RESUMO
Phosphorylation affects several biological functions of connexin43 (Cx43), although its role on Cx43-mediated inhibition of DNA synthesis is not known. Previous studies showed increased Cx43 phosphorylation on serine in response to growth factor stimulation of cardiomyocytes, mediated by protein kinase C-epsilon (PKCepsilon). Here we report that activation of PKCepsilon is also necessary for stimulation of cardiomyocyte DNA synthesis and mitosis. We have investigated the participation of specific serine residues that are putative PKC targets in producing phosphorylated Cx43 species and also in regulating DNA synthesis in cardiomyocytes. Interference with the PKC signaling system and/or the phosphorylation of specific amino-acids of Cx43 may allow regulation of the mitogenic response.
Assuntos
Conexina 43/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Mitose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Animais , Células Cultivadas , Clonagem Molecular , Conexina 43/genética , Replicação do DNA , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Mutação , Miócitos Cardíacos/metabolismo , Proteína Quinase C-épsilon , Ratos , Serina/metabolismoRESUMO
Fibroblast growth factor-2 (FGF-2) is a mitogen found in CUG-initiated 21-25 kDa ("hi") or AUG-initiated 16-18 kDa ("lo") forms. Previously we demonstrated that "hi"-but not "lo"-FGF-2 caused a distinct nuclear phenotype characterized by apparently condensed chromatin present as separate clumps in the nucleus of cardiac myocytes. In this manuscript we investigated whether these effects were related to apoptosis or mitosis and whether they reflected a direct effect of "hi" FGF-2 on chromatin. Myocytes overexpressing "hi" FGF-2 and presenting the clumped chromatin phenotype: (i) were not labeled above background with antibodies to phosphorylated histones H1 and H3 used as indicators of mitotic chromatin condensation; (ii) did not stain positive for TUNEL; (iii) their nuclear lamina, visualized by anti-laminB immunofluorescence, appeared intact; (iv) neither caspase inhibitors, nor Bcl-2 or "lo" FGF-2 overexpression prevented the manifestation of the compacted nuclear phenotype. Purified recombinant "hi" FGF-2 was more potent than "lo" FGF-2 in promoting the condensation/aggregation of chick erythrocyte chromatin partially reconstituted with histone H1 in vitro. We conclude that the DNA phenotype induced by "hi" FGF-2 in cardiac myocytes likely reflects a direct effect on chromatin structure that does not require the engagement of mitosis or apoptosis. By affecting chromatin compaction "hi" FGF-2 may contribute to the regulation of gene expression.
Assuntos
Cromatina/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Miocárdio/citologia , Miocárdio/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Sequência de Bases , Células Cultivadas , Galinhas , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , Primers do DNA , Eritrócitos/fisiologia , Eritrócitos/ultraestrutura , Fator 2 de Crescimento de Fibroblastos/genética , Histonas/metabolismo , Humanos , Cinética , Mitose , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , Isoformas de Proteínas/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
We have used adenoviral vectors to express dominant negative variants of protein kinase C epsilon (PKCepsilon) or mitogen kinase kinase 1 (MKK1) to investigate their involvement in phorbol ester-induced connexin-43 (Cx43) phosphorylation in cardiomyocytes. Stimulation of cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) increased the fraction of the slower migrating (> or = 45 kDa) and more extensively phosphorylated Cx43 species. Expression of dominant negative MKK1 did not prevent the effect of PMA on Cx43 phosphorylation. Selective inhibition of PKCE significantly decreased baseline levels of Cx43 phosphorylation and the PMA-induced accumulation of > or = 45 kDa Cx43. Thus, production of the more extensively phosphorylated species of Cx43 in cardiomyocytes by PMA requires activation of PKCepsilon.
Assuntos
Conexina 43/metabolismo , Isoenzimas/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Células Cultivadas , Vetores Genéticos , Isoenzimas/genética , MAP Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C-épsilon , Proteínas Serina-Treonina Quinases/genética , RatosRESUMO
Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKCepsilon displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCepsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCepsilon. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCepsilon but not PKCalpha coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCepsilon, suggesting increased association between PKCepsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCepsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKCepsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.
