RESUMO
PURPOSE: Growth hormone (GH)-producing pituitary adenomas (PAs) in childhood or young adulthood are rare, and the details surrounding these tumors remain enigmatic. We present the clinical, pathological and genetic features of this disease. METHODS: We identified 25 patients aged 20 years or younger with GH-producing PAs who underwent surgery between 2003 and 2016 at Toranomon Hospital in Tokyo. We retrospectively reviewed the clinical data, treatment outcomes and pathological features of these patients to shed light on childhood acromegaly. RESULTS: The cohort comprised 14 male and 11 female patients whose average age at the time of surgery was 17.3 years. Germline AIP mutations were present in 5 of 13 patients examined, and Carney complex was identified in 2 of 25 patients. The mean maximum tumor diameter was 26.7 mm, and total resection assessed during surgery was achieved in 17 patients. Based on their respective pathological findings, patients were divided into the following 4 groups: sparsely granulated adenomas (5), densely granulated (DG) adenomas (6), plurihormonal adenomas (9), and silent subtype 3 (SS3) adenomas (5). During the mean follow-up period of 50.3 months, complete endocrinological remission was achieved in 14 of 25 patients (56%) by surgery alone and in 19 patients (76%) after postoperative adjuvant therapy. CONCLUSIONS: GH-producing PAs in young patients are intriguing and difficult to treat due to their distinct tumor characteristics, including a lower incidence of the DG subtype and a higher incidence of SS3 adenomas and genetic abnormalities. Therefore, multi-modal therapies are essential to achieve optimal clinical outcomes.
Assuntos
Adenoma , Complexo de Carney , Adenoma Hipofisário Secretor de Hormônio do Crescimento , Acromegalia/etiologia , Adenoma/complicações , Adenoma/genética , Adenoma/patologia , Adenoma/cirurgia , Adolescente , Biomarcadores Tumorais/genética , Complexo de Carney/complicações , Complexo de Carney/genética , Complexo de Carney/patologia , Complexo de Carney/cirurgia , Quimioterapia Adjuvante , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Adenoma Hipofisário Secretor de Hormônio do Crescimento/complicações , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/cirurgia , Humanos , Hipofisectomia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Fenótipo , Radioterapia Adjuvante , Estudos Retrospectivos , Fatores de Tempo , Tóquio , Resultado do Tratamento , Carga Tumoral , Adulto JovemRESUMO
In adipose tissue, D-dopachrome tautomerase (DDT), a cytokine with structural similarity to macrophage migration inhibitory factor, is mainly expressed in adipocytes rather than preadipocytes and acts as an anti-obesity adipokine in an autocrine manner. However, its transcriptional regulation is largely unknown. In order to explore molecules affecting DDT transcription, a chemical library screening using HEK293 cells stably expressing a DDT promoter-reporter construct was performed. Several derivatives of 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, were identified as transcriptional activators of the DDT gene. Furthermore, DDT mRNA levels were reduced in SGBS adipocytes treated with compound C, an AMPK inhibitor, suggesting involvement of AMPK in DDT transcription. Overexpression of the FOXO1 constitutive active form reduced transcriptional activity of the DDT gene in SGBS cells, but increased it in HEK293 cells. Cell-type specific effects were also observed in the DDT gene expression of cells treated with AS1842856, a FOXO1 inhibitor. Finally, involvement of the mammalian target of rapamycin (mTOR) signaling in DDT transcription in SGBS adipocytes was investigated. Rapamycin, an inhibitor of mTOR, increased DDT mRNA levels and attenuated the inhibitory effects of compound C on DDT mRNA levels in SGBS adipocytes. In conclusion, DDT transcription may be regulated in a cell-dependent manner, and were enhanced by AMPK activation in SGBS adipocytes through inhibiting the mTOR signaling.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/fisiologia , Diferenciação Celular , Oxirredutases Intramoleculares/genética , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica , Adipócitos/efeitos dos fármacos , Linhagem Celular , Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Transdução de Sinais , Sirolimo/farmacologiaRESUMO
