RESUMO
There is increasing evidence to suggest a beneficial neuroprotective effect of growth hormone (GH) in the nervous system. While our previous studies have largely focused on retinal ganglion cells (RGCs), we have also found conclusive evidence of a pro-survival effect of GH in cells of the inner nuclear layer (INL) as well as a protective effect on the dendritic trees of the inner plexiform layer (IPL) in the retina. The administration of GH in primary neuroretinal cell cultures protected and induced neural outgrowths. Our results, both in vitro (embryo) and in vivo (postnatal), showed neuroprotective actions of GH against kainic acid (KA)-induced excitotoxicity in the chicken neuroretina. Intravitreal injections of GH restored brain derived neurotrophic factor (BDNF) expression in retinas treated with KA. In addition, we demonstrated that GH over-expression and exogenous administration increased BDNF and neurotrophin-3 (NT3) gene expression in embryonic neuroretinal cells. Thus, GH neuroprotective actions in neural tissues may be mediated by a complex cascade of neurotrophins and growth factors which have been classically related to damage prevention and neuroretinal tissue repair.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Hormônio do Crescimento/farmacologia , Ácido Caínico/farmacologia , Fármacos Neuroprotetores/farmacologia , Neurotrofina 3/metabolismo , Retina/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Modelos Animais de Doenças , Neuroproteção/efeitos dos fármacos , Retina/embriologia , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Daytime restricted feeding promotes the re-alignment of the food entrained oscillator (FEO). Endocrine cues which secretion is regulated by the transition of fasting and feeding cycles converge in the FEO. The present study aimed to investigate the ghrelin, growth hormone (GH) and insulin-like growth factor (IGF)-1 system because their release depends on rhythmic and nutritional factors, and the output from the system influences feeding and biochemical status. In a daily sampling approach, rats that were fed ad lib. were compared with rats on a reversed (daytime) and restricted feeding schedule by 3 weeks (dRF; food access for 2 h), also assessing the effect of acute fasting and refeeding. We undertook measurements of clock protein BMAL1 and performed somatometry of peripheral organs and determined the concentration of total, acylated and unacylated ghrelin, GH and IGF-1 in both serum and in its main synthesising organs. During dRF, BMAL1 expression was synchronised to mealtime in hypophysis and liver; rats exhibited acute hyperphagia, stomach distension with a slow emptying, a phase shift in liver mass towards the dark period and decrease in mass perigonadal white adipose tissue. Total ghrelin secretion during the 24-h period increased in the dRF group as a result of elevation of the unacylated form. By contrast, GH and IGF-1 serum concentration fell, with a modification of GH daily pattern after mealtime. In the dRF group, ghrelin content in the stomach and pituitary GH content decreased, whereas hepatic IGF-1 remained equal. The daily patterns and synthesis of these hormones had a rheostatic adaptation. The endocrine adaptive response elicited suggests that it may be associated with the regulation of metabolic, behavioural and physiological processes during the paradigm of daytime restricted feeding and associated FEO activity.
Assuntos
Adaptação Fisiológica/fisiologia , Ingestão de Alimentos/fisiologia , Grelina/fisiologia , Hormônio do Crescimento/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Fatores de Transcrição ARNTL/metabolismo , Aclimatação , Animais , Ritmo Circadiano/fisiologia , Grelina/sangue , Hormônio do Crescimento/sangue , Masculino , Neuro-Hipófise/metabolismo , Ratos , Ratos WistarRESUMO
Protein hormones from the anterior pituitary gland have well-established endocrine roles in their peripheral target glands. It is, however, now known that these proteins are also produced within many of their target tissues, in which they act as local autocrine or paracrine factors, with physiological and/or pathophysiological significance. This emerging concept is the focus of this brief review.
