RESUMO
The only light source for chickens in environmentally controlled houses is an artificial one. Thus, source, spectra, intensity and regimen of light supplementation became major factors in modern meat type bird management. Light spectra affect growth in meat type birds both in ovo and post hatch. Broilers photostimulated in ovo with green light gained significantly more weight than birds incubated under dark conditions. Furthermore, we defined the cellular and molecular events associated with the effect of in ovo green photostimulation on muscle growth. We found that in ovo photostimulation have a stimulatory effect on the proliferation and differentiation of satellite cells and a promoting effect on the uniformity of the muscle fibers in the early post-hatch period. How does in ovo photostimulation affect intracellular events, such as proliferation and differentiation of muscle cells, leading to post-hatch muscle growth? It is possible that the monochromatic green light penetrates the eggshell and has a direct effect on the embryo's muscle. We were unable to detect any proliferative effect of monochromatic green light on cultured myoblasts derived from standard (un-illuminated) E17 embryos and 3-day-old chicks. A more likely explanation is that green light indirectly affects myoblast proliferation by activating the endocrine system; the latter receives photic cues from the retinal or extra-retinal photoreceptors. We gathered some evidence to support these findings; we have shown a higher expression of growth hormone (GH) receptor mRNA in satellite cells derived from green light illuminated chicks. In addition, plasma GH levels and IGF-I levels in muscle tissue, were higher in the green group relative to the dark one in early post-hatch. Another possible explanation for this phenomenon could be that growth factor secretion is activated in response to green light photostimulation. Both retinal and extra-retinal photoreceptors are active during embryogenesis and can be first detected at E14. Combinations of in ovo and post-hatch green light photostimulation to broilers and turkeys did not cause synergetic effect on growth. In a recent study, we found that in ovo green light photostimulation suppresses the green and red opsin receptors gene expression in the last three days before hatching, while red light enhances their expression. Furthermore, we found that the down-regulation of the green and red opsins in response to incubation under monochromatic green lighting lasted up to 9days post hatch, suggesting a possible epigenetic effect.
Assuntos
Aves/fisiologia , Células Fotorreceptoras de Vertebrados/metabolismo , Animais , Aves/metabolismo , Peso Corporal/fisiologia , Galinhas , Masculino , Receptores da Somatotropina/metabolismoRESUMO
Sonic Hedgehog (Shh) has been shown to promote adult myoblast proliferation and differentiation and affect Akt phosphorylation via its effector Smoothened (Smo). Here, the relationship between Shh and insulin-like growth factor I (IGF-I) was examined with regard to myogenic differentiation via signaling pathways which regulate this process. Each factor enhanced Akt and MAPK/ERK (p42/44) phosphorylation and myogenic factor expression levels in a dose-responsive manner, while combinations of Shh and IGF-I showed additive effects. Blockage of the IGF-I effects by neutralizing antibody partially reduced Shh's effects on signaling pathways, suggesting that IGF-I enhances, but is not essential for Shh effects. Addition of cyclopamine, a Smo inhibitor, reduced Shh- and IGF-I-induced Akt phosphorylation in a similar manner, implying that Shh affects gain of the IGF-I signaling pathway. This implication was also examined via a genetic approach. In cultures derived from Smo(mut) (MCre;Smo(flox/flox)) mice lacking Smo expression specifically in hindlimb muscles, IGF-I-induced Akt and p42/44 phosphorylation was significantly reduced compared to IGF-I's effect on Smo(cont) cells. Moreover, remarkable inhibition of the stimulatory effect of IGF-I on myogenic differentiation was observed in Smo(mut) cultures, implying that intact Smo is required for IGF-I effects in myoblasts. Immunoprecipitation assays revealed that tyrosine-phosphorylated proteins, including the regulatory unit of PI3K (p85), are recruited to Smo in response to Shh. Moreover, IGF-IR was found to associate with Smo in response to Shh and to IGF-I, suggesting that Shh and IGF-I are already integrated at the receptor level, a mechanism by which their signaling pathways interact in augmenting their effects on adult myoblasts.