We previously identified D-dopachrome tautomerase (DDT) as a novel adipokine whose mRNA levels in adipocytes are negatively correlated with obesity-related clinical parameters, and which acts on adipocytes to regulate lipid metabolism. Here we investigated functions of DDT on preadipocytes. Recombinant DDT (rDDT) enhanced both the expression and secretion of interleukin-6 (IL-6) in SGBS cells, a human preadipocyte cell line. Treatment with rDDT increased levels of phosphorylated ERK1/2, but not p38, in SGBS cells, and rDDT-induced IL-6 mRNA expression was attenuated by pretreatment with an ERK inhibitor, U0126. Knockdown of CD74, but not CD44, inhibited rDDT-induced IL-6 mRNA expression in SGBS cells. These results suggested that the rDDT-induced IL-6 expression in preadipocytes occurred through the CD74-ERK pathway. Furthermore, in SGBS cells subjected to adipogenic induction, rDDT decreased the amount of triacylglycerol, number of cells with oil droplets, and levels of mRNA encoding adipocyte marker proteins. Increased expression of CCAAT/enhancer binding protein families and peroxisome proliferator-activated receptor γ2 during adipogenesis was inhibited in the cells treated with rDDT. These results suggested DDT to inhibit adipogenesis by suppressing the expression of genes encoding adipogenic regulators in preadipocytes.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-6/metabolismo , Oxirredutases Intramoleculares/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Butadienos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Receptores de Hialuronatos/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Nitrilas/farmacologia , PPAR gama/biossíntese , Fosforilação , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Proteínas Recombinantes/metabolismo , Triglicerídeos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The aim of this study was to identify genes that are specifically expressed in pancreatic islet ß-cells (hereafter referred to as ß-cells). Large-scale complementary DNA-sequencing analysis was performed for 3,429 expressed sequence tags derived from murine MIN6 ß-cells, through homology comparisons using the GenBank database. Three individual ESTs were found to code for protease serine S1 family member 53 (Prss53). Prss53 mRNA is processed into both a short and long form, which encode 482 and 552 amino acids, respectively. Transient overexpression of myc-tagged Prss53 in COS-7 cells showed that Prss53 was strongly associated with the luminal surfaces of organellar membranes and that it underwent signal peptide cleavage and N-glycosylation. Immunoelectron microscopy and western blotting revealed that Prss53 localized to mitochondria in MIN6 cells. Short hairpin RNA-mediated Prss53 knockdown resulted in Ppargc1a downregulation and Ucp2 and Glut2 upregulation. JC-1 staining revealed that the mitochondria were depolarized in Prss53-knockdown MIN6 cells; however, no change was observed in glucose-stimulated insulin secretion. Our results suggest that mitochondrial Prss53 expression plays an important role in maintaining the health of ß-cells.
Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Proteínas Mitocondriais , Serina Proteases/genética , Animais , Glucose , Insulina , Camundongos , Proteínas Mitocondriais/genéticaRESUMO
Insulin-responsive aminopeptidase (IRAP) and GLUT4 are two major cargo proteins of GLUT4 storage vesicles (GSVs) that are translocated from a postendosomal storage compartment to the plasma membrane (PM) in response to insulin. The cytoplasmic region of IRAP is reportedly involved in retention of GSVs. In this study, vimentin was identified using the cytoplasmic domain of IRAP as bait. The validity of this interaction was confirmed by pull-down assays and immunoprecipitation in 3T3-L1 adipocytes. In addition, it was shown that GLUT4 translocation to the PM by insulin was decreased in vimentin-depleted adipocytes, presumably due to dispersing GSVs away from the cytoskeleton. These findings suggest that the IRAP binding protein, vimentin, plays an important role in retention of GSVs.
Assuntos
Cistinil Aminopeptidase/metabolismo , Vesículas Citoplasmáticas/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Vimentina/metabolismo , Células 3T3-L1 , Animais , Técnicas de Silenciamento de Genes , Camundongos , Transporte Proteico , Vimentina/genéticaRESUMO
BACKGROUND/AIM: D-dopachrome tautomerase (DDT) is a macrophage migration inhibitory factor (MIF) homolog that promotes cell growth via CD74, a MIF cell surface receptor, in some types of tumors. We previously found that DDT acts as an anti-obesity adipokine independent of MIF. To understand the intrinsic properties of these two cytokines, a comparison of their actions in various tissues is necessary. In this study, we investigated the involvement of DDT in HepG2 cell (a human hepatoma cell line) proliferation, which is known to be promoted by MIF. MATERIALS AND METHODS: Cell proliferation and gene expression were evaluated in HepG2 cells expressing short hairpin RNA against the DDT gene. RESULTS: Inhibition of cell proliferation and reduced expression levels of cyclin D1 were observed in DDT-knockdown HepG2 cells. The inhibited proliferation was restored by administration of recombinant DDT. CONCLUSION: DDT promotes cell proliferation in HepG2 cells; therefore, its action may be similar to that of MIF.