Assuntos
Conexina 43/metabolismo , Isoenzimas/fisiologia , Miocárdio/metabolismo , Proteína Quinase C/fisiologia , Alcaloides , Animais , Benzofenantridinas , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Junções Comunicantes/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Miocárdio/citologia , Fenantridinas/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-épsilon , Ratos , Proteínas Recombinantes/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Distribuição TecidualRESUMO
Gap junctions (GJ), composed of connexins, are membrane channels that connect and enable communication between neighboring cells and which, in addition to being essential for the coordinated electrical and contractile activity of the heart, may regulate intercellular transmission of signals affecting proliferative growth. Alterations in GJ permeability that have been associated with the regulation of growth can occur acutely through phosphorylation of connexins: fibroblast growth factor-2 (FGF-2) causes decreased coupling and increased phosphorylation of a major cardiomyocyte connexin, connexin43 (Cx43), while stimulating proliferation of cardiomyocytes. On the other hand, transforming growth factor-ß (TGFß) prevents the effects of FGF-2 on Cx43 phosphorylation, as well as canceling the FGF-2-induced proliferation. Parallel to its link with growth regulation, Cx43 phosphorylation emerges as a functionally important end point for delineating cardiac signal transduction pathways elicited by diverse physiologic or pathologic stimuli.
RESUMO
Fibroblast growth factor-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (15% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.
Assuntos
Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Contração Miocárdica/efeitos dos fármacos , Fenantridinas/farmacologia , Proteína Quinase C/fisiologia , Sarcolema/enzimologia , Alcaloides , Animais , Benzofenantridinas , Western Blotting , Cálcio/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Células Cultivadas , Citosol/efeitos dos fármacos , Densitometria , Inibidores Enzimáticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Imunofluorescência , Masculino , Membranas/efeitos dos fármacos , Perfusão , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
A sequence-specific monoclonal antibody directed against the gap junction protein connexin43 (Cx43) is shown here to be specific for the unphosphorylated form of this protein. In tissues and cultured cells containing different phosphorylated and unphosphorylated forms of Cx43, the antibody detected only the latter as shown by Western blotting of native and alkaline phosphatase-treated samples. Immunohistochemically, this monoclonal antibody did not recognize gap junctions in the vast majority of cultured cardiac myocytes, where nearly all detectable Cx43 is phosphorylated. In contrast, it was able to detect some intracellular Cx43 in tracheal smooth muscle cells and an epithelial cell line (Cl-9 cells), producing patterns of labeling consistent with those seen using a polyclonal antibody that recognizes both phosphorylated and unphosphorylated forms of Cx43. Immunostaining of gap junctions in the cultured cells indicates that both phosphorylated and unphosphorylated Cx43 are present in some assembled gap junctions, suggesting that assembled junctions do not contain exclusively the phosphorylated form of the protein. Annular gap junctions, believed to form as part of the pathway for internalization and degradation of gap junctions, were only occasionally and sparsely labeled by the monoclonal antibody, indicating that complete protein dephosphorylation is not required for uptake and degradation of gap junctions. Furthermore, the ability of this antibody to recognize only unphosphorylated Cx43, and not any of the phosphorylated forms present in the tissues and cell types examined, suggests that a unique phosphorylation site, perhaps present in the epitope recognized by this antibody, must be phosphorylated prior to phosphorylation of Cx43 at other sites.