Assuntos
Proteínas Hedgehog/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Desenvolvimento Muscular/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Proteínas Hedgehog/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Desenvolvimento Muscular/genética , Mioblastos/citologia , Mioblastos/metabolismo , Miogenina/metabolismo , Fator de Transcrição PAX7/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Cross-Talk , Receptor IGF Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Receptor SmoothenedRESUMO
Chicks are commonly fasted for the first 36 to 72 h posthatch because of the logistics of commercial production. Fasting for 48 to 72 h posthatch results in retarded BW, delayed intestinal development, and lower pectoral muscle weight. This study is focused on the first 36 h of fasting and its interaction with feeding before hatch. Four treatment groups, differing in time of first feed, 6 h [early feeding (EF)] or 36 h [standard feeding procedure (SP)] posthatch, with or without in ovo feeding (IOF) with dextrin and ß-hydroxy-ß-methylbutyrate-calcium salt in a saline solution, were examined for glycogen status in the liver and pectoral muscle, myogenic cell proliferation, and myofiber diameter in embryos and chickens on various days posthatch. In addition, chicken BW, ADG, pectoral muscle weight, and pectoral muscle percentage of BW until 35 d of age were recorded. Results showed that delaying the first feed for 36 h posthatch (SP group) led to an irreversibly reduced growth rate compared with the EF group. However, IOF affected the growth of chickens in the SP group, whereas the control embryos had depleted glycogen reserves in the liver; IOF-treated embryos had elevated hepatic glycogen contents on embryonic day (E) 19, E20, and the day of hatch. In addition, on d 2 posthatch, although hatchlings in the SP group showed the predicted low levels of glycogen in their livers, birds in the EF group exhibited more than 30-fold and 3-fold increases in liver and muscle glycogen, respectively. In ovo-fed birds in the SP group also exhibited higher glycogen reserves, BW, pectoral muscle weight, and BW gain than control birds in the SP group. In ovo feeding had an immediate effect on promoting myoblast proliferation on E19, whereas on d 3 posthatch, the effect was pronounced only in the EF groups. On d 5, although myoblast proliferation in all groups declined, it remained higher in both IOF groups. These effects were expressed on d 3 and 35 by myofiber diameter. Together, IOF had a long-term supportive effect on BW and posthatch muscle growth when first feed was delayed by 36 h.
Assuntos
Criação de Animais Domésticos/métodos , Embrião de Galinha/metabolismo , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Métodos de Alimentação/veterinária , Glicogênio/metabolismo , Animais , Peso Corporal/fisiologia , Dextrinas/metabolismo , Ingestão de Alimentos/fisiologia , Feminino , Análise dos Mínimos Quadrados , Fígado/metabolismo , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Tamanho do Órgão/fisiologia , Valeratos/metabolismoRESUMO
In its wild-type form, the protein p53 can interfere with neoplastic processes. Tumor-derived cells often express mutant p53. Full-length mutant forms of p53 isolated so far from transformed mouse cells exhibit three common properties in vitro: loss of transformation-suppressing activity, gain of pronounced transforming potential, and ability to bind the heat shock protein cognate hsc70. A tumor-derived mouse p53 variant is now described, whose site of mutation corresponds to a hot spot for p53 in human tumors. While absolutely nonsuppressing, it is only weakly transforming and exhibits no detectable hsc70 binding. The data suggest that the ability of a p53 mutant to bind endogenous p53 is not the sole determinant of its oncogenic potential. The data also support the existence of gain-of-function p53 mutants.
