RESUMEN
Members of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left-right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger-Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left-right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory.
Asunto(s)
Tipificación del Cuerpo , Péptidos y Proteínas de Señalización Intercelular , Glicoproteínas de Membrana , Proteínas/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Pollo , Coturnix/embriología , Factor de Crecimiento Epidérmico/genética , Proteínas Ligadas a GPI , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento/genética , Corazón/embriología , Humanos , Factores de Determinación Derecha-Izquierda , Mesodermo , Ratones , Modelos Biológicos , Morfogénesis , Proteínas de Neoplasias/genética , Proteína Nodal , Notocorda , Organizadores EmbrionariosRESUMEN
Advances in molecular biology and retinoic acid receptor research have significantly contributed to the understanding of the role of vitamin A during vertebrate development. Examination of the function of this vitamin during very early developmental stages using the completely vitamin A-depleted avian embryo has revealed that the vitamin A requirement begins at the time of formation of the primitive heart, circulation and specification of hindbrain. The lack of vitamin A at this critical time results in gross abnormalities and early embryonic death. In rodent models, vitamin A deficiency can be targeted to later gestational windows and documents the need for vitamin A for more advanced stages of development. Major target tissues of vitamin A deficiency include the heart, central nervous system and structures derived from it, the circulatory, urogenital and respiratory systems, and the development of skull, skeleton and limbs. These abnormalities are also evident in mice mutants from retinoid receptor knockouts; they have revealed both morphological and molecular aspects of vitamin A function during development. Retinoic acid receptors (RAR) in partnership with retinoid X receptor (RXR)alpha appear to be the important retinoid receptor transcription factors regulating vitamin A function at the gene level during development via the physiologic ligand all-trans-retinoic acid. Homeostasis of retinoic acid is maintained by developmentally regulated vitamin A metabolism enzyme systems. Inadequate vitamin A nutrition during early pregnancy may account for some pediatric congenital abnormalities.
Asunto(s)
Desarrollo Embrionario y Fetal/fisiología , Vitamina A/fisiología , Animales , Humanos , Ratones , Ratones Noqueados , Modelos Animales , Codorniz/embriología , Ratas , Receptores de Ácido Retinoico/fisiología , Tretinoina/metabolismo , Deficiencia de Vitamina A/fisiopatologíaRESUMEN
AIM: To study the abnormal expression of beta-catenin gene and its relationship ith invasiveness of primary hepatocellular carcinoma among Chinese people. METHODS: Thirty-four hepatocellular carcinoma (HCC) specimens and adjacent para-cancerous tissues, 4 normal liver tissues were immunohistochemically stained to study subcellular distribution of beta-catenin. Semiquantitive analysis of expression of beta-catenin gene exon 3 mRNA was examined by RT-PCR and in situ hybridization. The relationship between expressions of both beta-catenin protein, mRNA and clinicopathological characteristics of HCC was also analyzed. RESULTS: Immuno-histochemistry showed that all normal liver tissues and para-cancerous tissues examined displayed membranous type staining for beta-catenin protein, occasionally with weak expression in the cytoplasm. While 21 cases (61.8%) of HCC examined showed accumulated type in cytoplasms or nuclei. The accumulated type Labling Index (LI) of cancer tissue and para-cancerous tissue was (59.9 +/- 26.3) and (18.3 +/- 9.7) respectively (P<0.01). Higher accumulated type LI was closely related with invasiveness of HCC. Results of RT-PCR showed the beta-catenin gene exon 3 mRNA Expression Index (EI) of 34 HCCs was higher than that of para-cancerous tissue and normal liver tissue. Using in situ hybridization, the signal corresponding to beta-catenin gene exon 3 mRNA was particularly strong in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Over expression of beta-catenin exon 3 was also found to be correlated with high metastatic potential of HCC. CONCLUSION: Abnormal expression of beta-catenin gene may contribute importantly to the invasiveness of HCC among Chinese people.
