RESUMO
Indoleamine 2 3-dioxygenase (IDO) is a protein usually described in mammals, which, among other functions, participates in the maternal-fetal tolerance process. The blue-shark, Prionace glauca (Linnaeus, 1758) is a viviparous placentary species in which the yolk sac develops during the pregnancy, turning into a placenta for matrotrophic nutrition of the embryo. The purpose of this study was to investigate the expression of IDO in the P. glauca maternal-fetal interface along three gestation phases and describe its distribution and the meaning of its presence. The results showed IDO labelling during the yolk sac/placenta development in the ectoderm on the three development phases and in the endoderm at the two first phases. In the uterine epithelium, IDO was observed in the last two phases. These interface tissues are major contact areas between the mother and the semiallogeneic conceptus and this relation could induce an immunological response against the fetus. Therefore, the presence of IDO may indicate that it could have a similar role in the mechanism of maternal-fetal tolerance in the P. glauca placental interface, as described in eutherian mammals.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/análise , Tubarões/crescimento & desenvolvimento , Saco Vitelino/enzimologia , Animais , Feminino , Viviparidade não MamíferaRESUMO
Carboxyl ester lipase (Cel), is a lipolytic enzyme secreted by the pancreas, which hydrolyzes various species of lipids in the gut. Cel is also secreted by mammary gland during lactation and exists in breast milk. It facilitates dietary fat digestion and absorption, thus contributing to normal infant development. This study aimed to examine whether the Cel in zebrafish embryos has a similar role of maternal lipid utilization as in human infants, and how Cel contributes to the utilization of yolk lipids in zebrafish. The cel1 and cel2 genes were expressed ubiquitously in the blastodisc and yolk syncytial layer before 24 hpf, and in the exocrine pancreas after 72 hpf. The cel1 and cel2 morphants exhibited developmental retardation and yolk sac retention. The total cholesterol, cholesterol ester, free cholesterol, and triglyceride were reduced in the morphants' body while accumulated in the yolk (except triglyceride). The FFA content of whole embryos was much lower in morphants than in standard controls. Moreover, the delayed development in cel (cel1/cel2) double morphants was partially rescued by FFA and cholesterol supplementation. Delayed and weakened cholesterol ester transport to the brain and eyes was observed in cel morphants. Correspondingly, shrunken midbrain tectum, microphthalmia, pigmentation-delayed eyes as well as down-regulated Shh target genes were observed in the CNS of double morphants. Interestingly, cholesterol injections reversed these CNS alterations. Our findings suggested that cel genes participate in the lipid releasing from yolk sac to developing body, thereby contributing to the normal growth rate and CNS development in zebrafish.
Assuntos
Carboxilesterase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transtornos do Crescimento/genética , Saco Vitelino/enzimologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Carboxilesterase/genética , Sistema Nervoso Central/embriologia , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero , Desenvolvimento Embrionário , Técnicas de Silenciamento de Genes , Transtornos do Crescimento/embriologia , Transtornos do Crescimento/enzimologia , Proteínas Hedgehog/metabolismo , Humanos , Metabolismo dos Lipídeos , Morfolinos/administração & dosagem , Morfolinos/genética , Pâncreas Exócrino/embriologia , Pâncreas Exócrino/enzimologia , Triglicerídeos/metabolismo , Saco Vitelino/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Urea is an essential osmolyte for marine cartilaginous fishes. Adult elasmobranchs and holocephalans are known to actively produce urea in the liver, muscle and other extrahepatic organs; however, osmoregulatory mechanisms in the developing cartilaginous fish embryo with an undeveloped urea-producing organ are poorly understood. We recently described the contribution of extraembryonic yolk sac membranes (YSM) to embryonic urea synthesis during the early developmental period of the oviparous holocephalan elephant fish (Callorhinchus milii). In the present study, to test whether urea production in the YSM is a general phenomenon among oviparous Chondrichthyes, we investigated gene expression and activities of ornithine urea cycle (OUC) enzymes together with urea concentrations in embryos of the elasmobranch cloudy catshark (Scyliorhinus torazame). The intracapsular fluid, in which the catshark embryo develops, had a similar osmolality to seawater, and embryos maintained a high concentration of urea at levels similar to that of adult plasma throughout development. Relative mRNA expressions and activities of catshark OUC enzymes were significantly higher in YSM than in embryos until stage 32. Concomitant with the development of the embryonic liver, the expression levels and activities of OUC enzymes were markedly increased in the embryo from stage 33, while those of the YSM decreased from stage 32. The present study provides further evidence that the YSM contributes to embryonic urea homeostasis until the liver and other extrahepatic organs become fully functional, and that urea-producing tissue shifts from the YSM to the embryonic liver in the late developmental period of oviparous marine cartilaginous fishes.