Assuntos
Proliferação de Células/genética , Oxirredutases Intramoleculares/genética , Sobrevivência Celular , Ciclina D1/genética , Técnicas de Silenciamento de Genes , Células Hep G2 , HumanosRESUMO
Craniopharyngioma (CP) is mainly classified into two pathological subtypes: adamantinomatous (ACP) and papillary (PCP). CTNNB1 (ß-catenin) mutations are detected in ACPs, and the BRAF V600E mutation is detected in PCPs. However, genetic analysis is not always possible in general medical practice. In this study, we investigated whether immunohistochemistry could replace genetic analysis as an aid in subtype diagnosis. Here, 38 CP patients who had undergone their first tumor resection were included. Among the 38 cases, 22 were morphologically diagnosed as ACP, 10 cases were diagnosed as PCP, and six cases were diagnosed as undetermined CP that were morphologically difficult to classify as either ACP or PCP. Results of immunohistochemistry and genetic analysis and clinical features were compared. Based on the immunohistochemistry, 26 (22 ACPs and four undetermined CPs) showed nuclear ß-catenin expression, 11 (nine PCPs and two undetermined CPs) exhibited positive BRAF V600E immunostaining, and one PCP showed membranous ß-catenin expression and negative BRAF V600E immunostaining. Among the 26 nuclear ß-catenin expression cases, 11 had CTNNB1 mutations; however, 15 cases had mutations of neither CTNNB1 nor BRAF V600E. All 11 BRAF V600E immunopositive cases had BRAF V600E mutations. When comparing clinical features, pediatric patients and those with tumor calcification and less solid components on MRI more commonly had nuclear ß-catenin expression tumors than BRAF V600E immunopositive tumors, reflecting the differences in clinical features between ACP and PCP. Accordingly, immunohistochemistry can replace genetic analysis as an aid to determine the subtype diagnosis of CP in general medical practice.
Assuntos
Biomarcadores Tumorais/análise , Craniofaringioma/diagnóstico , Imuno-Histoquímica/métodos , Neoplasias Hipofisárias/diagnóstico , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Craniofaringioma/genética , Craniofaringioma/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas B-raf/genética , Adulto Jovem , beta Catenina/genéticaRESUMO
Obesity is considered a chronic low-grade inflammatory status and the stromal vascular fraction (SVF) cells of adipose tissue (AT) are considered a source of inflammation-related molecules. We identified YKL-40 as a major protein secreted from SVF cells in human visceral AT. YKL-40 expression levels in SVF cells from visceral AT were higher than in those from subcutaneous AT. Immunofluorescence staining revealed that YKL-40 was exclusively expressed in macrophages among SVF cells. YKL-40 purified from SVF cells inhibited the degradation of type I collagen, a major extracellular matrix of AT, by matrix metalloproteinase (MMP)-1 and increased rate of fibril formation of type I collagen. The expression of MMP-1 in preadipocytes and macrophages was enhanced by interaction between these cells. These results suggest that macrophage/preadipocyte interaction enhances degradation of type I collagen in AT, meanwhile, YKL-40 secreted from macrophages infiltrating into AT inhibits the type I collagen degradation.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Colágeno Tipo I/metabolismo , Glicoproteínas/metabolismo , Macrófagos/metabolismo , Obesidade/metabolismo , Adipócitos/patologia , Adipocinas , Tecido Adiposo/patologia , Adulto , Idoso , Células Cultivadas , Proteína 1 Semelhante à Quitinase-3 , Técnicas de Cocultura , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Lectinas , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Pessoa de Meia-Idade , Obesidade/patologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Cyclin-dependent kinase inhibitors represented by the INK4 family comprising p16(INK4A), p15(INK4B), p18(INK4C), and p19(INK4D) are regulators of the cell cycle shown to be aberrant in many types of cancer. Mice lacking p18(Ink4c) exhibit a series of phenotypes including the development of widespread organomegaly and pituitary adenomas. The objective of our study is to examine the role of p18(INK4C) in the pathogenesis of human pituitary tumors. The protein and mRNA levels of p18(INK4C) were examined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction, respectively. The methylation status of the p18(INK4C) gene promoter and somatic mutations of the p18(INK4C) gene were also investigated. p18(INK4C) protein expression was lost or significantly reduced in 64% of pituitary adenomas compared with levels in normal pituitary glands. p18(INK4C) mRNA levels were low in all ACTH adenomas and non-functioning (NF)-FSH and in 42%, 70% and 66% of GH, PRL, and subtype 3 adenomas, respectively. p18(INK4C) mRNA levels were significantly associated with p18(INK4C) protein levels. Neither methylated promoters in pituitary adenomas, except in one NF-FSH adenoma, nor somatic mutations of the p18(INK4C) gene in any pituitary adenomas were detected. The down-regulation of p18(INK4C) expression may contribute to the tumorigenesis of pituitary adenomas.