Assuntos
Comunicação Autócrina/fisiologia , Comunicação Parácrina/fisiologia , Hormônios Adeno-Hipofisários/metabolismo , Animais , Encéfalo/metabolismo , Trato Gastrointestinal/metabolismo , Humanos , Pulmão/metabolismo , Pele/metabolismoRESUMO
Endocrine actions of growth hormone (GH) have been implicated during the development of adult testicular function in several mammalian species, and recently intracrine, autocrine, and paracrine effects have been proposed for locally expressed GH. Previous reports have shown the distribution of GH mRNA and the molecular heterogeneity of GH protein in both adult chicken testes and vas deferens. This study provides evidence of the presence and distribution of GH and its receptor (GHR) during all stages of spermatogenesis in adult chicken testes. This hormone and its receptor are not restricted to the cytoplasm; they are also found in the nuclei of spermatogonia, spermatocytes, and spermatids. The pattern of GH isoforms was characterized in the different, isolated germ cell subpopulations, and the major molecular variant in all subpopulations was 17 kDa GH, as reported in other chicken extra-pituitary tissues. Another molecular variant, the 29 kDa moiety, was found mainly in the enriched spermatocyte population, suggesting that it acts at specific developmental stages. The co-localization of GH with the proliferative cell nuclear antigen PCNA (a DNA replication marker present in spermatogonial cells) was demonstrated by immunohistochemistry. These results show for the first time that GH and GHR are present in the nuclei of adult chicken germinal cells, and suggest that GH could participate in proliferation and differentiation during the complex process of spermatogenesis.
Assuntos
Galinhas/metabolismo , Hormônio do Crescimento/metabolismo , Testículo/metabolismo , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Proliferação de Células , Hormônio do Crescimento/análise , Hormônio do Crescimento/genética , Imuno-Histoquímica , Masculino , Antígeno Nuclear de Célula em Proliferação/análise , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/metabolismo , Receptores da Somatotropina/análise , Receptores da Somatotropina/metabolismo , Maturidade Sexual , Espermatogênese , Testículo/citologia , Testículo/ultraestruturaRESUMO
Although growth hormone (GH) is mainly synthesized and secreted by pituitary somatotrophs, it is now well established that the GH gene can be expressed in many extrapituitary tissues, including the central nervous system (CNS). Here we studied the expression of GH in the chicken cerebellum. Cerebellar GH expression was analyzed by in situ hybridization and cDNA sequencing, as well as by immunohistochemistry and confocal microscopy. GH heterogeneity was studied by Western blotting. We demonstrated that the GH gene was expressed in the chicken cerebellum and that its nucleotide sequence is closely homologous to pituitary GH cDNA. Within the cerebellum, GH mRNA is mainly expressed in Purkinje cells and in cells of the granular layer. GH-immunoreactivity (IR) is also widespread in the cerebellum and is similarly most abundant in the Purkinje and granular cells as identified by specific neuronal markers and histochemical techniques. The GH concentration in the cerebellum is age-related and higher in adult birds than in embryos and juveniles. Cerebellar GH-IR, as determined by Western blot under reducing conditions, is associated with several size variants (of 15, 23, 26, 29, 35, 45, 50, 55, 80 kDa), of which the 15 kDa isoform predominates (>30% among all developmental stages). GH receptor (GHR) mRNA and protein are also present in the cerebellum and are similarly mainly present in Purkinje and granular cells. Together, these data suggest that GH and GHR are locally expressed within the cerebellum and that this hormone may act as a local autocrine/paracrine factor during development of this neural tissue.