Assuntos
Elasmobrânquios/embriologia , Fígado/embriologia , Ureia/metabolismo , Saco Vitelino/metabolismo , Animais , Sequência de Bases , Clonagem Molecular , Elasmobrânquios/genética , Enzimas/genética , Regulação da Expressão Gênica no Desenvolvimento , Fígado/enzimologia , Homologia de Sequência do Ácido Nucleico , Saco Vitelino/enzimologiaRESUMO
Mg2+ regulates many physiological processes and signalling pathways. However, little is known about the mechanisms underlying the organismal balance of Mg2+. Capitalizing on a set of newly generated mouse models, we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6. We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development. In adult mice, TRPM6 is required in the intestine to maintain organismal Mg2+ balance, but is dispensable in the kidney. Trpm6 inactivation in adult mice leads to a shortened lifespan, growth deficit and metabolic alterations indicative of impaired energy balance. Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice, but also may extend the lifespan of wildtype mice. Hence, maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood.
Assuntos
Desenvolvimento Embrionário , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Magnésio/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Feminino , Técnicas de Inativação de Genes , Camundongos , Placenta/enzimologia , Placenta/metabolismo , Gravidez , Análise de Sobrevida , Canais de Cátion TRPM/genética , Saco Vitelino/enzimologia , Saco Vitelino/metabolismoRESUMO
We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 °C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.
Assuntos
Endoderma/citologia , Células Epiteliais/citologia , Saco Vitelino/citologia , Animais , Proliferação de Células , Células Cultivadas , Coturnix , Células Epiteliais/enzimologia , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Esterol O-Aciltransferase/genética , Saco Vitelino/enzimologiaRESUMO
The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Saco Vitelino/irrigação sanguínea , Saco Vitelino/enzimologia , Animais , Autofagia/genética , Autofagia/fisiologia , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Endoderma/irrigação sanguínea , Endoderma/citologia , Endoderma/enzimologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mesoderma/irrigação sanguínea , Mesoderma/citologia , Mesoderma/enzimologia , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Neovascularização Fisiológica/genética , Gravidez , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Saco Vitelino/citologia , Saco Vitelino/embriologiaRESUMO
This paper provides new information related to the mechanism of OPI (organophosphorus insecticides) teratogenesis. The COMFA (comparative molecular field analysis) and COMSIA (comparative molecular similarity indices analysis) suggest that the electrostatic and steric fields are the best predictors of OPI structural requirements to inhibit in ovo chicken embryo yolk sac membrane kynurenine formamidase, the proposed target for OPI teratogens. The dominant electrostatic interactions are localized at nitrogen-1, nitrogen-3, nitrogen of 2-amino substituent of the pyrimidinyl of pyrimidinyl phosphorothioates, and the oxygen of crotonamide carbonyl in crotonamide phosphates. Bulkiness of the substituents at carbon-2 and carbon-6 of the pyrimidinyls and/or N-substituents and carbon-3 substituents of crotonamides are the steric structural components that contribute to superiority of those OPI as in ovo inhibitors of kynurenine formamidase.