Assuntos
Transformação Celular Neoplásica , Mutação , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/genética , Animais , Clonagem Molecular , Humanos , Camundongos , Plasmídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Ratos , Transfecção , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and terminal withdrawal from the cell cycle. This cell cycle arrest in the G0 phase requires the retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a skeletal muscle-specific transcriptional regulator, activated the expression of the Cdk inhibitor p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated induction of p21 did not require the tumor suppressor protein p53 and correlated with cell cycle withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle differentiation by increasing the expression of p21.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Ciclo Celular , Diferenciação Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/biossíntese , Proteínas de Ligação a DNA , Músculo Esquelético/citologia , Proteína MyoD/fisiologia , Proteínas Supressoras de Tumor , Animais , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/genética , Fatores de Transcrição E2F , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fase de Repouso do Ciclo Celular , Proteína do Retinoblastoma/fisiologia , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologiaRESUMO
This study aimed to elucidate the effects of thermal manipulations (TM) of broiler embryos, during the development of the thyroid and adrenal axis, on embryo development and metabolism. Cobb eggs were divided into 3 treatments: control, 24H-continuous TM at 39.5 degrees C and 65% RH from embryonic day 7 to 16 inclusive, and 12H-intermittent TM for 12 h/d in the same period. Only the 24H treatment negatively affected embryo growth and development, with lower relative weights of embryo, liver, and pipping muscle. During TM, eggshell temperature, heart rate, and oxygen consumption were elevated as embryos were in their ectothermic phase, but from the end of the TM until hatch, these parameters were significantly lower in both treatments than in the control. Moreover, plasma concentrations of the thyroid hormones were significantly lower in the 2 treatments during and after TM, until hatch. Plasma corticosterone concentration of the TM-treated embryos was significantly lower after the TM but significantly higher at hatch. It was concluded that TM during the development of the thyroid and adrenal axis lowered their functional set point, thus lowering metabolic rate during embryogenesis and at hatch.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Embrião de Galinha/fisiologia , Temperatura , Adaptação Fisiológica , Animais , Corticosterona , Desenvolvimento Embrionário/fisiologia , Consumo de Oxigênio , Hormônios Tireóideos/metabolismoRESUMO
Rapid growth rate has presented broiler chickens with serious difficulties when called on to thermoregulate efficiently in hot environmental conditions. Altering the incubation temperature may induce an improvement in the acquisition of thermotolerance (AT). This study aimed to elucidate the effect of thermal manipulations (TM) during the development of the thyroid and adrenal axis of broiler embryos on the potential of broilers to withstand acute thermal stress at marketing age. Cobb broiler embryos were subjected to TM at 39.5 degrees C and 65% RH from embryonic day 7 to 16 (inclusive), either continuously (24 h) or intermittently (12 h). After hatching chicks were raised under standard conditions to 35 d of age and then subjected to thermal challenge (35 degrees C for 5 h). Continuous TM caused a significant decline in hatchability, coupled with significantly lower BW and body temperature at hatching. The intermittent (12-h) chicks showed results similar to the controls but had significantly lower body temperature. Thermal challenge at marketing age demonstrated a significant improvement in AT in both the 12- and 24-h TM-treated broilers, which was characterized by a significantly lower level of stress (as evidenced by the level of plasma corticosterone) and rate of mortality. It was concluded that TM during the portion of embryogenesis when the thyroid and adrenal axis develop and mature had a long-lasting effect and improved the AT of broiler chickens. Whereas intermittent TM had no significant effect on hatchability and performance parameters, continuous TM negatively affected these parameters.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Embrião de Galinha/embriologia , Desenvolvimento Embrionário/fisiologia , Glândulas Suprarrenais/fisiologia , Animais , Peso Corporal/fisiologia , Corticosterona/sangue , Feminino , Sistema Hipotálamo-Hipofisário/fisiologia , Masculino , Glândula Tireoide/fisiologia , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
The transformation-related protein p53 is normally very labile. The stability of p53 is significantly increased in a number of fibrosarcoma cell lines derived from mouse tumors induced by treatment with physical or chemical agents. In many instances, p53 stabilization is correlated with the ability to form a stable complex with the heat shock protein cognate hsc70. We describe a line in which p53 is very stable yet has no detectable interaction with hsc70. The inability to form such a complex probably resides in the primary structure of the endogenous p53, since introduction of other p53 variants into those cells resulted in the appearance of a p53-hsc70 complex. The factors affecting p53 stability were investigated by stable transfection experiments. The results indicated that the primary structure of the p53 protein is a major determinant of its turnover rate; different p53 variants were degraded at distinct and characteristic rates in a number of transformed cell types. However, at least one p53 variant was degraded differently in nontransformed BALB/c-3T3 than in transformed fibrosarcoma cells, demonstrating that the specific cellular environment can also affect the stability of p53.