Assuntos
Hormônio do Crescimento/biossíntese , Envelhecimento , Sequência de Aminoácidos , Animais , Sequência de Bases , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Galinhas , Células de Purkinje/metabolismo , RNA Mensageiro/metabolismo , Receptores da Somatotropina/biossíntese , Alinhamento de SequênciaRESUMO
Growth hormone (GH) is expressed in the chicken bursa of Fabricius (BF), an organ that undergoes three distinct developmental stages: rapid growth (late embryogenesis until 6-8 weeks of age [w]), plateaued growth (between 10 and 15w), and involution (after 18-20w). The distribution and abundance of GH-immunoreactivity (GH-IR) and GH mRNA expression in stromal and non-stromal bursal cells during development, as well as the potential anti-apoptotic effect of GH in bursal cell survival were the focus of this study. GH mRNA expression was mainly in the epithelial layer and in epithelial buds at embryonic day (ED) 15; at 2w it was widely distributed within the follicle and in the interfollicular epithelium (IFE); at 10w it clearly diminished in the epithelium; whereas at 20w it occurred in only a few cortical cells and in the connective tissue. Parallel changes in the relative proportion of GH mRNA expression (12, 21, 13, 1%) and GH-IR (19, 18, 11, <3%) were observed at ED 15, 2w, 10w, and 20w, respectively. During embryogenesis, GH-IR co-localized considerably with IgM-IR, but scarcely with IgG-IR, whereas the opposite was observed after hatching. Significant differences in bursal cell death occurred during development, with 9.3% of cells being apoptotic at ED 15, 0.4% at 2w, 0.23% at 10w, and 21.1% at 20w. Addition of GH increased cultured cell survival by a mechanism that involved suppression (up to 41%) of caspase-3 activity. Results suggest that autocrine/paracrine actions of bursal GH are involved in the differentiation and proliferation of B lymphocytes and in BF growth and cell survival in embryonic and neonatal chicks, whereas diminished GH expression in adults may result in bursal involution.
Assuntos
Bolsa de Fabricius/embriologia , Galinhas/fisiologia , Hormônio do Crescimento/fisiologia , Animais , Apoptose/fisiologia , Bolsa de Fabricius/citologia , Bolsa de Fabricius/fisiologia , Sobrevivência Celular/fisiologia , Embrião de Galinha , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Hormônio do Crescimento/genética , Imunoglobulina G/fisiologia , Imunoglobulina M/fisiologia , Imuno-Histoquímica/veterinária , Hibridização In Situ/veterinária , Marcação In Situ das Extremidades Cortadas/veterinária , Masculino , RNA Mensageiro/química , RNA Mensageiro/genética , Organismos Livres de Patógenos Específicos , Células Estromais/citologia , Células Estromais/metabolismo , Células Estromais/fisiologiaRESUMO
Expression of growth hormone (GH) and GH receptor (GHR) genes in the bursa of Fabricius of chickens suggests that it is an autocrine/paracrine site of GH production and action. The cellular localization of GH and GH mRNA within the bursa was the focus of this study. GH mRNA was expressed mainly in the cortex, comprised of lymphocyte progenitor cells, but was lacking in the medulla where lymphocytes mature. In contrast, more GH immunoreactivity (GH-IR) was present in the medulla than in the cortex. In non-stromal tissues, GH-IR and GH mRNA were primarily in lymphocytes, and also in macrophage-like cells and secretory dendritic cells. In stromal tissues, GH mRNA, GH and GHR were expressed in cells near the connective tissue (CT) between follicles and below the outer serosa. In contrast, GH (but not GH mRNA or GHR), was present in cells of the interfollicular epithelium (IFE), the follicle-associated epithelium (FAE) and the interstitial corticoepithelium. This mismatch may reflect dynamic temporal changes in GH translation. Co-expression of GHR-IR, GH-IR, GH mRNA and IgG was found in immature lymphoid cells near the cortex and in IgG-IR CT cells, suggesting an autocrine/paracrine role for bursal GH in B-cell differentiation.