Assuntos
Arilformamidase/antagonistas & inibidores , Inseticidas/química , Inseticidas/toxicidade , Compostos Organofosforados/química , Compostos Organofosforados/toxicidade , Relação Quantitativa Estrutura-Atividade , Teratogênicos/toxicidade , Saco Vitelino/enzimologia , Animais , Embrião de Galinha , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Leptofós/química , Leptofós/toxicidade , Modelos Moleculares , Paration/química , Paration/toxicidade , Eletricidade Estática , Teratogênicos/química , Saco Vitelino/efeitos dos fármacosRESUMO
Prostaglandins are key components of the parturition cascade; however, the mechanisms that regulate prostaglandin concentrations in the uterus during pregnancy are largely unknown. The purpose of this study was to determine the intrauterine expression of the chief prostaglandin-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase (PGDH), during gestation and labor in the guinea pig, an animal model in which the endocrine control of pregnancy and parturition is analogous to that of women. PGDH messenger RNA (mRNA) abundance decreased significantly in the visceral yolk sac membrane (VYS, the anatomical equivalent of the human chorion laeve) and the amnion throughout the last third of pregnancy. PGDH protein was robustly expressed in the VYS epithelium and mesoderm, correlated strongly with PGDH mRNA levels and exhibited a nadir at term prior to labor onset. PGDH protein was not detected in the amnion. PGDH mRNA and protein levels in the placenta and myoendometrium were variable throughout late gestation. In the placenta, PGDH protein was concentrated in the parietal yolk sac membrane (PYS) lining the placental surface and in placental blood vessels. We observed strong expression of PGDH protein in the endometrial epithelium with comparably little expression in the myometrium. These data indicate that metabolic inactivation of prostaglandins in the pregnant guinea pig uterus takes place in the VYS, PYS, and endometrium. Decreased PGDH expression in the fetal membranes may contribute to the increase in intrauterine prostaglandin concentrations at term, stimulating the onset of labor.
Assuntos
Hidroxiprostaglandina Desidrogenases/biossíntese , Parto/metabolismo , Útero/metabolismo , Animais , Córion/citologia , Córion/enzimologia , Feminino , Cobaias , Humanos , Início do Trabalho de Parto/metabolismo , Mesoderma/citologia , Mesoderma/enzimologia , Modelos Animais , Placenta/citologia , Placenta/enzimologia , Gravidez , Útero/citologia , Saco Vitelino/citologia , Saco Vitelino/enzimologiaRESUMO
DiGeorge Critical Region 8 (DGCR8) is a double-stranded RNA-binding protein that interacts with Drosha and facilitates microRNA (miRNA) maturation. However, the role of DGCR8 in vascular smooth muscle cells (VSMCs) is not well understood. To investigate whether DGCR8 contributes to miRNA maturation in VSMCs, we generated DGCR8 conditional knockout (cKO) mice by crossing VSMC-specific Cre mice (SM22-Cre) with DGCR8(loxp/loxp) mice. We found that loss of DGCR8 in VSMCs resulted in extensive liver hemorrhage and embryonic mortality between embryonic days (E) 12.5 and E13.5. DGCR8 cKO embryos displayed dilated blood vessels and disarrayed vascular architecture. Blood vessels were absent in the yolk sac of DGCR8 KOs after E12.5. Disruption of DGCR8 in VSMCs reduced VSMC proliferation and promoted apoptosis in vitro and in vivo. In DGCR8 cKO embryos and knockout VSMCs, differentiation marker genes, including αSMA, SM22, and CNN1, were significantly down-regulated, and the survival pathways of ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated. Knockout of DGCR8 in VSMCs has led to down-regulation of the miR-17/92 and miR-143/145 clusters. We further demonstrated that the miR-17/92 cluster promotes VSMC proliferation and enhances VSMC marker gene expression, which may contribute to the defects of DGCR8 cKO mutants. Our results indicate that the DGCR8 gene is required for vascular development through the regulation of VSMC proliferation, apoptosis, and differentiation.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , MicroRNAs/metabolismo , Músculo Liso Vascular/embriologia , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas/metabolismo , Animais , Perda do Embrião/genética , Perda do Embrião/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Proteínas/genética , Proteínas de Ligação a RNA , Saco Vitelino/irrigação sanguínea , Saco Vitelino/citologia , Saco Vitelino/enzimologiaRESUMO
Rho-associated coiled-coil-forming protein serine/threonine kinase (ROCK) consisting of two isoforms, ROCK-I and ROCK-II, functions downstream of the small GTPase Rho for assembly of actomyosin bundles. To examine the role of ROCK isoforms in vivo, we previously generated and examined mice deficient in each of the two isoforms individually. Here, we further examined the in vivo role of ROCK isoforms by generating mice deficient in both isoforms. Cross-mating of ROCK-I(+/-) ROCK-II(+/-) double heterozygous mice showed that all of the ROCK-I(-/-) ROCK-II(-/-) homozygous mice die in utero before 9.5 days post-coitum (dpc) and ROCK-I(-/-) ROCK-II(+/-) homo-heterozygous or ROCK-I(+/-) ROCK-II(-/-) hetero-homozygous mice die during a period from 9.5 to 12.5 dpc, whereas mice of other genotypes survive until 12.5 dpc with the expected Mendelian ratio. All of the ROCK-I(+/-) ROCK-II(-/-) or ROCK-I(-/-) ROCK-II(+/-) mice showed impaired body turning and defective vascular remodeling in the yolk sac. Impairment of vascular remodeling was also observed in wild-type embryos treated ex vivo with a ROCK inhibitor, Y-27632. These results suggest that ROCK isoforms function redundantly during embryogenesis and play a critical role in vascular development.
