc antibody. In the resting cells, almost all of myc-RhoA was observed in the cytosol. When the cells were stimulated with phorbol ester or hepatocyte growth factor, membrane rufflings were induced and myc-RhoA was translocated to the membrane ruffling area. Moreover, myc-RhoA was translocated from the cytosol to the cell-cell adhesion sites when the cells were transferred from a low to normal Ca2+ medium. RhoA was also concentrated to the cleavage furrows during cytokinesis in Swiss 3T3 cells. Translocation of myc-RhoA to the membrane ruffling area was inhibited by prior microinjection into the cells of Rho GDI, a negative regulator of Rho which inhibits activation of Rho, or of C3, an exoenzyme of Clostridium botulinum which ADP-ribosylates Rho and inhibits its functions, indicating that both activation and functioning of Rho are essential for the translocation of Rho. The ERM (Ezrin, Radixin, Moesin) family members were colocalized with RhoA at all of these sites. However, RhoA was not apparently observed at the focal adhesion plaque where vinculin was localized. These results suggest that at least one of the functioning sites of Rho is the ERM family-controlled actin filament/plasma membrane association sites.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Células 3T3 , Animais , Transporte Biológico , Adesão Celular , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Complemento C3/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Cães , Fator de Crescimento de Hepatócito/farmacologia , Camundongos , Microinjeções , Proteínas Proto-Oncogênicas c-myc/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Proteína rhoA de Ligação ao GTPRESUMO
The Rho small G protein family consists of the Rho, Rac, and Cdc42 subfamilies and regulates various cell functions through reorganization of the actin cytoskeleton. We previously showed that the Rho subfamily regulates the formation of stress fibers and focal adhesions whereas the Rac subfamily regulates the E-cadherin-based cell-cell adhesion in MDCK cells. We studied here the function of the Cdc42 subfamily, consisting of two members, Cdc42Hs and G25k, in cell adhesion, migration, and morphology of MDCK cells. For this purpose, we made and used MDCK cell lines stably expressing each of dominant active mutants of Cdc42Hs (sMDCK-Cdc42HsDA) and G25K (sMDCK-G25KDA). Actin filaments at the cell-cell adhesion sites increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Both E-cadherin and beta-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Electron microscopic analysis revealed that sMDCK-Cdc42HsDA cells tightly contacted with each other throughout the lateral membranes. Moreover, both the HGF- and TPA-induced disruption of the cadherin-based cell-cell adhesion and the subsequent cell migration were inhibited in both sMDCK-Cdc42HsDA and -G25KDA cells. Co-expression of the dominant negative mutant of Rac1, a member of the Rac subfamily, with the dominant active mutant of Cdc42Hs did not inhibit the increased accumulation of actin filaments at the cell-cell adhesion sites. These results suggest that the Cdc42 subfamily is involved in the cadherin-based cell-cell adhesion in a manner independent of the Rac subfamily. Furthermore, the cells were frequently enveloped by the large multinuclear cells in both sMDCK-Cdc42HsDA and -G25KDA cells. Video microscopic analysis revealed that the cells were engulfed by the large cells during cytokinesis.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Transativadores , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular/genética , Adesão Celular/fisiologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular/patologia , Linhagem Celular/ultraestrutura , Movimento Celular/genética , Tamanho Celular/genética , Proteínas do Citoesqueleto/metabolismo , Cães , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Rim/citologia , Rim/patologia , Rim/ultraestrutura , Mutagênese Sítio-Dirigida , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional , beta Catenina , Proteína cdc42 de Ligação ao GTP , Proteínas rac de Ligação ao GTPRESUMO
Hepatocyte growth factor (HGF) induced motility of cultured mouse keratinocytes (308R cells). This HGF-induced cell motility was inhibited by microinjection of either rho GDI, an inhibitory GDP/GTP exchange protein for rho p21 small GTP-binding protein, or a botulinum exoenzyme C3 which is known to selectively impair the function of rho p21 by ADP-ribosylating its effector domain. The rho GDI action was prevented by comicroinjection with the guanosine 5'-(3-0-thio)triphosphate (GTP gamma S)-bound active form of rhoA p21, and the C3 action was prevented by comicroinjection with a rhoA p21 mutant (rhoAIle41 p21) which is resistant to the C3 action. The HGF-induced cell motility was not inhibited by microinjection of a dominant negative rac1 p21 mutant (rac1Asn17 p21) or a dominant negative Ki-ras p21 mutant (Ki-rasAsn17 p21). Microinjection of the GTP gamma S-bound form of rac1 p21 or a dominant active Ki-ras p21 mutant (Ki-rasVal12 p21) did not induce cell motility. These results indicate that both rho p21 and rho GDI, but neither rac p21 nor ras p21, are involved in the HGF-induced cell motility. However, microinjection of the GTP gamma S-bound form of rhoA p21 alone did not induce cell motility in the absence of HGF, suggesting that activation of rho p21 is necessary but not sufficient for the HGF-induced cell motility. The HGF-induced cell motility was mimicked by 12-0-tetradecanoyl-phorbol-13-acetate, a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore. The phorbol ester-induced cell motility was also inhibited by microinjection of rho GDI or C3. These results indicate that both rho p21 and rho GDI are also involved in the phorbol ester-induced cell motility.