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Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/fisiopatología , Proteínas del Citoesqueleto/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/fisiopatología , Transactivadores , Cadherinas/genética , Carcinoma Hepatocelular/genética , China , Proteínas del Citoesqueleto/análisis , Exones , Humanos , Inmunohistoquímica , Hígado/química , Hígado/patología , Neoplasias Hepáticas/genética , Invasividad Neoplásica , ARN Mensajero/análisis , beta CateninaRESUMEN
BACKGROUND: Retinoic acid (RA) is necessary for normal differentiation of the tail bud into the secondary neural tube. Excess RA, however, is teratogenic and causes neural tube defects (NTDs). The way in which RA modulates secondary neurulation is unclear but probably involves RA-regulated downstream genes such midkine (MK), which encodes a growth factor implicated in tail bud mesenchymal-neuroepithelial conversion. Our objective was to determine whether RA-deficiency would produce similar defects and if MK is involved. METHODS: Citral, a drug that blocks endogenous RA formation, as well as a neutralizing antibody, were used to block RA activity in chick embryos. Immunohistochemistry and in situ hybridization were used to localize RA and MK in the tail bud. Competitive RT-PCR was used to examine the effects of excess RA and RA deficiency due to citral on the expression of MK mRNA. RESULTS: Citral-induced NTDs displayed a morphological resemblance to those caused by excess RA. However, citral treatment did not significantly increase embryonic mortality, and RA rescue of citral-treated embryos proved unsuccessful. MK mRNA was detected in the differentiating tail bud by in situ hybridization. Competitive RT-PCR showed that excess RA decreased MK expression by 60%. Doses of citral that caused a comparable incidence of defects, however, caused only a 25% decrease. CONCLUSIONS: The results show that excess RA and RA deficiency both cause defects of secondary neurulation. While excess RA decreased MK expression, RA deficiency had minimal effects. However, whether or not MK is an intermediary in the developmental phenomena regulated physiologically or pathologically by RA remains to be elucidated.
Asunto(s)
Anomalías Inducidas por Medicamentos , Proteínas Portadoras/toxicidad , Citocinas , Queratolíticos/toxicidad , Monoterpenos , Factores de Crecimiento Nervioso/toxicidad , Defectos del Tubo Neural/inducido químicamente , Teratógenos , Tretinoina/fisiología , Tretinoina/toxicidad , Deficiencia de Vitamina A/complicaciones , Monoterpenos Acíclicos , Animales , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Inmunohistoquímica , Hibridación in Situ , Midkina , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cola (estructura animal)/embriología , Cola (estructura animal)/metabolismo , Terpenos/farmacología , Factores de Tiempo , Tretinoina/antagonistas & inhibidoresRESUMEN
Vitamin A-deficient (VAD) quail embryos have severe abnormalities, including a high incidence of reversed cardiac situs. Using this model we examined in vivo the physiological function of vitamin A in the left/right (L/R) cardiac asymmetry pathway. Molecular analysis reveals the expression of early asymmetry genes activin receptor IIa, sonic hedgehog, Caronte, Lefty-1, and Fgf8 to be unaffected by the lack of retinoids, while expression of the downstream genes nodal-related, snail-related (cSnR), and Pitx2 is altered. In VAD embryos nodal expression in left lateral plate mesoderm (LPM) is severely downregulated and the expression domain altered during neurulation. Similarly, the expression of cSnR in the right LPM and of Pitx2 in the left side posterior heart-forming region (HFR) is downregulated in the VAD embryos. The lack of retinoids does not cause randomization or ectopic expression of nodal, cSnR, or Pitx2. At the six- to eight-somite stage nodal is expressed transiently in the left posterior HFR of normal quail embryos; this expression is missing in VAD embryos and may be linked to the loss of Pitx2 expression in this region of VAD quail embryos. Administration of retinoids to VAD embryos prior to the six-somite stage rescues the expression of nodal, cSnR, and Pitx2 as well as the randomized VAD cardiac phenotype. There is an absolute requirement for retinoids at the four- to five-somite developmental window for cardiogenesis and cardiac L/R specification to proceed normally. We conclude that retinoids do not regulate the left/right-specific sidedness assignments for expression of genes on the vertebrate cardiac asymmetry pathway, but are required during neurulation for the maintenance of adequate levels of their expression and for the development of the posterior heart tube and a loopable heart. Cardiac asymmetry may be but one of several critical events regulated by retinoid signaling in the retinoid-sensitive developmental window.