Assuntos
Saco Vitelino/irrigação sanguínea , Saco Vitelino/enzimologia , Quinases Associadas a rho/deficiência , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese/genética , Mutação/genética , Neovascularização Patológica/enzimologia , Neovascularização Patológica/genética , Fenótipo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
The iodotyrosine dehalogenase1 (DEHAL1) enzyme is a transmembrane protein that belongs to the nitroreductase family and shows a highly conserved N-terminal domain. DEHAL1 is present in the liver, kidney and thyroid of mammals. DEHAL1 is known to act on diiodotyrosine (DIT) and monoiodotyrosine (MIT), and is involved in iodine recycling in relation to thyroglobulin. Here, we show the distribution of DEHAL1 during gastrulation to neurulation in developing chick. Immunocytochemistry using an anti-serum directed against the N-terminal domain (met(27)-trp(180) fragment) of human DEHAL1 revealed labelled cells in the embryonic ectoderm, embryonic endoderm, neural plate and in the yolk platelets of the chick embryo at gastrulation stage. Distinct DEHAL1 positive cells were located in the presumptive head ectoderm, presumptive neural crest, head mesenchymal cells and in the dorsal, lateral and ventral parts of neural tube during neurulation. Some cells located at the margin of the developing notochord and somites were also DEHAL1-positive. While the functional significance of this observation is not known, it is likely that DEHAL1 might serve as an agent that regulates cell specific deiodination of MIT and DIT before the onset of thyroidal secretion. The presence of DEHAL1 in different components of the chick embryo suggests its involvement in iodine turnover prior to the formation of functional thyroid.
Assuntos
Embrião de Galinha/enzimologia , Di-Iodotirosina/metabolismo , Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Monoiodotirosina/metabolismo , Glândula Tireoide/embriologia , Animais , Padronização Corporal , Ectoderma/enzimologia , Endoderma/enzimologia , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Hidrolases/genética , Immunoblotting , Iodo/metabolismo , Mesoderma/enzimologia , Crista Neural/enzimologia , Placa Neural/enzimologia , Tubo Neural/enzimologia , Neurulação , Glândula Tireoide/enzimologia , Saco Vitelino/enzimologiaRESUMO
We report that angiotensin-converting enzyme (ACE), a critical physiologic regulator of blood pressure, angiogenesis, and inflammation, is a novel marker for identifying hemangioblasts differentiating from human embryonic stem cells (hESC). We demonstrate that ACE+CD45-CD34+/- hemangioblasts are common yolk sac (YS)-like progenitors for not only endothelium but also both primitive and definitive human lymphohematopoietic cells. Thrombopoietin and basic fibroblast growth factor are identified as critical factors for the proliferation of human hemangioblasts. The developmental sequence of human embryoid body hematopoiesis is remarkably congruent to the timeline of normal human YS development, which occurs during weeks 2 to 6 of human gestation. Furthermore, ACE and the renin-angiotensin system (RAS) directly regulate hemangioblast expansion and differentiation via signaling through the angiotensin II receptors AGTR1 and AGTR2. ACE enzymatic activity is required for hemangioblast expansion, and differentiation toward either endothelium or multipotent hematopoietic progenitors is dramatically augmented after manipulation of angiotensin II signaling with either AGTR1- or AGTR2-specific inhibitors. The RAS can therefore be exploited to direct the hematopoietic or endothelial fate of hESC-derived hemangioblasts, thus providing novel opportunities for human tissue engineering. Moreover, the initial events of human hematoendotheliogenesis can be delineated in a manner previously impossible because of inaccessibility to early human embryonic tissues.