Assuntos
Toxinas Botulínicas , Proteínas de Ligação ao GTP/fisiologia , Fator de Crescimento de Hepatócito/farmacologia , ADP Ribose Transferases/fisiologia , Animais , Movimento Celular/efeitos dos fármacos , Camundongos , Microinjeções , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas c-met , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Proteína rhoA de Ligação ao GTPRESUMO
We have recently isolated two novel actin filament-binding proteins, l-afadin and neurabin-II and shown that they are localized at cell-cell adherens junction (AJ) in epithelial cells. We found here that l-afadin, neurabin-II, ZO-1, and E-cadherin showed similar and different behavior during the formation and destruction of cell-cell AJ in MDCK cells. In MDCK cells, the accumulation of both l-afadin and E-cadherin, but not that of ZO-1, changed in parallel depending on Rac small G protein activity. Dissociation of MDCK cells by culturing the cells at 2 microM Ca2+ caused rapid endocytosis of E-cadherin, but not that of l-afadin or ZO-1. Addition of phorbol 12-myristate 13-acetate to these dissociated cells formed a tight junction-like structure where ZO-1 and l-afadin, but not neurabin-II or E-cadherin, accumulated. We furthermore found that, in non-epithelial EL cells, which expressed E-cadherin and attached to each other, l-afadin, neurabin-II, ZO-1 and E-cadherin were all localized at AJ. In cadherin-deficient L cells, I-afadin was mainly localized at cell-cell contact sites, but ZO-1 was mainly localized at the tip area of cell processes. Neurabin-II did not accumulate at the plasma membrane area. Neither l-afadin nor neurabin-II significantly interacted with alpha-, beta-catenin, E-cadherin, ZO-1 or occludin.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Junções Íntimas/metabolismo , Transativadores , Sequência de Aminoácidos , Animais , Caderinas/metabolismo , Cálcio/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Cromatografia de Afinidade , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Cães , Endocitose/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Rim , Cinesinas , Células L , Proteínas de Membrana/fisiologia , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Miosinas , Fosfoproteínas/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Junções Íntimas/efeitos dos fármacos , Proteína da Zônula de Oclusão-1 , alfa Catenina , beta Catenina , Proteínas rac de Ligação ao GTPRESUMO
Specific receptors for brain-gut peptide hormones, cholecystokinin (CCK) and gastrin, are expressed in a variety of human tumor cells. CCK and gastrin promote the growth of NIH3T3 cells into which the CCK-B/gastrin receptor had been introduced via a eukaryotic expression vector. In this study, we have examined the effect of CCK-8 on the actin cytoskeleton by using two mouse fibroblast cell lines expressing human CCK-B/gastrin receptors. Treatment with very low concentration of CCK-8 (10(-10) M) induced the formation of actin stress fibers within one minute. Stress fiber formation increased for 30 min. In contrast, a potent mitogen for fibroblasts, platelet-derived growth factor (PDGF), initially induced membrane ruffling and, later, a weak formation of stress fibers. Microinjection of rho GDP dissociation inhibitor or Clostridium botulinum ADP-ribosyltransferase C3 which is known to impair the function of a small GTP-binding protein, rho p21, inhibited the stress fiber formation by CCK-8 as well as by PDGF. These results indicate that CCK-B/gastrin receptor could regulate stress fiber formation in a rho p21-dependent manner. The signals from CCK-B/gastrin receptor might affect cell growth as well as cell motility or adhesion by regulating the actin cytoskeleton.