Assuntos
Adenoma/genética , Inibidor de Quinase Dependente de Ciclina p18/genética , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hipofisárias/genética , Adenoma/metabolismo , Adenoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Metilação de DNA , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , RNA Mensageiro/metabolismoRESUMO
CONTEXT: Mutations have been identified in the aryl hydrocarbon receptor-interacting protein (AIP) gene in familial isolated pituitary adenomas (FIPA). It is not clear, however, how this molecular chaperone is involved in tumorigenesis. OBJECTIVE: AIP sequence changes and expression were studied in FIPA and sporadic adenomas. The function of normal and mutated AIP molecules was studied on cell proliferation and protein-protein interaction. Cellular and ultrastructural AIP localization was determined in pituitary cells. PATIENTS: Twenty-six FIPA kindreds and 85 sporadic pituitary adenoma patients were included in the study. RESULTS: Nine families harbored AIP mutations. Overexpression of wild-type AIP in TIG3 and HEK293 human fibroblast and GH3 pituitary cell lines dramatically reduced cell proliferation, whereas mutant AIP lost this ability. All the mutations led to a disruption of the protein-protein interaction between AIP and phosphodiesterase-4A5. In normal pituitary, AIP colocalizes exclusively with GH and prolactin, and it is found in association with the secretory vesicle, as shown by double-immunofluorescence and electron microscopy staining. In sporadic pituitary adenomas, however, AIP is expressed in all tumor types. In addition, whereas AIP is expressed in the secretory vesicle in GH-secreting tumors, similar to normal GH-secreting cells, in lactotroph, corticotroph, and nonfunctioning adenomas, it is localized to the cytoplasm and not in the secretory vesicles. CONCLUSIONS: Our functional evaluation of AIP mutations is consistent with a tumor-suppressor role for AIP and its involvement in familial acromegaly. The abnormal expression and subcellular localization of AIP in sporadic pituitary adenomas indicate deranged regulation of this protein during tumorigenesis.
Assuntos
Adenoma/genética , Neoplasias Hipofisárias/genética , Proteínas/fisiologia , Acromegalia/genética , Acromegalia/metabolismo , Adenoma/metabolismo , Adolescente , Adulto , Idoso , Proliferação de Células , Criança , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Testes Genéticos , Hormônio do Crescimento Humano/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/metabolismo , Ligação Proteica , Proteínas/genética , Proteínas/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Previous analysis of PCR products derived from total RNA from the abdominal gland of the male newt, Cynops pyrrhogaster, inhabiting the Nara area of Japan led to the identification of a gene encoding [Val(8)]sodefrin, as well as the female-attracting peptide pheromone, sodefrin. In this study, purification of this sodefrin variant from the abdominal glands of male newts from the Nara area was accomplished using gel-filtration chromatography and reversed-phase HPLC. Amino acid sequence analysis and mass spectrometry confirmed that the final product was [Val(8)]sodefrin. A full-length cDNA encoding the biosynthetic precursor of [Val(8)]sodefrin was cloned and characterized. The deduced amino acid sequence of prepro[Val(8)]sodefrin showed 86.2% identity with that of the sodefrin precursor. The [Val(8)]sodefrin variant potently attracted females from the Nara area, but the variant was much less or not effective in attracting females captured in the Niigata and Chiba areas. The term aonirin ("aoni" from "aoni-yoshi", the conventional epithet of Nara) is proposed to designate this region-specific pheromone. It is speculated that the coevolution of a novel pheromone and its complementary receptor in the Nara newts may lead to reproductive isolation and eventual differentiation into a separate species.