Assuntos
Proteínas de Choque Térmico HSP70 , Proteínas Oncogênicas/metabolismo , Fosfoproteínas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Fibrossarcoma , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico/metabolismo , Camundongos , Plasmídeos , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53RESUMO
Patterns of p53 expression were investigated in chemically induced fibrosarcoma tumors and cell lines. Most, if not all, cell lines were found to carry alterations at the protein level, reflected in the overproduction of greatly stabilized p53 proteins. In many cases, this was accompanied by formation of complexes with hsc70. Hence, all of these lines may be expressing one sort or another of mutant p53. The mutant nature of the p53 gene was directly verified, in a number of cases, by PCR-amplified cDNA cloning. In one line, no p53 protein was made at all; this turned out to be because of a mutation in a splice donor site, resulting in the production of an aberrant mRNA. In all other cases, mRNAs carrying mis-sense mutations were present, and were sometimes expressed along with wt p53 mRNA. When tested in an in vitro transformation assay, all cloned mutants possessed a discrete oncogenic activity, while having lost the ability to interfere with oncogene-mediated transformation. The system described here could potentially be very helpful in elucidating the significance of p53 mutations.
Assuntos
Fibrossarcoma/genética , Mutagênese , Sarcoma Experimental/genética , Proteína Supressora de Tumor p53/genética , Animais , Sequência de Bases , Linhagem Celular , Transformação Celular Neoplásica , DNA de Neoplasias/genética , Fibrossarcoma/induzido quimicamente , Metilcolantreno , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Splicing de RNA , RNA Mensageiro/genética , Sarcoma Experimental/induzido quimicamenteRESUMO
Prostaglandin and hydroxyeicosatetraenoic acid (HETE) production from arachidonic acid in bovine seminal vesicles and kidney as influenced by the addition of beta-carotene, retinol or alpha-tocopherol was studied. The major product formed was prostaglandin E2 (approximately 85% prostaglandin E2 of control), and its proportion decreased with increasing concentration of the additives, while the proportion of HETE increased. Prostaglandin and HETE production was considerably inhibited by beta-carotene and retinol, and to a lesser extent by alpha-tocopherol; HETE production was inhibited less than that of prostaglandin. It appears that beta-carotene, retinol and alpha-tocopherol influence both the cyclooxygenase and lipoxygenase pathways; this modulation of arachidonic acid oxidation by physiological compounds may have important in vivo implications.
Assuntos
Ácidos Araquidônicos/metabolismo , Carotenoides/farmacologia , Vitamina A/farmacologia , Vitamina E/farmacologia , Animais , Ácido Araquidônico , Bovinos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Técnicas In Vitro , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Oxirredução , Prostaglandinas/biossíntese , Glândulas Seminais/efeitos dos fármacos , Glândulas Seminais/metabolismo , beta CarotenoRESUMO
The effect of halofuginone--a plant alkaloid used as a coccidiostat in birds--on collagen metabolism was studied in various avian and mammalian cell cultures. In avian skin fibroblasts halofuginone attenuated the incorporation of [3H]proline into collagenase-digestible proteins (CDP) at concentrations as low as 10(-11) M, without affecting production of [3H]collagenase-nondigestible proteins (NCDP), cell proliferation or collagen degradation. Halofuginone depressed specifically the expression of alpha 1 gene of collagen type I but not that of collagen type II. This was demonstrated in skin fibroblasts and growth-plate chondrocytes using probes containing inserts sequences corresponding to the alpha 1(I) and alpha 1(II) mRNAs. A slight inhibition of the expression of alpha 2(I) was observed in avian skin fibroblasts but not in growth-plate chondrocytes. The inhibition of gene expression of both polypeptides of collagen type I in skin fibroblasts resulted in a decrease in synthesis, as demonstrated by immunoprecipitation with specific type I collagen antiserum. In primary cultures of mouse skin fibroblasts, avian epiphyseal growth plate chondrocytes and a rat embryo cell line--all of which produce and secrete collagen type I--halofuginone inhibited the incorporation of [3H]proline into CDP, the Rat-1 line being the most sensitive to the drug. These results suggest that halofuginone affects specifically type I collagen synthesis by repressing gene-expression. The need for extremely low concentrations of halofuginone to inhibit collagen type I synthesis, regardless of the tissue or animal species, contributes to the potential usefulness of the substance in studying collagen metabolism.