Assuntos
Células 3T3/metabolismo , Actinas/biossíntese , Toxinas Botulínicas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina , Receptores da Colecistocinina/fisiologia , Células 3T3/ultraestrutura , ADP Ribose Transferases/farmacologia , Animais , Proteínas de Ligação ao GTP/fisiologia , Humanos , Camundongos , Sincalida/antagonistas & inibidores , Sincalida/farmacologia , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Both E-cadherin, a cell-cell adhesion molecule, and c-Met, the hepatocyte growth factor (HGF)/scatter factor (SF) receptor, were colocalized at cell-cell adhesion sites of MDCK cells. HGF/SF or a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced disruption of cell-cell adhesion, which was accompanied by endocytosis of both E-cadherin and c-Met. Reduction of medium Ca2+ to a micromolar range showed the same effects. Re-increase in medium Ca2+ to a millimolar range formed cell-cell adhesion, which was accompanied by exocytosis of E-cadherin and c-Met, followed by their re-colocalization at the cell-cell adhesion sites. These results suggest that E-cadherin and c-Met are colocalized at cell-cell adhesion sites and undergo co-endo-exocytosis. We have previously shown that TPA does not induce disruption of cell-cell adhesion and subsequent scattering of MDCK cells stably expressing a dominant active mutant of RhoA or Rac1 small G protein or a dominant negative mutant of Rab5 small G protein. In these cell lines, the HGF- or TPA-induced coendocytosis of E-cadherin and c-Met was inhibited, but the coendocytosis of E-cadherin and c-Met in response to reduction of medium Ca2+ was not affected. Wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, inhibited the HGF-induced disruption of cell-cell junction and endocytosis of E-cadherin and c-Met, but not the TPA-induced ones. These results suggest that disruption of cell-cell adhesion is involved in the HGF- or TPA-induced coendocytosis of E-cadherin and c-Met in MDCK cells, and that the Rho and Rab family members indirectly regulate this coendocytosis. In addition, coendocytosis of E-cadherin and c-Met in response to HGF is partly mediated by PI 3-kinase. The cross-talk between cell-cell and cell-matrix adherens junctions is discussed.
Assuntos
Caderinas/metabolismo , Adesão Celular/fisiologia , Endocitose/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Meios de Cultura , Cães , Inibidores Enzimáticos/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various actin cytoskeleton-dependent cell functions. The Rho subfamily members regulate ERM (ezrin, radixin and moesin)-dependent association of the actin cytoskeleton with the plasma membrane. Moreover, the N-terminal regions of ERM interact with Rho GDI, an inhibitory regulator of all the Rho family members, and reduce its inhibitory action, finally initiating the activation of the Rho family members. We show here that the N-terminal region of radixin furthermore interacts with Dbl, a stimulatory GDP/GTP exchange protein of the Rho family members. This interaction does not affect the Dbl activity to stimulate the GDP/GTP exchange reaction of RhoA, a member of the Rho subfamily. Dbl does not interact with radixin which is precomplexed with Rho GDI, and Rho GDI displaces Dbl from radixin. Thus, radixin plays an important role in activation of the Rho family members by recruiting their positive and negative regulators.
Assuntos
Proteínas Sanguíneas/química , Proteínas do Citoesqueleto , Inibidores de Dissociação do Nucleotídeo Guanina , Proteínas de Membrana/química , Proteínas Oncogênicas de Retroviridae/química , Sítios de Ligação , Proteínas Sanguíneas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Oncogênicas de Retroviridae/metabolismo , Fator Rho/química , Fator Rho/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
A simple and versatile solution-processing method based on molecular self-assembly is used to fabricate organic single crystal microwires of a low bandgap quinoidal oligothiophene derivative. Individual single crystal microwire transistors present well-balanced ambipolar behaviour with hole and electron mobilities as high as 0.4 and 0.5 cm(2) V(-1) s(-1), respectively.