Asunto(s)
Proteínas Aviares , Tipificación del Cuerpo , Corazón/embriología , Proteínas Nucleares , Retinoides/metabolismo , Transactivadores , Vitamina A/metabolismo , Receptores de Activinas Tipo II , Animales , Proteínas de Unión al ADN/aislamiento & purificación , Factor 8 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/aislamiento & purificación , Cardiopatías Congénitas/etiología , Proteínas Hedgehog , Proteínas de Homeodominio/aislamiento & purificación , Factores de Determinación Derecha-Izquierda , Proteína Nodal , Factores de Transcripción Paired Box , Proteínas/aislamiento & purificación , Codorniz , Receptores de Factores de Crecimiento/aislamiento & purificación , Transducción de Señal , Somitos , Distribución Tisular , Factores de Transcripción/aislamiento & purificación , Factor de Crecimiento Transformador beta/aislamiento & purificación , Deficiencia de Vitamina A , Proteína del Homeodomínio PITX2RESUMEN
Vitamin A deficiency and excess both cause abnormalities in mammalian longitudinal bone growth. Because all-trans retinoic acid (RA) is synthesized from vitamin A, we hypothesized that RA regulates growth plate chondrogenesis. Consistent with this hypothesis, a single oral dose of RA reduced the height of the rat proximal tibial growth plate. To determine whether RA acts directly on growth plate, fetal rat metatarsal bones were cultured in the presence of RA. In this system, RA inhibited longitudinal bone growth by three mechanisms: 1) decreased chondrocyte proliferation, (assessed by 3H-thymidine incorporation), particularly in the proliferative zone of the growth plate; 2) decreased matrix synthesis (assessed by 35SO4 incorporation into glycosaminoglycans); and 3) decreased cell hypertrophy (determined histologically). The growth-inhibiting effects of RA were completely reversed by a retinoic acid receptor (RAR) antagonist. In the absence of exogenous RA, this antagonist accelerated bone growth, as did an RA-specific neutralizing antibody, suggesting that endogenous RA negatively regulates growth plate chondrogenesis. We conclude that RA, acting through RARs, negatively regulates longitudinal bone growth by inhibiting growth plate chondrocyte proliferation, chondrocyte hypertrophy, and matrix synthesis.
Asunto(s)
Desarrollo Óseo/fisiología , Condrogénesis/fisiología , Placa de Crecimiento/fisiología , Tretinoina/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Huesos/anatomía & histología , Huesos/citología , Huesos/enzimología , Placa de Crecimiento/anatomía & histología , Placa de Crecimiento/enzimología , Histocitoquímica , Masculino , Huesos Metatarsianos/citología , Naftalenos/farmacología , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Receptores de Ácido Retinoico/antagonistas & inhibidores , Sulfatos/metabolismo , Timidina/metabolismo , Tretinoina/antagonistas & inhibidoresRESUMEN
Vitamin A is an essential micronutrient throughout the life cycle. Its active form, retinoic acid via retinoid receptors, is involved in signal transduction pathways regulating development. Both the lack and excess of vitamin A during embryonic development result in congenital malformations. Approaches to examine the function of vitamin A in embryonic development have included treatment with excess retinoids and the use of retinoid receptor knock-out mice, which have provided important insights into the complexity of the retinoid signaling system. A recently explored model is the retinoid ligand knock-out, i.e., the vitamin A-deficient embryo. Early development can be successfully examined in the vitamin A-deficient avian embryo, in which bioactive retinoids can rescue the deficient genotype as well as phenotype. In this model it has been possible to unequivocally link the physiological function of vitamin A to development of heart, embryonal circulatory and central nervous systems and the regulation of heart asymmetry. Several developmental genes regulated by endogenous vitamin A during early embryogenesis have been identified. Retinoid receptors and their endogenous ligands, the vitamin A-active forms, are present in the early embryo. It is the developmentally regulated biogeneration of the vitamin A-active forms via distinct spatio-temporal metabolic pathways that is critically linked to the initiation of retinoid signal transduction during embryonic development.