Assuntos
Bolsa de Fabricius/imunologia , Galinhas/imunologia , Galinhas/metabolismo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Hormônio do Crescimento/genética , Hormônio do Crescimento/imunologia , Animais , Bolsa de Fabricius/metabolismo , Galinhas/genética , Hormônio do Crescimento/metabolismo , Imunoglobulina G/imunologia , RNA Mensageiro/genética , Receptores da Somatotropina/metabolismoRESUMO
Growth hormone (GH) expression is not confined to the pituitary and occurs in many extrapituitary tissues. Here, we describe the presence of GH-like moieties in chicken lymphoid tissues and particularly in the bursa of Fabricius. GH-immunoreactivity (GH-IR), determined by ELISA, was found in thymus, spleen, and in bursa of young chickens, but at concentrations <1% of those in the pituitary gland. Although the GH concentration in the spleen and bursa was approximately 0.82 and 0.23% of that in the pituitary at 9-weeks of age, because of their greater mass, the total GH content in the spleen, bursa, and in thymus were 236, 5.18, and 31.5%, respectively, of that in the pituitary gland. This GH-IR was associated with several proteins of different molecular size, as in the pituitary gland, when analyzed by SDS-PAGE under reducing conditions. While most of the GH-IR in the pituitary was associated with the 26 kDa monomer (40%), the putatively glycosylated 29 kDa variant (16%), the 52 kDa dimer (14%) and the 15 kDa submonomeric isoform (16%), GH-IR in the lymphoid tissues was primarily associated (27-36%) with a 17 kDa moiety, although bands of 14, 26, 29, 32, 37, 40, and 52 kDa were also identified in these tissues. The heterogeneity pattern and relative abundance of bursal GH-IR bands were determined during development between embryonic day 13 (ED13) and 9-weeks of age. The relative proportion of the 17 kDa GH-like band was higher (45-58%) in posthatched birds than in the 15 and 18-day old embryos (21 and 19%, respectively). The 26 kDa isoform was minimally present in embryos (<4% of total GH-IR) but in posthatched chicks it increased to 12-20%. Conversely, while GH-IR of 37, 40, and 45 kDa were abundantly present in embryonic bursa ( approximately 30% at ED13 and approximately 52-55% at ED15 and ED18, respectively), in neonatal chicks and juveniles they accounted for less than 5%. These ontogenic changes were comparable to those previously reported for similar GH-IR proteins in the chicken testis during development. In summary, these results demonstrate age-related and tissue-specific changes in the content and composition of GH in immune tissues of the chicken, in which GH is likely to be an autocrine or paracrine regulator.
Assuntos
Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Hormônio do Crescimento/metabolismo , Tecido Linfoide/metabolismo , Animais , Bolsa de Fabricius/metabolismo , Embrião de Galinha , Imuno-Histoquímica , Hipófise/metabolismo , Baço/metabolismo , Timo/metabolismoRESUMO
There has been extensive research of the anterior pituitary gland of livestock and poultry due to the economic (agricultural) importance of physiological processes controlled by it including reproduction, growth, lactation and stress. Moreover, farm animals can be biomedical models or useful in evolutionary/ecological research. There are for multiple sites of control of the secretion of anterior pituitary hormones. These include the potential for independent control of proliferation, differentiation, de-differentiation and/or inter-conversion cell death, expression and translation, post-translational modification (potentially generating multiple isoforms with potentially different biological activities), release with or without a specific binding protein and intra-cellular catabolism (proteolysis) of pituitary hormones. Multiple hypothalamic hypophysiotropic peptides (which may also be produced peripherally, e.g. ghrelin) influence the secretion of the anterior pituitary hormones. There is also feedback for hormones from the target endocrine glands. These control mechanisms show broadly a consistency across species and life stages; however, there are some marked differences. Examples from growth hormone, prolactin, follicle stimulating hormone and luteinizing hormone will be considered. In addition, attention will be focused on areas that have been neglected including the role of stellate cells, multiple sub-types of the major adenohypophyseal cells, functional zonation within the anterior pituitary and the role of multiple secretagogues for single hormones.