Assuntos
Elétrons , Tiofenos/química , Transistores Eletrônicos , Cristalização , Nanofios/química , Nanofios/ultraestrutura , SoluçõesRESUMO
Cholesterol efflux (CE) is the initial and important step of reverse cholesterol transport (RCT), a major protective system against atherosclerosis. However, most of the molecular mechanism for CE still remains to be clarified. In the present study, cDNA subtraction revealed that the expression of a member of the Rho GTPase family, Cdc42Hs, was markedly decreased in both passaged fibroblasts and macrophages (Mφ) from patients with Tangier disease (TD), a rare lipoprotein disorder with reduced CE. This small G protein is known to have many cell biological activities such as rearrangement of actin cytoskeleton and vesicular transport, however the association between this molecule and lipid transport has never been reported. We demonstrate that MDCK cells expressing the dominant negative form of Cdc42Hs had reduced CE, inversely ones expressing the dominant active form had increased CE. From these observations, we would like to raise a novel hypothesis that this type of small G protein may play a role in some steps of CE. To our knowledge, the present study is the first demonstration that the expression of this molecule is altered in cells from human disease.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Doença de Tangier/enzimologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cães , Fibroblastos/enzimologia , Biblioteca Gênica , Humanos , Macrófagos/enzimologia , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/genética , Pele/enzimologia , Doença de Tangier/genética , Transfecção , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Behavioral and electrophysiological methods were used to investigate the hyperalgesia and allodynia, and functional changes in lumbar spinal dorsal horn neurons, in a model of neuropathic pain (Selzer et al. 1990) involving ligation of one-third to one-half of one sciatic nerve in rats. One and 5 weeks following ligation, there was a significant reduction in hind limb withdrawal latency to noxious radiant heat on the operated side and, to a lesser degree, on the unoperated side. By 16 weeks, heat withdrawal latencies were reduced about equally (approximately 40%) on both sides. Withdrawal threshold to mechanical pressure was markedly reduced within 1 week on the operated side, and decreased in a time-dependent manner on the unoperated side. Heat withdrawal latency and von Frey withdrawal thresholds were not significantly affected in sham-operated rats. The same rats were tested in a paradigm measuring the isometric force of hind limb withdrawals elicited by graded noxious contact heat stimuli (38-52 degrees C, 5 sec). Withdrawal force increased monotonically with stimulus temperature starting at a threshold of approximately 40 degrees C. Stimulus-response functions were not significantly different between a sham-operated group and groups tested 5 (acute) and 16 weeks (chronic) after partial sciatic nerve ligation. Following behavioral testing, the animals were deeply anesthetized with pentobarbital sodium to allow electrophysiological recording of responses of single lumbar dorsal horn wide-dynamic range-type neurons to mechanical and noxious thermal stimulation of the hind paw. Recordings were made from 6 sham-operated rats (26 neurons ipsilateral and 31 contralateral to the operated leg), from 7 rats receiving partial sciatic nerve ligation 5 weeks previously (29 ipsilateral and 29 contralateral to ligation), and from 7 rats receiving partial sciatic ligation 16 weeks previously (18 ipsilateral, 29 contralateral to ligation). In several ligated rats we were unable to find heat-responsive neurons with cutaneous receptive fields on the hind paw ipsilateral to the ligation. For the neurons that were sensitive to heat, responses increased monotonically from a threshold of 40-42 degrees C. Neuronal stimulus-response functions for heat were not significantly different between ipsi- and contralateral (to operated) sides in the sham, 5-week or 16-week post-ligation groups, or between sham and 5- or 16-week post-ligation groups. Mechanical receptive field areas were not significantly different between ipsi- and contralateral sides in the sham and 5-week post-ligation groups, or between sham and 5-week post-ligation groups. However, receptive field areas were significantly larger in the 16-week post-ligation group (both ipsi- and contralateral to ligation) compared to sham and 5-week post-ligation groups. The results suggest that allodynia may be associated with a chronic enhancement of neuronal mechanosensitivity, but that the thermal hyperalgesia is not associated with enhanced neuronal responsiveness or force of withdrawal.