Asunto(s)
Desarrollo Embrionario y Fetal/fisiología , Vitamina A/fisiología , Animales , Humanos , Ratones , CodornizRESUMEN
We examined the effect of consumption of graded increases of dietary fiber (soft white wheat bran) on the development of mammary gland carcinomas in intact female Sprague-Dawley rats during the promotion stage of carcinogenesis, induced with 7,12-dimethylbenz(a)anthracene (DMBA). The percent of rats with mammary carcinomas, the total number of mammary carcinomas and the mean number of mammary carcinomas per rat were reduced significantly at all fiber levels examined compared to rats fed a control diet. Inclusion of 9.6% fiber in the diets of ovariectomized rats that had been treated with a single i.v. dose of 2.5 mg DMBA/100 g body weight 2 weeks prior to removal of the ovaries resulted in a significant decrease of carcinomatous and benign mammary tumors compared to ovariectomized rats fed a control diet. Development of spontaneous mammary carcinomas in virgin C3H/HeOuJ female mice and growth of a transplantable mammary gland tumor in such mice were reduced by inclusion of 9.6% fiber in the diet, a reduction that was significant or just barely missed significance, depending on the source of the fiber. Our observations provide evidence that inclusion of soft white wheat bran in the diet is effective in the suppression of mammary gland tumorigenesis in an array of experimental animal models.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno , Carcinógenos , Fibras de la Dieta/uso terapéutico , Neoplasias Mamarias Experimentales/prevención & control , Neoplasias Hormono-Dependientes/prevención & control , Ovario/fisiología , Animales , División Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C3H , Trasplante de Neoplasias , Neoplasias Hormono-Dependientes/inducido químicamente , Neoplasias Hormono-Dependientes/patología , Ovariectomía , Ovario/cirugía , Ratas , Ratas Sprague-Dawley , Triticum , Células Tumorales CultivadasRESUMEN
The effect of ethanol on early avian cardiovascular development was investigated in stage 8 quail embryos grown in culture for 24 hr. When the culture medium contained 1% ethanol, 50% of the embryos developed abnormalities of the cardiovascular system, some of which resembled vitamin A deficiency. Only 15% of the embryos grown in control media developed abnormalities attributed to the manipulation of the embryo. When all-trans-retinoic acid, the active form of vitamin A, was added at 10(-8) M to the ethanol-containing medium, the cardiovascular development was similar to that of untreated controls. Inclusion of 4-methylpyrazole and citral, enzyme inhibitors for the conversion of retinol to retinoic acid, produced cardiovascular abnormalities in embryos similar to those observed in vitamin A deficiency. These abnormalities were partially prevented by the presence of 10(-8) M all-trans-retinoic acid in the medium. Immunohistochemical studies using antibodies specific for the heart muscle myosin heavy chain (MF-20) and quail endothelial cells (QH-1) revealed that looping of the heart of ethanol-treated embryos was prevented, and the embryonal circulation had no or minimal vascular connections to the extraembryonic circulation. Our studies provide indirect evidence that ethanol is producing vitamin A deficiency during embryonic cardiovascular development and that these effects are specifically prevented by the presence of retinoic acid. These findings may explain some of the symptoms of fetal alcohol syndrome.
Asunto(s)
Etanol/antagonistas & inhibidores , Trastornos del Espectro Alcohólico Fetal/embriología , Cardiopatías Congénitas/embriología , Tretinoina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Embrión no Mamífero/efectos de los fármacos , Etanol/toxicidad , Codorniz , Deficiencia de Vitamina A/embriologíaRESUMEN
Retinoids were analyzed in 11-day chick embryos and eggs from white Leghorn hens (Gallus domesticus) fed environmentally-derived polychlorinated biphenyls (PCB) in carp (Cyprinus carpio) from Saginaw Bay. The yolks and the embryos contained all-trans-retinol, 3,4-didehydroretinol and retinyl esters. There was no significant difference in the total retinoid content in the yolks of 11-day incubated eggs among hens fed for 7 weeks diets containing 0.5-6.6 mg PCB/kg diet. However, the proportional amount of retinols in the high (6.6 mg) PCB group was significantly less than that in low (0.5 mg) PCB controls, while the amount of retinyl palmitate in the high PCB group was significantly greater than that in the controls. Retinoids in the embryos were not affected by any of the PCB levels fed to hens for 7 weeks prior to laying the eggs. The 50% reduction in the molar ratio of retinols to retinyl palmitate in the yolks of eggs as the result of the high PCB level fed to hens for 7 weeks can serve as an indicator for chronic exposure to PCB contamination at the level of 6.6 mg or higher PCB/kg diet.