Assuntos
Células-Tronco Embrionárias/enzimologia , Células-Tronco Embrionárias/imunologia , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/imunologia , Peptidil Dipeptidase A/metabolismo , Células-Tronco Pluripotentes/enzimologia , Células-Tronco Pluripotentes/imunologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Sinergismo Farmacológico , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Células Endoteliais/enzimologia , Células Endoteliais/imunologia , Substâncias de Crescimento/administração & dosagem , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Pluripotentes/citologia , Sistema Renina-Angiotensina , Trombopoetina/administração & dosagem , Saco Vitelino/citologia , Saco Vitelino/enzimologia , Saco Vitelino/imunologiaRESUMO
Intraembryonic hematopoiesis occurs at two different sites, the floor of the aorta and subaortic patches (SAPs) of the para-aortic splanchnopleura (P-Sp)/aorta-gonad-mesonephros (AGM) region. Notch1 and RBP-jkappa are critical for the specification of hematopoietic stem cells (HSCs) in Notch signal-receiving cells. However, the mechanism by which Notch signaling is triggered from the Notch signal-sending cells to support embryonic hematopoiesis remains to be determined. We previously reported that Mind bomb-1 (Mib1) regulates Notch ligands in the Notch signal-sending cells (B. K. Koo, M. J. Yoon, K. J. Yoon, S. K. Im, Y. Y. Kim, C. H. Kim, P. G. Suh, Y. N. Jan, and Y. Y. Kong, PLoS ONE 2:e1221, 2007). Here, we show that intraembryonic hematopoietic progenitors were absent in the P-Sp of Mib1(-/-) embryos, whereas they were partly preserved in the Tie2-cre; Mib1(f)(/f) P-Sps, suggesting that Mib1 plays a role in the endothelium and the SAPs. Interestingly, dll1 and dll4/Jag1 are expressed in the SAPs and the endothelium of the AGM, respectively, where mib1 is detected. Indeed, Notch signaling was activated in the nascent HSCs at both sites. In the P-Sp explant culture, the overexpression of Dll1 in OP9 stromal cells rescued the failed production of hematopoietic progenitors in the Mib1(-/-) P-Sp, while its activity was abolished by Mib1 knockdown. These results suggest that Mib1 is important for intraembryonic hematopoiesis not only in the aortic endothelium but also in the SAPs.
Assuntos
Aorta/embriologia , Aorta/enzimologia , Embrião de Mamíferos/enzimologia , Endotélio Vascular/embriologia , Endotélio Vascular/enzimologia , Hematopoese , Ubiquitina-Proteína Ligases/metabolismo , Animais , Aorta/citologia , Proteínas de Ligação ao Cálcio , Linhagem Celular , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Células Endoteliais/enzimologia , Gônadas/citologia , Gônadas/embriologia , Gônadas/enzimologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Mesonefro/citologia , Mesonefro/embriologia , Mesonefro/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptor TIE-2/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Células Estromais/enzimologia , Saco Vitelino/enzimologiaRESUMO
To address the role of PKC isoforms in hyperglycemia-induced apoptosis and malformations in the embryos of diabetic pregnancies, expression of PKCalpha, beta1, beta 2, gamma, delta, epsilon, and zeta was examined in the neural tube of rat embryos and showed to overlap with the regions of increased apoptosis. Levels of activated (phosphorylated) PKCalpha , beta2, and delta were increased in the embryos of diabetic dams whereas those of PKCepsilon and zeta were decreased when compared with those in control groups. Cytosolic phospholipase A(2) (cPLA(2)) was also activated. Blocking the activity of PKCalpha , beta2, and delta using isoform-specific inhibitors in embryos cultured in hyperglycemia (40 mM) reduced malformation rates when compared with those in untreated hyperglycemic and euglycemic (8.3 mM) groups. These observations demonstrate that PKCalpha, beta2, and delta play an essential role in diabetic embryopathy. Activation of cPLA(2) was also decreased, suggesting that PKCs mediate the hyperglycemic effects through the cPLA(2)-phospholipid peroxidation pathway.