Assuntos
Oligopeptídeos/isolamento & purificação , Feromônios/isolamento & purificação , Salamandridae/metabolismo , Atrativos Sexuais/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , DNA Complementar/química , DNA Complementar/genética , Feminino , Japão , Masculino , Dados de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/farmacologia , Feromônios/genética , Feromônios/farmacologia , Salamandridae/genética , Análise de Sequência de DNA , Análise de Sequência de Proteína , Atrativos Sexuais/genética , Atrativos Sexuais/farmacologia , Comportamento Sexual Animal/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177-186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile(187)-Ser(188)-Ala(189) and flanked at its N-terminus by Leu(174)-Gly(175)-Arg(176). This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbo-nyl (Boc)-Leu-Gly-Arg-4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45 degrees C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40 degrees C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males.
Assuntos
Anfíbios/metabolismo , Oligopeptídeos/biossíntese , Animais , Concentração de Íons de Hidrogênio , Leucina/análogos & derivados , Masculino , TemperaturaRESUMO
Anuran amphibians, animals that spend a terrestrial life after metamorphosis, exhibit a marked development of hindlimbs during and after metamorphosis. In order to see whether changes occur in the muscle protein components in the course of postmetamorphic development, we subjected gastrocnemius muscle extracts from growing froglets to two-dimensional electrophoresis (2DE). As a result, we found two proteins to undergo a change in level. One spot, indicating a molecular mass of approximately 12 kDa and an isoelectric point (pI) of 5.0 first became detectable at 45 days after metamorphosis. Another spot, corresponding to a protein of 11 kDa and pI 4.8, was prominent until the former spot appeared. N-terminal amino acid sequence analysis and comparison of the spots with those of parvalbumin (PA) revealed that these two proteins were PA alpha and PA beta. Northern blot analysis using PA alpha and PA beta cDNAs as probes revealed that the PA beta mRNA level declined whereas that of PA alpha mRNA rose as the frogs grew.
Assuntos
Músculo Esquelético/metabolismo , Parvalbuminas/biossíntese , Rana catesbeiana/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/biossíntese , Eletroforese em Gel Bidimensional , Feminino , Membro Posterior , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Dados de Sequência Molecular , Músculo Esquelético/crescimento & desenvolvimento , Parvalbuminas/análise , Parvalbuminas/genética , RNA Mensageiro/análise , Rana catesbeiana/crescimento & desenvolvimento , Extratos de Tecidos/análiseRESUMO
Amphibian sex pheromones of 3 urodele (Cynops pyrrhogaster, C. ensicauda, and Plethodon jordani) and 1 anuran (Litoria splendida) species have been isolated and characterized and found to be either small peptides or larger proteins. Each pheromone secreted by the male acts on conspecific females. Endocrine control of pheromone secretion has been best studied in Cynops. The C. pyrrhogaster pheromone, sodefrin, and the C. ensicauda pheromone, silefrin, are generated from their precursor proteins. The sodefrin and silefrin precursor mRNA levels in the abdominal gland of the cloaca are elevated by prolactin and androgen. An increase in the level of both immunoassayable pheromones caused by treatment with these hormones has also been demonstrated. Receptors for both of these hormones have been localized in the abdominal gland. The discharge of sodefrin into the water is elicited by arginine vasotocin. The responsiveness of the female vomeronasal epithelial cells to sodefrin, as estimated by electro-olfactography, is enhanced markedly by a combination of prolactin and estrogen. Sodefrin elevates intracellular calcium levels in vomeronasal epithelial cells. The population of the sodefrin-responsive cells increases during the breeding period.