RESUMEN
Both Hensen's node, the organizer center in chick embryo, and exogenous retinoic acid are known to induce limb duplication when grafted or applied to the host chick limb bud. Retinoic acid is known to be present in the node and has been proposed as the putative morphogen for chick limb development. Here, we report that Hensen's node from vitamin A-deficient quail embryo induces limb duplication in the host chick embryo similar to that induced by the node from vitamin A-sufficient control embryos. We also demonstrate that the expression of Sonic hedgehog (Shh), recently shown to be the mediator of polarizing activity in the chick limb bud, is not affected by the endogenous vitamin A status of the embryo. Furthermore, whole-mount in situ hybridization revealed asymmetry of Shh expression in the Hensen's node of both vitamin A-sufficient and -deficient quail embryos. Retinoids were not detectable in the eggs from which vitamin A-deficient embryos were obtained. Extracts from normal embryos induced a level of expression of reporter gene equivalent to the presence of 3.4 pg of active retinoids per embryo, while those from vitamin A-deficient embryos induced a baseline level of reporter gene expression similar to that of the controls. Our studies suggest that endogenous retinoic acid is not involved in Shh expression nor in regulating its asymmetry during normal early avian embryogenesis and support the current view that endogenous retinoic acid may not be a direct morphogen for limb bud duplication.
Asunto(s)
Inducción Embrionaria , Proteínas/metabolismo , Transactivadores , Tretinoina/metabolismo , Deficiencia de Vitamina A , Animales , Bioensayo , Embrión de Pollo , Cromatografía Líquida de Alta Presión , Coturnix , Extremidades/embriología , Genes Reporteros , Proteínas Hedgehog , Hibridación in Situ , Óvulo/química , Proteínas/genética , Retinoides/análisis , Trasplante de TejidosRESUMEN
The early embryo is initially bilaterally symmetrical. One of the first distinct indications of asymmetry in the embryo occurs during heart looping. The midline tubular heart begins to bend to the right to form a C-shaped structure around 30 hr of development in the avian model. A molecular basis for heart asymmetry and direction of looping is not known, although factors inherent to the myocardium are believed to underlie looping. A left-right asymmetric localization of a specific molecule in the bilateral heart forming regions has not been reported previously. One molecule that we are calling flectin (flectere, in L., to bend or to loop) shows a bilateral asymmetric localization early in the heart forming mesoderm and continues to be expressed asymmetrically in a highly organized manner in the cardiac jelly during heart looping. This large extracellular matrix molecule has been identified using a monoclonal antibody F-22 (Mieziewska et al., 1994a,b). Flectin shows a discrete spatiotemporal pattern of extracellular matrix expression during avian heart development. An asymmetric expression of flectin is observed during heart development at stage 7+/8- (approximately at 24 hr of development around the 3-somite stage). It is predominantly expressed in the left precardiac mesoderm at this developmental period. Between stages 12 and 14, flectin continues to be asymmetrically expressed in the myocardium and is localized at high levels on the basal side of the myocardium and within the cardiac jelly extending to the endocardial cell surfaces. In the same plane of the looping part of the heart it is differentially organized within the cardiac jelly on the convex side and in the outer loop areas. A reduced expression is apparent anteriorly and posteriorly along the tubular heart. The initial asymmetry of localization is maintained throughout the tubular heart. At stage 22 (Embryonic Day 3.5), intensity of immunolocalization of flectin is significantly decreased, with left-right asymmetry becoming less discernible or absent. It again is expressed in Day 10 embryonic hearts. Flectin expression appears to be modulated by retinoids. In vitamin A-deficient quail embryonic hearts that do not loop (Dersch and Zile, 1993; Twal et al., 1995), flectin protein expression is decreased and disorganized, as are other extracellular matrix components comprising the cardiac jelly.