Assuntos
Animais Domésticos/fisiologia , Adeno-Hipófise/fisiologia , Animais , Modelos Animais , Adeno-Hipófise/citologia , Hormônios Adeno-Hipofisários/biossíntese , Hormônios Adeno-Hipofisários/metabolismo , Isoformas de Proteínas , PesquisaRESUMO
Glycosylation is one of the post-translational modifications that growth hormone (GH) can undergo. This has been reported for human, rat, mouse, pig, chicken and buffalo GH. The nature and significance of GH glycosylation remains to be elucidated. This present study further characterizes glycosylated chicken GH (G-cGH) and examines changes in the pituitary concentration of G-cGH during embryonic development and post hatching growth. G-cGH was purified from chicken pituitaries by affinity chromatography (Concanavalin A-Sepharose and monoclonal antibody bound to Sepharose). Immunoreactive G-cGH has a MW of 26 kDa or 29 kDa as determined by SDS-PAGE, respectively, under non-reducing and reducing conditions. Evidence that it is N-glycosylated comes from its susceptibility to peptide N-glycosidase F, and its resistance to O-glycosidase. Based on the ability of G-cGH to bind Concanavalin A or wheat germ agglutinin but not other lectins and its susceptibility to peptide N-glycosidase F, a hybrid or biantennary type glycopeptide (GlcNac2, Man) structure is proposed. Some G-cGH can be observed in the pituitary at most ages examined (from 15-day embryo to adult). Moreover, electron microscopy revealed the presence of both immuno-reactive GH and Concanavalin A-reactive sites in the same secretory granules in the somatotrope. There were marked changes in the level and relative proportion of G-cGH in the pituitary gland during development and growth, the proportion of G-cGH rising during late embryonic development (e.g., between 15 and 18 days of development) and with further increases between 9 weeks and 15 weeks old. G-cGH was able to bind to chicken liver membrane preparations with less affinity than non-glycosylated monomer; on the other hand, however, G-cGH stimulated cell proliferation on Nb2 lymphoma bioassay whereas the non-glycosylated monomer was uncapable to do it.
Assuntos
Embrião de Galinha/crescimento & desenvolvimento , Embrião não Mamífero , Hormônio do Crescimento/metabolismo , Hipófise/fisiologia , Animais , Proliferação de Células , Galinhas , Cromatografia de Afinidade , Glicosilação , Hormônio do Crescimento/biossíntese , Microscopia Eletrônica , Hipófise/embriologia , Hipófise/crescimento & desenvolvimento , Isoformas de ProteínasRESUMO
Growth hormone (GH) gene expression is not restricted to pituitary somatotrophs and has recently been demonstrated in a variety of extrapituitary sites in mammals and the domestic chicken. The possibility that GH gene expression occurs in the male reproductive system of chickens was therefore examined, since GH has established roles in male reproductive function and GH immunoreactivity is present in the chicken testis. Using RT-PCR and oligonucleotide primers for pituitary GH cDNA, GH mRNA was shown to be present in the testes and vas deferens of adult cockerels. Although testicular GH mRNA was of low abundance (not detectable by Northern blotting), a 690 bp fragment of the amplified testicular GH cDNA was cloned and had a nucleotide sequence 99.6% homologous with pituitary GH cDNA. GH mRNA was localized by in situ hybridization in spermatogonia and primary spermatocytes of the seminiferous tubules, but unlike testicular GH-immunoreactivity, GH mRNA was not present in secondary spermatocytes, spermatids or spermatozoa. The presence of Pit-1 mRNA in the male reproductive tract may indicate Pit-1 involvement in GH expression in these tissues. The presence of GH receptor mRNA in the testis and vas deferens also suggests they are target sites for GH action. These results demonstrate, for the first time, expression of the pituitary GH gene in the testis, in which GH mRNA was discretely localized in primary spermatocytes. The local expression of the GH gene in these cells suggests autocrine or paracrine actions of GH during spermatogenesis.