Assuntos
Anuros/fisiologia , Glândulas Endócrinas/metabolismo , Atrativos Sexuais/metabolismo , Urodelos/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Glândulas Endócrinas/fisiologia , Feminino , Masculino , Dados de Sequência Molecular , Atrativos Sexuais/genéticaRESUMO
Sodefrin (SIPSKDALLK) is a female-attracting pheromone that is secreted by the abdominal gland of the male red-bellied newt. We found that mRNA encoding a sodefrin variant, [Val(8)] sodefrin, is expressed exclusively in specimens captured in the Nara area of Japan. The synthetic peptide was tested for its activity. It attracted females from Nara, but not those from other regions, suggesting that there is a geographic variation in the pheromone molecule and in the responsiveness to the pheromone. Employing an abdominal gland extract and synthetic substrates, the possibility of generation of the putative pheromone, [Val(8)] sodefrin, from the precursor molecule was demonstrated.
Assuntos
Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Salamandridae/genética , Salamandridae/metabolismo , Atrativos Sexuais/genética , Atrativos Sexuais/metabolismo , Cavidade Abdominal/fisiologia , Animais , Glândulas Exócrinas/metabolismo , Feminino , Variação Genética , Masculino , Processamento de Proteína Pós-TraducionalRESUMO
Mechanical stimuli regulate fundamental cell processes such as proliferation, differentiation, and morphogenesis. We attempted to identify microRNA (miRNA) whose expression is changed during compressive treatment in MC3T3-E1, a pre-osteoblastic cell line. Microarray analysis followed by reverse transcription-quantitative polymerase chain reaction revealed that compressive force at 294 Pa for 24 h in MC3T3-E1 cells increased levels of miR-494-3p, miR-146a-5p, miR-210-3p, and miR-1247-3p. Among these miRNAs, miR-494-3p was found to inhibit cell proliferation in MC3T3-E1 cells. Furthermore, cells subjected to compressive force showed slower cell growth compared with control cells. Levels of mRNA for fibroblast growth factor receptor 2 (FGFR2) and Rho-associated coiled-coil kinase 1 (ROCK1), which were predicted to be targets of miR-494-3p, were decreased by compressive force or overexpression of miR-494-3p mimics in MC3T3-E1 cells. Furthermore, binding sites of miR-494-3p within 3'-untranslated regions of Fgfr2 and Rock1 were determined using luciferase reporter assay. In conclusion, compressive force affected expressions of several miRNAs including miR-494-3p in MC3T3-E1 cells. Compressive force might inhibit cell proliferation in osteoblasts by up-regulating miR-494-3p followed by FGFR2 and ROCK1 gene repressions.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/biossíntese , MicroRNAs/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Estresse Mecânico , Regiões 3' não Traduzidas/genética , Linhagem Celular , Proliferação de Células/genética , Regulação para Baixo , Humanos , Luciferases/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Quinases Associadas a rho/genéticaRESUMO
Peptides derived from the post-translational processing of preprosodefrin were isolated from an extract of the abdominal glands of male red-bellied newts Cynops pyrrhogaster obtained 5 months prior to the onset of the breeding season. Structural characterization of the peptides showed that the pheromone sodefrin (SIPSKDALLK) is stored in a biologically inactive COOH-terminally extended form (SIPSKDALLKISA). It follows, therefore, that the activation of a protease that cleaves at a Lys-Ile bond to generate the active pheromone must occur by the time of onset of reproductive behavior. Additional peptides (representing preprosodefrin-(146-175)-peptide and preprosodefrin-(159-173)-peptide), that are derived from the precursor by cleavage at monobasic and dibasic processing sites, were also purified from the extract. The isolation of paralogs of these peptides, including an inactive COOH-terminally extended form of [Asn10]sodefrin, provides evidence for the expression of multiple genes encoding preprosodefrin. PCR products derived from total RNAs from the abdominal gland of individual newts collected from three different regions of Japan were analyzed. The data confirm the existence of multiple genes encoding sodefrin and its variants whose expression varied according to the individuals and the regions. However, genes encoding sodefrin were found to be expressed in all the specimens sampled.