Assuntos
Coccidiostáticos/farmacologia , Colágeno/biossíntese , Quinazolinas/farmacologia , Animais , Aves , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Colágeno/genética , Embrião de Mamíferos , Embrião não Mamífero , Fibroblastos , Expressão Gênica/efeitos dos fármacos , Lâmina de Crescimento , Inibidores de Metaloproteinases de Matriz , Camundongos , Piperidinas , Prolina , Quinazolinonas , Ratos , Especificidade da EspécieRESUMO
The role of hepatocyte growth factor (HGF) and its receptor, c-met, in proliferation and differentiation of satellite cells was studied in primary cultures of chicken skeletal muscle satellite cells and a myogenic C2 cell line. HGF mRNA was expressed mainly in the myotubes of both cultures. The addition of conditioned medium derived from those cultures had a scattering effect on the canine kidney epithelial cell line, MDCK. In contrast, c-met mRNA levels decreased during cell differentiation of C2 and primary satellite cells. Application of exogenous HGF to chicken myoblasts resulted in their enhanced DNA synthesis. Among several growth factors, HGF was the first to induce DNA synthesis in quiescent satellite cells, thereby driving them into the cell cycle. Ectopic expression of chicken HGF in primary satellite cells suppressed the activation of muscle-regulatory gene reporter constructs MCK-CAT, MRF4-CAT, MEF2-CAT and 4Rtk-CAT, as well as the gene expression of MyoD and myogenin, and MHC protein expression. Ectopic MyoD reversed HGF's inhibitory effect on MCK transactivation. These data suggest that HGF inhibits cell differentiation by inhibiting the activity of basic helix-loop-helix (bHLH)/E protein heterodimers, thus inhibiting myogenic determination factor activity and subsequent muscle-specific protein expression. During muscle growth and regeneration, HGF plays a dual role in satellite-cell myogenesis, affecting both the proliferation and differentiation of these cells in a paracrine fashion.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Fator de Crescimento de Hepatócito/fisiologia , Músculo Esquelético/citologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Galinhas , DNA/biossíntese , Cães , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento de Hepatócito/biossíntese , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Rim , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/biossíntese , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteínas Proto-Oncogênicas c-met/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/farmacologia , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rat's age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.
Assuntos
Lasers , Músculo Esquelético/citologia , Músculo Esquelético/efeitos da radiação , Fatores Etários , Animais , Diferenciação Celular/efeitos da radiação , Divisão Celular/efeitos da radiação , Fusão Celular/efeitos da radiação , Células Cultivadas , Ciclinas/metabolismo , Expressão Gênica/efeitos da radiação , Técnicas In Vitro , Complexo Principal de Histocompatibilidade , Camundongos , Músculo Esquelético/fisiologia , Ratos , Regeneração/efeitos da radiaçãoRESUMO
The effect of halofuginone (a plant alkaloid) on collagen alpha 1(I) gene expression and collagen synthesis was evaluated in human skin fibroblasts from patients with chronic graft-versus-host disease (cGvHD) or scleroderma and from a normal individual. Halofuginone caused a dose-dependent inhibition in collagen alpha 1(I) gene expression and collagen synthesis in all cultures tested, the cGvHD fibroblasts being the least sensitive. In normal and scleroderma fibroblasts, concentrations of halofuginone as low as 10(-10) M and 10(-9) M were sufficient to cause a significant reduction in collagen alpha 1(I) gene expression and collagen synthesis, respectively. In addition, halofuginone also inhibited the transforming growth factor beta-induced collagen synthesis. Three days after halofuginone removal, collagen gene expression returned to control levels. The reduction of collagen mRNA transcript levels by halofuginone appeared to be dependent on new protein synthesis because simultaneous treatment of fibroblasts with protein synthesis inhibitors prevents the suppressive effect of halofuginone on collagen alpha 1(I) mRNA gene expression. The ability of extremely low concentrations of halofuginone to inhibit collagen alpha 1(I) synthesis specifically and transiently at the transcriptional level suggests that this material may be an important tool for studying collagen alpha 1(I) gene regulation and may be used as a novel and promising antifibrotic therapy.