Assuntos
Galinhas/fisiologia , Hormônio do Crescimento/biossíntese , Testículo/fisiologia , Ducto Deferente/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hormônio do Crescimento/genética , Masculino , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatócitos/fisiologia , Espermatogônias/fisiologiaRESUMO
Growth hormone (GH) gene expression is not confined to pituitary somatotrophs and occurs in many extrapituitary tissues. In this study, we describe the presence of GH moieties in the chicken testis. GH-immunoreactivity (GH-IR), determined by ELISA, was found in the testis of immature and mature chickens, but at concentrations <1% of those in the pituitary gland. The immunoassayable GH concentration in the testis was unchanged between 4 and 66 weeks of age, and approximately 10-fold higher than that at 1-week of age and 25-fold higher than that in 1-day-old chicks and perinatal (embryonic day 18) embryos. This immunoreactivity was associated with several proteins of different molecular size, as in the pituitary gland, when analyzed by SDS-PAGE under reducing conditions. However, while most of the GH-IR in the pituitary ( approximately 40 and 15%, respectively) is associated with monomer (26 kDa) or dimer (52 kDa) GH moieties GH-IR in the testis is primarily (30-50%) associated with a 17 kDa moiety. GH bands between 32 and 45 kDa are also relatively more abundant in the testis than in the pituitary. During ontogeny the relative abundance of a 14 kDa GH and 40 kDa GH moieties in the testis significantly declined, whereas the relative abundance of the 17 and 45 kDa moieties increased with advancing age. In adult birds, GH-IR was widespread and intense in the seminiferous tubules. Although the GH-IR was not present in the basal compartment of Sertoli cells, nor in spermatogonia and primary spermatocytes, it was abundantly present in secondary spermatocytes and spermatids in the luminal compartments of the tubules as well as in some surrounding myocytes and interstitial cells. In summary, immunoreactive GH moieties are present in the chicken testis but at concentrations far less than in the pituitary. Age-related changes in the relative abundance of testicular GH variants may be related to local (autocrine/paracrine) actions of testicular GH. The localization of GH in spermatocytes and spermatids suggests hitherto unsuspected roles in gamete development.
Assuntos
Galinhas/fisiologia , Genitália Masculina/crescimento & desenvolvimento , Genitália Masculina/fisiologia , Hormônio do Crescimento/metabolismo , Maturidade Sexual/fisiologia , Envelhecimento/fisiologia , Animais , Western Blotting , Embrião de Galinha , Densitometria , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Hormônio do Crescimento/biossíntese , Hormônio do Crescimento/química , Imuno-Histoquímica , Masculino , Peso Molecular , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Túbulos Seminíferos/crescimento & desenvolvimento , Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismo , Desenvolvimento Sexual , Espermatócitos/metabolismo , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Ducto Deferente/crescimento & desenvolvimento , Ducto Deferente/metabolismoRESUMO
Chicken embryonic adipofibroblasts (CEA) accumulate intracytoplasmic lipids when cultured in medium containing chicken serum (CS), but not in medium with fetal bovine serum (FBS). To characterize this process of lipid accumulation, we evaluated the expression of the enzyme glycerol-3-phosphate dehydrogenase (E.C.1.1.1.8) (GPDH), first in chicken tissues and then in CEA cultured under diverse conditions. GPDH activity in adipose depots from 4-wk-old broiler chickens was similar or higher than that shown by liver, the main organ for fatty acid synthesis in chickens, while skeletal muscle had the lowest levels of GPDH. In vitro, GPDH activity increased in CEA cultured in the presence of CS but not in medium with FBS, paralleling the lipid accumulation by these cells. Both lipid accumulation and GPDH activity were further increased in CEA cultured in the presence of embryonic CS. Our results show that GPDH is highly expressed in avian tissues related to lipid metabolism and therefore can be a reliable marker for avian adipogenesis, and suggest that ECS is an optimum source for the purification of avian adipogenic factors.