Assuntos
Doenças Fetais/enzimologia , Hiperglicemia/complicações , Defeitos do Tubo Neural/enzimologia , Tubo Neural/enzimologia , Fosfolipases A2 Citosólicas/metabolismo , Proteína Quinase C/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Diabetes Mellitus Experimental , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/fisiologia , Feminino , Doenças Fetais/etiologia , Doenças Fetais/prevenção & controle , Masculino , Tubo Neural/efeitos dos fármacos , Tubo Neural/embriologia , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/prevenção & controle , Fosfolipases A2 Citosólicas/fisiologia , Fosforilação , Gravidez , Isoformas de Proteínas , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Saco Vitelino/embriologia , Saco Vitelino/enzimologiaRESUMO
Assessment of cholinesterase (ChE) inhibition is widely used as a specific biomarker for evaluating the exposure and effects of non-target organisms to anticholinesterase agents. Cholinesterase and carboxylesterase activities have been measured in larvae of gilthead seabream, Sparus aurata, during the endogenous feeding stage, and ChE was characterized with the aid of diagnostic substrates and inhibitors. The results of the present study showed that whole-body ChE of yolk-sac seabream larvae possesses typical properties of acetylcholinesterase (AChE) with a apparent affinity constant (K(m)) of 0.163+/-0.008 mM and a maximum velocity (V(max)) of 332.7+/-2.8 nmol/min/mg protein. Moreover, sensibility of this enzyme was investigated using the organophosphorus insecticide azinphosmethyl. Static-renewal toxicity tests were conducted over 72 h and larval survival and AChE inhibition were recorded. Mean mortality of seabream larvae increased with increasing concentrations of azinphosmethyl and exposure duration. The estimated 72-h LC50 value to azinphosmethyl was 4.59 microg/l (95% CI=0.46-8.71 microg/l) and inhibition of ChE activity gave an IC50 of 3.04 microg/l (95% CI=2.73-3.31 microg/l). Larvae exposed to azinphosmethyl for 72h showed a 70% inhibition of the whole-body acetylcholinesterase activity at approximately the LC50. In conclusion, the results of the present study suggested that monitoring ChE activity is a valuable tool indicating OP exposure in S. aurata larvae and that acetylthiocholine is the most appropriate substrate for assessing ChE inhibition in this early-life stage of the fish.
Assuntos
Azinfos-Metil/farmacologia , Inibidores da Colinesterase/farmacologia , Colinesterases/metabolismo , Inseticidas/farmacologia , Dourada/metabolismo , Animais , Azinfos-Metil/toxicidade , Benzenamina, 4,4'-(3-oxo-1,5-pentanodi-il)bis(N,N-dimetil-N-2-propenil-), Dibrometo/farmacologia , Inibidores da Colinesterase/toxicidade , Concentração Inibidora 50 , Inseticidas/toxicidade , Cinética , Larva , Fisostigmina/farmacologia , Especificidade por Substrato , Tetraisopropilpirofosfamida/farmacologia , Saco Vitelino/efeitos dos fármacos , Saco Vitelino/enzimologiaRESUMO
We aimed to investigate the extent to which maternal diabetes with or without folic acid (FA) supplementation affects mRNA levels and protein distribution of ROS scavenging enzymes, vascular endothelial growth factor-A (Vegf-A), folate binding protein-1 (Folbp-1), and apoptosis-associated proteins in the yolk sacs of rat embryos on gestational days 10 and 11. Commencing at conception and throughout pregnancy, half of the streptozotocin-diabetic and half of the control rats received daily FA injections. Maternal diabetes impaired vascular morphology and decreased CuZnSOD and GPX-1 gene expression in yolk sacs. Maternal diabetes also increased the levels of CuZnSOD protein, increased the Bax/Bcl-2 protein ratio and decreased Vegf-A protein distribution. FA treatment normalized vascular morphology, decreased mRNA levels of all three SOD isoforms and increased Vegf-A mRNA levels, rectified CuZnSOD protein distribution and Bax/Bcl-2 ratio. A teratogenic diabetic environment produces a state of vasculopathy, oxidative stress, and mild apoptosis in the yolk sac. FA administration normalizes vascular morphology, diminishes apoptotic rate, and increases Vegf-A gene expression and protein distribution in the yolk sac of diabetic rats.