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Proteínas de la Matriz Extracelular/aislamiento & purificación , Matriz Extracelular/química , Corazón/embriología , Miocardio/química , Animales , Anticuerpos Monoclonales , Embrión de Pollo , Epitelio/embriología , Proteínas de la Matriz Extracelular/inmunología , Inmunohistoquímica , Mesodermo , Distribución TisularRESUMEN
The vitamin A deficiency-associated lethal syndrome observed in avian embryos may be linked to dysfunction of vitamin A-dependent genes. We tested this hypothesis in a quail embryo model by examining the expression of retinoic acid receptors (RARs) and cytosolic retinoic acid binding protein (CRABP) in normal and vitamin A-deficient embryos during early development. RARα and RARγ mRNA were expressed at the same level in normal and vitamin A-deficient embryos during all stages studied. Expression of CRABP I was low in normal and vitamin A-deficient quail embryos during early development, but increased rapidly at later stages. Two transcripts of RARß, 3.2 and 3.5 kb, were detected in quail embryos during developmental stages 6-12. In normal emryos the level of the 3.2-kb isoform increased as embryonic development advanced. The expression of the 3.5-kb transcript was significantly decreased in vitamin A-deficient embryos, while the 3.2-kb transcript was undetectable by northern analysis. In situ hybridization of stage 7-8 normal quail embryos using a chicken RARß2 riboprobe revealed that RARß2 expression was predominantly associated with the cell populations in heart-forming regions, somites, neural folds, notochord and the presumptive thyroid. In stark contrast, in the vitamin A-deficient quail embryo RARß2 was not expressed in any of the above cell populations. We conclude that the expressions of RARß and CRABP I are developmentally regulated. Additionally, the expression of RARß2 during early embryogenesis is regulated by vitamin A status. We propose that RARß2 plays an important role in the mechanism of action of retinoids in early avian development. The lack of expression of RARß2 may be linked to abnormalities of the cardiovascular system.
RESUMEN
Nuclear retinoic acid receptor beta (RAR-beta) expression decreases in human premalignant oral lesions (POLs). RAR-beta suppression could result from a decrease in the cellular level of retinoids because RAR-beta gene transcription is enhanced by retinoids. To explore this hypothesis, we compared the binding of a monoclonal antibody (mAb) against all-transretinoic acid (RA; anti-RA mAbs) to normal oral tissue and POLs. All 7 normal specimens stained positive with the antibody compared to only 20 of 43 POLs; similarly, 7 of 7 normal specimens contained RAR-beta mRNA compared to only 14 of 43 POLs. Twenty-four specimens were available before and after a 3-month treatment with 13-cis-RA in vivo. Anti-RA mAb binding to these specimens increased from 10 of 24 before to 22 of 24 after treatment, and the expression of RAR-beta mRNA increased from 7 of 24 before to 21 of 24 after treatment, respectively. There was a strong agreement between the binding of anti-RA mAbs and the expression of RAR-beta. Thus, we propose that the binding of anti-RA mAbs reflects the level of retinoids in the tissues and that this level is related strongly to RAR-beta expression.
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Anticuerpos Monoclonales/metabolismo , Mucosa Bucal/metabolismo , Neoplasias de la Boca/metabolismo , Lesiones Precancerosas/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Humanos , Isotretinoína/uso terapéutico , Neoplasias de la Boca/tratamiento farmacológico , Lesiones Precancerosas/tratamiento farmacológico , ARN Mensajero/metabolismo , Tretinoina/inmunologíaRESUMEN
Vitamin A is required to rescue the vitamin A-deficient quail embryo from early death, but the endogenous presence of bioactive retinoids has not been documented in these embryos. The analysis of 2000 pooled stage 5-8 normal quail embryos described here provides for the first time direct evidence for the presence of endogenous all-trans-retinoic acid (5 nM) and 3,4-didehydroretinoic acid (4 nM), signaling molecules known to be potent ligands for nuclear retinoic acid receptors. The demonstration of all-trans-retinal (80 nM), all-trans-retinol (100 nM), 3,4-didehydroretinol (200 nM), and retinyl esters (100 nM) suggests the capability of the early avian embryo to generate in situ the vitamin A-bioactive molecules required for development. Analysis of 2100 pooled stage 5-8 quail embryos from vitamin A-deficient eggs revealed no vitamin A-like molecules, supporting the evidence that links vitamin A deficiency in these embryos to abnormalities and early death.