Assuntos
Tecido Adiposo/enzimologia , Galinhas/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Metabolismo dos Lipídeos , Tecido Adiposo/citologia , Tecido Adiposo/embriologia , Animais , Sangue , Células Cultivadas , Embrião de Galinha , Meios de Cultura , Ácidos Graxos/biossíntese , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Fígado/citologia , Fígado/enzimologia , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/enzimologiaRESUMO
There is evidence for a cleaved form of GH in the chicken pituitary gland. A 25 kDa band of immunoreactive-(ir-)GH, as well as the 22 kDa monomeric form and some oligomeric forms were observed when purified GH or fresh pituitary extract were subjected to SDS-PAGE under nonreducing conditions. Under reducing conditions, the 25 kDa ir-GH was no longer observed, being replaced by a 15 kDa band, consistent with reduction of the disulfide bridges of the cleaved form. The type of protease involved was investigated using exogenous proteases and monomeric cGH. Cleaved forms of chicken GH were generated by thrombin or collagenase. The site of cleavage was found in position Arg133-Gly134 as revealed by sequencing the fragments produced. The NH2-terminal sequence of 40 amino acid residues in the 15 kDa form was identical to that of the rcGH and analysis of the remaining 7 kDa fragment showed an exact identity with positions 134-140 of cGH structure. The thrombin cleaved GH and the 15 kDa form showed reduced activity (0.8% and 0.5% of GH, respectively) in a radioreceptor assay employing a chicken liver membrane preparation. However, this fragment had a clear bioactivity in an angiogenic bioassay and was capable to inhibit the activity of deiodinase type III in the chicken liver.
Assuntos
Galinhas/metabolismo , Hormônio do Crescimento/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Colagenases/metabolismo , Dimerização , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicosilação , Hormônio do Crescimento/análise , Hormônio do Crescimento/química , Iodeto Peroxidase/antagonistas & inibidores , Fígado/ultraestrutura , Masculino , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Hipófise/química , Ensaio Radioligante , Trombina/metabolismoRESUMO
BACKGROUND: Seabather's eruption (SBE) is a highly pruritic dermatosis affecting swimmers and divers in marine waters off Florida, in the Gulf of Mexico, and the Caribbean Sea. Its cause has been attributed to various organisms but recently to the larvae of the schyphomedusa, Linuche unguiculata. OBJECTIVE: We attempted to determine whether immature and adult Linuche cause SBE. METHODS: Episodes of SBE in the Cancun and Cozumel area of the Mexican Caribbean were evaluated during the season of high tourism (January-June). This time corresponds to the moments in the life cycle when the three swimming stages of L unguiculata-ephyrae, medusae, and larvae-can be sequentially observed. Our methods include (1) observations by divers, biologists, and students coinciding with stinging outbreaks and the onset of SBE; (2) serologic evaluation of individuals stung by L unguiculata; and (3) the demonstration of Linuche nematocysts on the affected skin. RESULTS: All 3 swimming Linuche stages can cause SBE. CONCLUSION: The offending stages of Linuche can be identified by the cutaneous lesion's morphology and the time of year.
Assuntos
Venenos de Cnidários/intoxicação , Prurido/etiologia , Dermatopatias Vesiculobolhosas/etiologia , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Humanos , Estágios do Ciclo de Vida , Masculino , Pessoa de Meia-Idade , Cifozoários/crescimento & desenvolvimento , NataçãoRESUMO
Somatostatin (SRIF) binding sites were characterized in goldfish brain. Binding of (125)I-[Tyr(11)]-SRIF-14 to a brain membrane preparation was found to be saturable, reversible, and time-, temperature-, and pH-dependent. Binding was also displaceable by different forms of SRIF. Under optimal conditions (22 degrees C, pH 7.2), the equilibrium binding of (125)I-[Tyr(11)]-SRIF-14 to goldfish brain membranes was achieved after 60 min incubation. Analysis of saturable equilibrium binding revealed a one-site model fit with K(a) of 1.3 nM. SRIF-14, mammalian SRIF-28, and salmon SRIF-25 displaced (125)I-[Tyr(11)]-SRIF-14 binding with similar affinity, whereas other neuropeptides, e.g., substance P, were unable to displace (125)I-[Tyr(11)]-SRIF-14. Autoradiography studies demonstrated that (125)I-[Tyr(11)]-SRIF-14 binding sites are found throughout the goldfish brain. A high density of (125)I-[Tyr(11)]-SRIF-14 binding sites was found in the forebrain, including the nucleus preopticus, nucleus preopticus periventricularis, nucleus anterioris periventricularis, nucleus lateralis tuberis, nucleus dorsomedialis thalami, nucleus dorsolateralis thalami, nucleus ventromedialis thalami, and nucleus diffusus lobi inferioris. In midbrain, (125)I-[Tyr(11)]-SRIF-14 binding sites were found in the optic tectum. The facial and vagal lobes and the mesencephalic-cerebellar tract were found to have a high density of binding sites. This study provides the first characterization and distribution of specific binding sites for SRIF in a fish brain.