Assuntos
Coccidiostáticos/farmacologia , Colágeno/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Doença Enxerto-Hospedeiro/tratamento farmacológico , Quinazolinas/farmacologia , Adulto , Colágeno/biossíntese , Relação Dose-Resposta a Droga , Feminino , Humanos , Pessoa de Meia-Idade , Piperidinas , Quinazolinonas , Fatores de TempoRESUMO
Avian skin fibroblasts were isolated, cultured and incubated with [3H]proline for 24 h. The cells exported radiolabeled collagenase-digestible (CDP) and non-collagenase-digestible (NCDP) proteins into the medium. Human, bovine and avian growth hormone (GH) as well as insulin-like growth factor I (IGF-I) attenuated the appearance of [3H]CDP in the medium without affecting [3H]NCDP. The appearance of [3H]CDP was not affected by prolactin. The effects of GH and IGF-I were enhanced by increasing concentrations of fetal calf serum (FCS). A synergism was observed between GH and IGF-I in their effect on CDP. Each peptide, at an ineffective concentration, increased the sensitivity of the cells to the other peptide. Collagenase activity in the medium was enhanced by IGF-I, but not modified by GH, FCS, or by their interaction with IGF-I. GH and IGF-I inhibition of type I procollagen gene expression was demonstrated with the aid of probes containing sequences corresponding to the mRNAs for avian alpha I and alpha II chains. The results suggest that GH and IGF-I cooperate in regulating collagen synthesis, but collagen degradation is affected by IGF-I and not by GH.
Assuntos
Colágeno/genética , Regulação da Expressão Gênica , Hormônio do Crescimento/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Animais , Bovinos , Células Cultivadas , Galinhas , Colágeno/biossíntese , Fibroblastos , Humanos , Colagenase Microbiana/metabolismo , Prolactina/fisiologiaRESUMO
Avian chondrocytes and fibroblasts, derived from epiphyseal growth-plate and skin, respectively, were cultured in vitro. In chondrocytes, epidermal growth factor (EGF) caused a dose-dependent stimulation of proliferation. EGF receptor mRNA was not detected with the v-erb B probe in chondrocytes cultured in the presence of 5% fetal calf serum (FCS). In the absence of FCS in the medium, a time-dependent increase in the level of EGF receptor mRNA was observed. Parallel changes were also observed in the level of EGF receptor, as demonstrated by immunofluorescence using antibodies directed against avian EGF receptor. In avian fibroblasts, EGF receptor mRNA and EGF receptor levels were not affected by FCS. Furthermore, FCS did not affect the level of thyroid hormone receptor mRNA (using v-erb A as a probe) in either chondrocytes or fibroblasts. Parathyroid hormone (PTH), which acts as a mitogen in avian chondrocytes attenuated--whereas atrial natriuretic peptide (ANP), a suppressor of chondrocyte proliferation, enhanced--EGF receptor mRNA. The present results show that avian growth-plate chondrocytes respond to EGF and bear EGF receptors. The levels of EGF mRNA and EGF receptor are inversely related to cell proliferation. The results also support previous suggestions that PTH and ANP play important roles in chondrocyte proliferation, possibly through their effect on the synthesis of the EGF receptor.
Assuntos
Fator Natriurético Atrial/farmacologia , Receptores ErbB/genética , Lâmina de Crescimento/metabolismo , Hormônio Paratireóideo/farmacologia , Animais , Aves , Sangue , Divisão Celular , Células Cultivadas , Imunofluorescência , Expressão Gênica , Lâmina de Crescimento/citologia , Humanos , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
The effect of chicken growth hormone (cGH) on the proliferation and differentiation of avian growth-plate chondrocyte was evaluated in culture. In culture, addition of ascorbic acid to the culture media caused cell differentiation. Treatment of proliferating chondrocytes with cGH caused a time-dependent increase in collagen type II gene expression together with a decrease in the appearance of osteopontin (OPN) in the medium. In addition, the ascorbic acid-dependent increase in alkaline phosphatase (AP) activity was inhibited by cGH. IGF-I, on the other hand, caused an increase in AP activity in the ascorbic acid-treated chondrocytes. In the presence of ascorbic acid, cGH did not affect collagen type II gene expression or the appearance of OPN in the medium. Proliferation of avian growth-plate chondrocytes, in contrast to mammalian chondrocytes, was not stimulated by GH alone, although the presence of cGH was essential for chondrocyte survival in long-term culture. cGH in combination with epidermal growth factor (EGF) stimulated cell proliferation. These results suggest that GH inhibits differentiation in avian growth-plate chondrocytes, thereby sustaining their proliferative state and maintaining their sensitivity to growth factors such as EGF.