Assuntos
Cavidade Abdominal , Peptídeos/metabolismo , Precursores de Proteínas/química , Sequência de Aminoácidos , Animais , Cromatografia em Gel , DNA Complementar/metabolismo , Masculino , Dados de Sequência Molecular , Peptídeos/química , Feromônios/química , Reação em Cadeia da Polimerase , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA/metabolismo , Salamandridae , Homologia de Sequência de AminoácidosRESUMO
This article reviews the current state of understanding of reproductive pheromones in amphibians, focusing mainly on the purification and characterization of peptide pheromones in newts of the genus Cynops, molecular cloning of cDNAs encoding the pheromone molecules, and hormonal control of secretion of these pheromones. Pheromones that attract sexually developed female Cynops pyrrhogaster and C. ensicauda newts were isolated from the male abdominal glands. The C. pyrrhogaster and C. ensicauda pheromones are peptides, designated sodefrin and silefrin, with the amino acid sequences SIPSKDALLK and SILSKDAQLK, respectively. Each pheromone attracts only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed the presence of precursor proteins that are considered to generate these pheromone peptides. Pheromone precursor mRNA levels and radioimmunoassayable pheromone concentrations in the abdominal glands were elevated by prolactin and androgen. Sexual dimorphism and hormone dependency of the responsiveness of vomeronasal epithelium to sodefrin were noted. Significance of pheromones in the form of peptide for those performing reproductive behavior in an aquatic environment was also discussed.
Assuntos
Peptídeos/fisiologia , Feromônios/fisiologia , Animais , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Hormônios/química , Masculino , Oligopeptídeos/química , Peptídeos/química , Feromônios/química , RNA Mensageiro/metabolismo , Salamandridae , Fatores Sexuais , Comportamento Sexual AnimalRESUMO
Purification, characterization and biological activity of urodele and anuran sex-pheromones were reviewed. Female-attracting pheromones obtained from the abdominal gland of Cynops pyrrhogaster and C. ensicauda males are peptides consisting of 10 amino acid residues being designated sodefrin and silefrin, respectively. Each pheromone attracted only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed that both are generated from precursor proteins. Synthesis of these pheromones is regulated by prolactin (PRL) and androgen. Responsiveness of the female vomeronasal epithelium to sodefrin is enhanced by PRL and estrogen. The submandibular gland of the male terrestrial salamander, Plethodon jardani secretes a 22-kD proteinaceous pheromone that enhances female receptivity. It was revealed that every salamander synthesizes multiple isoforms of this pheromone, Plethodontid receptivity factor. The magnificent tree frog, Litoria splendida breed in an aquatic environment. The skin glands of the male secrete a female-attracting peptide pheromone, splendipherin, comprising 25 amino acid residues. The significance of the structure of the amphibian sex-pheromone as peptide and protein is discussed in terms of their species specificity.
Assuntos
Anfíbios/metabolismo , Peptídeos/fisiologia , Feromônios/fisiologia , Androgênios/metabolismo , Animais , Bufonidae , Clonagem Molecular , DNA Complementar/metabolismo , Feminino , Masculino , Prolactina/metabolismo , Salamandridae , Atrativos Sexuais/metabolismo , Comportamento Sexual Animal , Pele/metabolismo , Especificidade da Espécie , Glândula Submandibular/metabolismo , UrodelosRESUMO
Although the cause of familial isolated pituitary adenoma (FIPA) remains unknown in many cases, germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene were identified in approximately 20 % of families with FIPA. We investigated the AIP gene mutation by a standard sequencing method in 12 members of a Japanese two-generation FIPA family, which includes 3 patients with early-onset acromegaly. Multiplex ligation-dependent probe amplification analysis in a tumor sample was attempted to examine the loss of heterozygosity (LOH) in the locus. The effect of the detected mutation on cell proliferation was investigated. A germline mutation of c.943C > T (p.Q315X) generating an AIP protein with the C-terminal end deleted was found in the FIPA family. Biallelic inactivation of AIP by a combination of the germline mutation and LOH at 11q13 was confirmed in the tumor. The nonsense mutation disrupted the ability to inhibit cell proliferation. We conclude that p.Q315X mutation in the AIP gene is a pathogenic variant and the C-terminal region of AIP plays an important role in the predisposition to pituitary adenomas.