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Ácido Fólico/farmacologia , Oxirredutases/metabolismo , Gravidez em Diabéticas/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Saco Vitelino/efeitos dos fármacos , Animais , Antioxidantes/uso terapêutico , Caspase 3/metabolismo , Catalase/metabolismo , Diabetes Mellitus Experimental/embriologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Feminino , Ácido Fólico/uso terapêutico , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Idade Gestacional , Glutationa Peroxidase/metabolismo , Glicina N-Metiltransferase/metabolismo , Oxirredutases/genética , Gravidez , Gravidez em Diabéticas/genética , Gravidez em Diabéticas/metabolismo , Gravidez em Diabéticas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Saco Vitelino/enzimologia , Saco Vitelino/patologia , Proteína X Associada a bcl-2/metabolismoRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9), recently cloned in several laboratories, including ours, causes a third form of autosomal dominant hypercholesterolemia. Its mechanism of action remains unclear. We studied the expression and subcellular localization of PCSK9 in fetal and adult rat tissues associated with cholesterol homeostasis using quantitative reverse transcriptase--PCR, Western blot analysis, subcellular fractionation, and confocal immunofluorescent microscopy. PCSK9 mRNA is most abundant in yolk sac and fetal liver, but the highest expression of the protein was found in differentiated hepatoma FAO-1 cell line, which also shows the highest expression of LDLR. In FAO-1 cells PCSK9 expression is downregulated by cholesterol and 25-hydroxycholesterol and upregulated in the absence of sterols following the same pattern of expression as HMG-CoA reductase, synthase, and LDLR. Subcellular fractionation, combined with Western blotting, showed that PCSK9 is localized in the ER and intermediate vesicular compartment of the cell but not in Golgi cisternae. The mature enzyme is secreted from the liver and hepatoma cells. Double labeling with antibodies to PCSK9 and LDLR or clathrin revealed some colocalization of PCSK9 with clathrin-coated vesicles and LDLR. In conclusion, our results show that PCSK9 is processed in the ER, and the mature convertase is secreted in the plasma.
Assuntos
Regulação Enzimológica da Expressão Gênica , Hepatócitos/enzimologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Brefeldina A/farmacologia , Células COS , Centrifugação com Gradiente de Concentração , Chlorocebus aethiops , Colesterol/farmacologia , DNA Complementar/genética , Feminino , Feto/enzimologia , Imunofluorescência , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Isoquinolinas/farmacologia , Fígado/enzimologia , Masculino , Gravidez , Pró-Proteína Convertase 9 , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Células Tumorais Cultivadas , Saco Vitelino/enzimologiaRESUMO
We have recently established a unique in vitro experimental model for mitochondrion-rich cell (MRC) research, a ;yolk-ball' incubation system, in which the yolk sac is separated from the embryonic body of Mozambique tilapia embryos and subjected to in vitro incubation. To evaluate the ion-transporting property of the yolk balls, we examined Cl- content and turnover in yolk balls incubated in freshwater and seawater for 48 h, and distribution patterns of three ion transporters, Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR), in MRCs in the yolk-sac membrane. The Cl- turnover rate measured by whole-body influx of 36Cl- was about 60 times higher in yolk balls in seawater than in freshwater, while there was no essential difference in Cl- content between them. Na+/K+-ATPase-immunoreactive MRCs were larger in yolk balls from seawater than yolk balls from freshwater. Distribution patterns of ion-transporting proteins allowed us to classify MRCs in freshwater yolk balls into three types: cells showing only basolateral Na+/K+-ATPase, cells showing basolateral Na+/K+-ATPase and apical NKCC, and cells showing basolateral Na+/K+-ATPase and basolateral NKCC. The seawater yolk balls, on the other hand, were characterized by the appearance of MRCs possessing basolateral Na+/K+-ATPase, basolateral NKCC and apical CFTR. Those seawater-type MRCs were considered to secrete Cl- through the CFTR-positive apical opening to cope with diffusional Cl- influx. These findings indicate that the yolk balls preserve the Cl- transporting property of intact embryos, ensuring the propriety of the yolk ball as an in vitro experimental model for the yolk-sac membrane that contains MRCs.