Assuntos
Anticorpos Monoclonais , Conexina 43/imunologia , Conexina 43/metabolismo , Músculo Liso/química , Miocárdio/química , Animais , Especificidade de Anticorpos , Western Blotting , Células Cultivadas , Conexina 43/análise , Cães , Imunofluorescência , Junções Comunicantes/química , Microscopia Imunoeletrônica , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Liso/citologia , Miocárdio/citologia , Fosforilação , Coelhos , Ratos , Ratos Sprague-Dawley , Traqueia/citologiaRESUMO
Cardiac gap junction (GJ) channels, composed of connexins, allow electrical and metabolic couplings between cardiomyocytes, properties important for coordinated action of the heart as well as tissue homeostasis and control of growth and differentiation. Fibroblast growth factor-2 (FGF-2) is an endogenous growth-promoting protein, believed to participate in the short- and long-term responses of the heart to injury. We have examined short-term effects of FGF-2 on cardiac myocyte GJ-mediated metabolic coupling, using cultures of neonatal rat cardiomyocytes. FGF-2 decreased coupling between cardiomyocytes assessed by scrape dye loading as well as microinjection and dye transfer within 30 minutes of administration. Genistein blocked the effects of FGF-2. To determine the mechanism, we next assessed the effect of FGF-2 on expression, distribution, and phosphorylation of connexin43 (Cx43), which is a major cardiomyocyte connexin. FGF-2 did not affect Cx43 mRNA or protein accumulation and synthesis, and it did not change Cx43 localization at sites of intercellular contact as assessed by immunostaining with a polyclonal anti-Cx43 antibody raised against a synthetic peptide containing residues 346 to 363 of Cx43. FGF-2, however, decreased staining intensity at sites of intermyocyte contact when a monoclonal anti-Cx43 antibody was used, suggesting a localized masking of epitope(s) recognized by the monoclonal but not the polyclonal antibody. These epitopes appear to reside within residues 261 to 270 of Cx43, as indicated by full quenching of monoclonal antibody staining with synthetic peptides. In addition, FGF-2 induced a more than twofold increase in Cx43 phosphorylation. Phosphoamino acid analysis indicated increased phosphorylation of Cx43 on serine residues. Although tyrosine phosphorylation of Cx43 was not detected in either treated or control cells, a fraction of Cx43 was immunoprecipitated with anti-phosphotyrosine-specific antibodies in FGF-2-treated myocytes, suggesting interaction (and hence coprecipitation) with phosphotyrosine-containing protein(s). In conclusion, we have identified Cx43 and intercellular communication as targets of FGF-2-triggered and tyrosine phosphorylation-dependent signal transduction in cardiac myocytes. It is suggested that phosphorylation of Cx43 on serine induced by FGF-2 contributes to decreased metabolic coupling between cardiomyocytes.
Assuntos
Comunicação Celular/efeitos dos fármacos , Conexina 43/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Miocárdio/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Conexina 43/imunologia , Epitopos/imunologia , Junções Comunicantes/metabolismo , Miocárdio/citologia , Fosforilação/efeitos dos fármacos , Ratos , Proteínas Recombinantes/farmacologiaAssuntos
Fator 2 de Crescimento de Fibroblastos/biossíntese , Expressão Gênica , Coração/fisiologia , Miocárdio/metabolismo , Receptores Proteína Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/biossíntese , Envelhecimento/fisiologia , Animais , Linhagem Celular , Galinhas , Desenvolvimento Embrionário e Fetal , Fator 2 de Crescimento de Fibroblastos/fisiologia , Variação Genética , Coração/embriologia , Coração/crescimento & desenvolvimento , Humanos , Peso Molecular , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Proteínas Recombinantes/biossíntese , TransfecçãoRESUMO
Gap junctions (GJ) are membrane specializations responsible for intercellular communication and for ensuring electrical and/or metabolic coupling between cells. They are composed of connexins, a family of related proteins. Connexin-43 (Cx43) is a major connexin of the rat heart, expressed by myocytes as well as non-muscle cells. In this communication we have examined expression of Cx43 by cardiac fibroblasts and regulation of its expression by an endogenous mitogen, basic fibroblast growth factor (bFGF). Recombinant human bFGF, administered to cultured cells which had been maintained in 0.5% serum for 48 h, induced dose-dependent and statistically significant increases in Cx43 mRNA as well as protein accumulation, at 6 h after addition. Intercellular communication was also increased at 6 h but not 30 min after bFGF treatment, as assessed using a scrape-loading protocol. It is concluded that the bFGF-induced stimulation of Cx43 expression caused increased coupling between cardiac fibroblasts. This would be of importance in injured myocardium, the increased bFGF content of which might stimulate electrical coupling involving fibroblasts of the scar tissue.