Assuntos
Hormônio do Crescimento/farmacologia , Lâmina de Crescimento/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Ácido Ascórbico/farmacologia , Desenvolvimento Ósseo/efeitos dos fármacos , Desenvolvimento Ósseo/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Colágeno/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lâmina de Crescimento/citologia , Lâmina de Crescimento/crescimento & desenvolvimento , Osteopontina , Sialoglicoproteínas/metabolismoRESUMO
Human growth hormone (hGH) inhibits alpha 1(I) collagen gene expression in cultured avian skin fibroblasts resulting in a decrease in the amount of collagenase-digestible proteins (CDP) in the medium. In addition, a synergism exists between GH and insulin-like growth factor-I (IGF-I) in their effect on CDP. Four N-terminal modified hGH analogs were tested for their ability to affect collagen metabolism in these cells. The truncated analog Des-7 hGH(R8M, D11A) was found to be a strong antagonist of the hGH-induced inhibition of the collagen synthesis but by itself did not inhibit collagen alpha 1(I) gene expression or modify the CDP appearance in the medium. Some synergism between Des-7 hGH and IGF-I was observed. The analog Met-hGH(R19H, L20P), in which Arg19 was replaced by histidine, and Leu20 by proline was only partially potent compared with the native hormone in causing inhibition of collagen gene expression, in attenuating CDP appearance in the medium, and in antagonizing hGH. However, this analog was as potent as hGH in its ability to synergize with IGF-I. The importance of His18 was assessed by testing the response to Met-hGH(H18D), in which His18 was replaced by Asp, and to Met-hGH(H18Q), in which His18 was replaced by glutamine (as in chicken GH sequence). Substitution of His18 by a negatively charged amino acid abolished all the hormone activities tested whereas substitution with glutamine restored only part of the activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Colágeno/biossíntese , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/análogos & derivados , Pele/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Galinhas , Sinergismo Farmacológico , Fibroblastos/metabolismo , Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Dados de Sequência Molecular , Pele/citologia , Pele/metabolismo , Relação Estrutura-AtividadeRESUMO
Several physical aspects of a high molecular weight lipid-protein aggregate separated by gel chromatography from chick and rat liver cytosol and its possible role in intracellular fatty acid metabolism were investigated. Electron microscopic examination of the high molecular weight lipid-protein aggregate indicated spherical particles with a diameter range of 200-600 A. This structure is consistent with a microemulsion particle of triglyceride encapsulated by phospholipid and protein. Uptake of fatty acids by microsomes occurred from the same lipid-protein aggregate, and the triglycerides synthesized in microsomes also became associated with these particles in the cytosol. The lipid-protein aggregate prepared by different homogenization methods showed identical ratios of components, but these ratios changed following incubation. These findings lend support to the concept that this aggregate plays a physiological role in intracellular lipid metabolism, and may be identifiable with previously reported subcellular fatty acid and triglyceride pools.
Assuntos
Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Fígado/análise , Proteínas/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Galinhas , Citosol/análise , Lipídeos/isolamento & purificação , Substâncias Macromoleculares , Masculino , Microscopia Eletrônica , Microssomos Hepáticos/metabolismo , Peso Molecular , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Tamanho da Partícula , Proteínas/isolamento & purificação , Ratos , Triglicerídeos/metabolismoRESUMO
A lipolytic zinc-copper protein has been isolated from the cytosol of chick liver. This material had a molecular weight of 6000 daltons, contained four atoms of zinc and one atom of copper per molecule. The 6000 dalton fraction aggregated at high ionic strength or in the presence of sodium dodecyl sulphate. Lipolytic activity was observed towards triolein, tripalmitin , phosphatidyl choline and retinyl palmitate, and was stimulated by cholate, Ca and high NaCl concentrations, and was inhibited by sulphydryl reagents, inhibitors of serine esterases, alkaline phosphatase and chelating agents. It appears that this copper-zinc protein is distinct from metallothionein which has no lipolytic activity.