Assuntos
Cloretos/metabolismo , Água Doce , Transporte de Íons/fisiologia , Água do Mar , Tilápia/embriologia , Tilápia/metabolismo , Saco Vitelino/metabolismo , Animais , Embrião não Mamífero/metabolismo , Modelos Biológicos , Moçambique , Simportadores de Cloreto de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Saco Vitelino/enzimologia , Saco Vitelino/ultraestruturaRESUMO
ALA-D (EC 4.2.1.24) is the first cytosolic enzyme in the haem metabolic pathway. Some metals compete with its major cofactor Zn(2+), modifying both enzyme structure and function. Our purpose was to contribute to the understanding of the biochemical role of metals such as Pb(2+), Cd(2+), Cu(2+), Mg(2+), Zn(2+), Na(+), K(+) and Li(+) on ALA-D, using chicken embryos as experimental model. Mg(2+) and Zn(2+) showed enzyme activation in yolk sac membrane (YSM) (113% at 10(-5) M Mg(2+) and from 10(-4) M Zn(2+)), and slight inactivation in liver. Cd(2+) and Cu(2+) caused a non allosteric inhibition in both tissues (100% from 10(-4) M). Surprisingly Pb(2+) was not such a strong inhibitor. Interference of cations during the Schiff base formation in enzymatic catalysis process is explained considering their Lewis acid-base capacity, coordination geometry and electron configuration of valence. Interactions among monovalent cations and biochemical substances are governed chiefly by its electrostatic potential. 0.1 M K(+) and 0.4 M Na(+) produced 30% of enzymatic inhibition by the interference on interactions among the functional subunits. Li(+) activated the YSM enzyme (130% at 10(-5) M) due to a more specific interaction. This study may contribute to elucidate for the first time the possible structural differences between the YSM and liver enzymes from chicken embryo.
Assuntos
Metais/farmacologia , Sintase do Porfobilinogênio/antagonistas & inibidores , Sintase do Porfobilinogênio/metabolismo , Animais , Cátions/farmacologia , Embrião de Galinha , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Fígado/enzimologia , Sintase do Porfobilinogênio/química , Bases de Schiff/química , Saco Vitelino/enzimologiaRESUMO
Inactivation of the gene encoding mouse angiotensin I-converting enzyme (ACE), which converts angiotensin I into angiotensin II, results in anemia in adult animals. This anemia is corrected by angiotensin II, demonstrating the involvement of angiotensin II in adult (definitive) erythropoiesis. We investigated the possible role of the renin-angiotensin system (RAS) in primitive erythropoiesis in the yolk sac of the chicken embryo. Enzymatically active ACE was detected in the yolk sac endoderm, concomitantly with the differentiation of blood islands in the adjacent yolk sac mesoderm. The simultaneous presence of all the other components of the RAS (renin, angiotensinogen, angiotensin II receptor) in the vicinity of the blood islands suggests that this system is involved in erythropoiesis. This role was confirmed by in vivo blockade of the RAS with fosinoprilate, a specific inhibitor of chicken ACE, which decreased hematocrit by 15%. A similar decrease in hematocrit was observed following treatment with the angiotensin II receptor antagonist Sar1-Ile8-Angiotensin II, suggesting that this effect was mediated by angiotensin II. Both treatments affected hematocrit by decreasing erythroblast proliferation. Thus, the RAS, and its effector peptide angiotensin II in particular, modulates primitive erythropoiesis.