Assuntos
Comunicação Celular , Conexina 43/biossíntese , Fator 2 de Crescimento de Fibroblastos/farmacologia , Junções Comunicantes/fisiologia , Regulação da Expressão Gênica , Miocárdio/metabolismo , Animais , Northern Blotting , Células Cultivadas , Conexina 43/genética , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Microscopia de Fluorescência , Miocárdio/citologia , RNA Mensageiro/análise , Ratos , Estimulação Química , Fatores de TempoRESUMO
Initiation of translation from alternate codons in the same mRNA results in multiple forms of basic fibroblast growth factor (bFGF). High molecular weight species of bFGF make use of leucine translation initiation sites located upstream of the methionine residue used to produce the 18 kiloDalton (kDa) form. Although the addition of exogenous 18 kDa bFGF is known to stimulate DNA synthesis and proliferation of several cell types including embryonic chicken cardiac myocytes, little is known about the role of high molecular weight forms of bFGF. We modified the rat bFGF cDNA to yield high (22/21.5 kDa) or low (18 kDa) molecular weight species of bFGF. Expression of 22/21.5 kDa or 18 kDa bFGF in transfected embryonic chicken ventricular myocyte cultures was confirmed by protein blotting. Expression of both high and low molecular weight species of bFGF was associated with (i) a three-fold increase in overall thymidine incorporation as well as cardiomyocyte labelling index (fraction of cardiomyocyte nuclei incorporating tritiated thymidine); (ii) a two- to three-fold increase in cell number; (iii) an eight-fold increase in protein synthesis; and (iv) a three-fold decrease in myosin accumulation. Subcellular localization of bFGF in the transfected myocyte cultures was also assessed by immunofluorescence microscopy. Over-expression of cDNAs yielding high molecular weight bFGF resulted in predominantly nuclear bFGF staining. By contrast, both cytoplasmic and nuclear staining were observed following over-expression of 18 kDa bFGF. Over-expression of 22/21.5 kDa bFGF was associated with the formation of multiple DNA-containing "clumps" resembling condensed chromatin in cardiac myocyte nuclei. These DNA "clumps" were not observed in cardiac myocyte cultures over-expressing 18 kDa bFGF. These data indicate that over-expression of high as well as low molecular weight forms of bFGF can stimulate cardiac myocyte proliferative potential and decrease myosin accumulation. However, these forms possess distinct subcellular localizations and can have different biological functions in the nucleus.
Assuntos
Núcleo Celular/ultraestrutura , Códon , Fator 2 de Crescimento de Fibroblastos/biossíntese , Regulação da Expressão Gênica , Proteínas Musculares/biossíntese , Miocárdio/metabolismo , Iniciação Traducional da Cadeia Peptídica , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Galinha , Replicação do DNA , DNA Complementar/genética , Fator 2 de Crescimento de Fibroblastos/genética , Genes Sintéticos , Microscopia de Fluorescência , Mitose , Dados de Sequência Molecular , Peso Molecular , Proteínas Musculares/genética , Miocárdio/citologia , Coelhos , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , TransfecçãoRESUMO
Control of cardiomyocyte growth and differentiation may be exercised, in part, at the level of expression of endogenous growth factors such as bFGF (basic fibroblast growth factor), believed to act locally, in an autocrine or paracrine fashion. Examination of bFGF accumulation by extract fractionation and immunoblotting indicated a 4-fold increase of an 18-kDa bFGF in adult compared to newborn rat heart ventricles. In contrast, a 22-kDa bFGF species, found to be the predominant form in newborn ventricles, displayed a 2.5-fold decrease in extracts from adult (compared to neonatal) ventricles. Since newborn rats are physiologically hypothyroid, the effect of thyroid status on the accumulation of 22-kDa bFGF in the heart was examined. A 21.5- to 22-kDa bFGF showed a 5-fold increase in extracts from hyperthyroid rat heart ventricles, compared to those from euthyroid controls. The 21.5- to 22-kDa bFGF was essentially unaffected by thyroid status in extracts from brain. Our data point to a correlation between immature or differentiated cardiac phenotype and the predominance of 21- to 22.5-kDa or 18-kDa bFGF species, respectively. Elevated levels of 21.5- to 22-kDa bFGF detected in cardiac ventricles in either physiological or experimentally induced hypothyroidism indicates that thyroid hormone may, directly or indirectly, down-regulate accumulation of the higher molecular mass forms of bFGF in the heart.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Ventrículos do Coração/metabolismo , Hipotireoidismo/metabolismo , Tiroxina/farmacologia , Animais , Animais Recém-Nascidos/fisiologia , Química Encefálica , Regulação para Baixo , Fator 2 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Ventrículos do Coração/química , Hipotireoidismo/sangue , Masculino , Peso Molecular , Fenótipo , Ratos , Ratos Sprague-Dawley , Tiroxina/sangueRESUMO