Assuntos
Química Encefálica , Carpa Dourada/metabolismo , Receptores de Somatostatina/análise , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Concentração de Íons de Hidrogênio , Hipotálamo/química , Hipotálamo/metabolismo , Radioisótopos do Iodo , Cinética , Área Pré-Óptica/química , Área Pré-Óptica/metabolismo , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Somatostatina/metabolismo , Somatostatina-28 , Temperatura , Distribuição TecidualRESUMO
There is considerable evidence for the existence of structural variants of growth hormone (GH). The chicken is a useful model for investigating GH heterogeneity as both size and charge immunoreactive-(ir) variants have been observed in the pituitary and plasma. The present study examined the size distribution of ir-GH in the pituitary gland of chicken, from late embryogenesis through adulthood. Pituitaries were homogenized in the presence of protease inhibitor, and the GH size variants were separated by SDS-PAGE, transferred by Western blotting, immunostained with a specific antiserum to chicken GH, and quantitated by chemiluminescence followed by laser densitometry (chemiluminescent assay). Under nonreducing conditions ir-GH bands of 15, 22, 25, 44, 50, 66, 80, 98, 105 and >110 kDa were observed. Both the relative proportion of the GH size variants and the total pituitary content varied with developmental stage and age. The proportion of the 15-kDa fragment was greatest in the embryonic stage, and then it decreased. The proportion of the monomeric 22-kDa form was lowest at 18 days of embryogenesis (dE) and highest at 20 dE. In contrast, the high MW forms (>/=66 kDa) were lowest in embryos, and they increased (P < 0.05) after hatching. The 22-, 44-, 66-, and 80-kDa forms were assayed for activity by radioreceptor assay following isolation by semipreparative SDS-PAGE. Only the 22-kDa GH variant showed radioreceptor activity. Under reducing conditions for SDS-PAGE, ir-GH bands of 13, 15, 18, 23, 26, 36, 39, 44, 48, 59 and 72 kDa were oberved, but most of the high MW form disappeared. There was a concomitant increase in the proportion of the monomeric band and of several submonomeric forms. The present data indicate that the expression, processing, and/or release of some if not all size variants are under some differential control during growth and development of the chicken.
Assuntos
Hormônio do Crescimento/química , Hormônio do Crescimento/metabolismo , Hipófise/metabolismo , Envelhecimento , Animais , Embrião de Galinha , Galinhas , Eletroforese em Gel de Poliacrilamida , Hormônio do Crescimento/isolamento & purificação , Medições Luminescentes , Peso Molecular , Hipófise/embriologia , Hipófise/crescimento & desenvolvimento , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismoRESUMO
A useful system to evaluate the angiogenic activity of hormones and growth factors is the chorioallantoic membrane (CAM) of chick embryos. The present studies examined the angiogenic activity of chicken anterior pituitary glands and both fibroblast growth factor (FGF) and growth hormone (GH). Grafts of anterior pituitary gland evoked an angiogenic response on the CAM which was lost if the adenohypophyseal tissue was first boiled. The magnitude of the angiogenic response to anterior pituitary glands increased with the age of the donor (from a minimum 15 days of embryonic development to a maximum between 2 and 6 weeks old). In view of the similarity of the profile of the angiogenic response and the reported changes in GH secretion, the angiogenic activity of GH was then examined. Considerable angiogenic responses were observed with GH; there being increases (P < 0.05) in number of new blood vessels on the CAM of chick embryos on which native chicken GH or native bovine GH or recombinant bovine GH were added. These data support GH having an angiogenic action.