Basic fibroblast growth factor (bFGF) is a ubiquitous and multifunctional polypeptide that is believed to have a role in tissue repair and to act as a morphogen in embryonic development. Here, we have used immunohistochemical and biochemical methods with antibodies directed against the amino-terminal domain of bFGF, designated IS2, which recognize native and denatured bFGF, to demonstrate that in addition to its known intracellular and extracellular localization in heart, bFGF is also associated with cardiomyocyte gap junctions. In tissue sections, IS2 labeled regions of intercalated discs, producing an immunofluorescence pattern virtually indistinguishable from that obtained with antibodies against the heart gap junction protein connexin-43. By electron microscopy, gap junctions but not other regions of plasma membrane were heavily immunolabeled with this antibody. By solid phase immunoassay, bFGF was found to be more concentrated in a fraction enriched in cardiac gap junctions than in whole sarcolemmal preparations. Finally, an 18-kDa protein was recognized by several different antibodies specific for bFGF on Western blots of heart subcellular fractions enriched in gap junctions. We suggest that bFGF-like peptides are either an integral part of, or exist in close association with, cardiac gap junctions and thus may play a role in modulating gap junctional intercellular communication.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Junções Intercelulares/ultraestrutura , Miocárdio/metabolismo , Animais , Western Blotting , Bovinos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Fatores de Crescimento de Fibroblastos/ultraestrutura , Imunofluorescência , Microscopia Eletrônica , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
Distribution of basic-fibroblast-growth-factor-like peptides in immature cultured cardiac myocytes was investigated using specific antisera and immunolocalization. Basic FGF was detected in association with the external surface of the cell membrane, with specialized intercellular junctions and with the myofibril Z lines in the cytoplasm. Intense, punctate nuclear anti-bFGF labeling was observed in a fraction of interphase myocytes of near-confluent, proliferating cultures. This staining pattern persisted even after the dissolution of the nuclear envelope in prophase myocytes. The pattern of cellular localization of bFGF indicates a continuous participation of this factor in myocyte physiology as well as a role in the cell cycle. Furthermore, the identification of bFGF not only in cell lysates but also in culture media after gentle mechanical disruption suggests that cardiac myocytes may release bFGF in vivo following tissue damage.
Assuntos
Fator 2 de Crescimento de Fibroblastos/fisiologia , Miocárdio/citologia , Animais , Western Blotting , Ciclo Celular , Núcleo Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Imunofluorescência , Traumatismos Cardíacos/patologia , Técnicas In Vitro , Ratos , CicatrizaçãoRESUMO
Extracts from bovine pituitaries and other tissues contained basic fibroblast growth factor-like peptides of 22-26 kda, co-fractionating with smaller, 16-20 kda bFGFs. Heparin-bound, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by tryptic proteolysis. In solution, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by an activity present in pituitary extracts. Calcium protected higher molecular weight pituitary bFGFs from truncation by the endogenous activity, which was not acid-activated, co-purified with bFGF during heparin-sepharose chromatography, remained operant at high salt concentrations and was inhibited by phenylmethan-sulfonyl fluoride.
Assuntos
Cálcio/farmacologia , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Hipófise/química , Tripsina/farmacologia , Animais , Western Blotting , Cromatografia de Afinidade , Heparina , Peso Molecular , Hipófise/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
The gene katG, encoding catalase HPI of Escherichia coli, was sequenced, predicting a 726-amino-acid protein. The sequence was confirmed by identification of potential regulatory elements and amino acid sequencing of peptides. HPI shows no homology to other catalases. The distances between katG, metF, and ppc were defined.