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Coturnix/embriología , Retinoides/metabolismo , Animales , Ligandos , Receptores de Ácido Retinoico/fisiología , Deficiencia de Vitamina A/embriologíaRESUMEN
Homeobox-containing genes may play an important role in establishing embryonic patterns during development of vertebrates. Retinoic acid is able to induce expression of Hox genes in cells in culture and to alter expression patterns in the developing vertebrate embryos. Using wholemount in situ hybridization, we have examined and compared the expression patterns of a homeobox-containing gene, Msx-1, in early normal and vitamin A-deficient quail embryos. At gastrulation stage, Msx-1 is primarily expressed in the posterior half of both normal and vitamin A-deficient embryos. However, the gene is expressed wider and stronger in the vitamin A-deficient embryos. At neurulation stages, Msx-1 is continuously expressed in the posterior region up to Hensen's node and in the edge of the neural fold in both normal and vitamin A-deficient embryos. Notably, in the vitamin A-deficient embryos, Msx-1 is expressed more strongly and is also expressed ectopically in the anterior and precardiac regions. These results provide evidence that endogenous retinoids are involved in the normal expression of Msx-1 in avian embryo and that the expression of Msx-1 is downregulated by endogenous and physiological retinoids in vivo during early avian embryogenesis.
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Coturnix/genética , Proteínas de Homeodominio/genética , Factores de Transcripción , Deficiencia de Vitamina A/genética , Animales , Coturnix/embriología , Embrión no Mamífero/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hibridación in Situ , Factor de Transcripción MSX1 , Tretinoina/farmacologíaRESUMEN
Avian cardiovascular development is vitamin A-dependent, and retinoic acid has been suggested to be active in this important developmental event. We report here that a monoclonal antibody against all-trans-retinoic acid blocks normal embryonic development in the quail causing cardiovascular abnormalities typical of avian vitamin A deficiency. In whole-mount preparations of stage 5 normal quail embryos the fluorescence associated with the antiretinoic acid monoclonal antibody localizes in Hensen's node and in caudal area. In stage 7-8 embryos fluorescence localizes in heart-forming areas as well as in head mesenchyme, in Hensen's node, in nephrotome, and in caudal area. These studies are the first to localize endogenous all-trans-retinoic acid during very early stages of normal avian development. We propose that all-trans-retinoic acid is biosynthesized in its target cells during early avian embryo-genesis and that the availability of this signal molecule is spatiotemporally regulated. We conclude that all-trans-retinoic acid or a closely related metabolite is the physiological form of vitamin A required for normal cardiovascular development and for other very early developmental events in the quail embryo.
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Codorniz/embriología , Tretinoina/análisis , Animales , Anticuerpos Monoclonales/farmacología , Sistema Cardiovascular/efectos de los fármacos , Sistema Cardiovascular/metabolismo , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Codorniz/metabolismo , Tretinoina/inmunologíaRESUMEN
In AEV-transformed erythroleukemic cells the v-erbA gene product is likely to antagonize the function of triiodothyronine (T3) and retinoic acid (RA) receptors and thereby to block cell differentiation. We have thus investigated the effects of T3 and RA on normal early erythrocytic progenitor cells. Here we show: (1) that either RA or T3 play an essential role during the early commitment to erythrocytic differentiation, (2) that both T3 and RA induce death by apoptosis and a strong inhibition of self-renewal in progenitor cells grown in the absence of differentiation-inducing agents and (3) that the v-erbA oncogene renders erythrocytic progenitor cells insensitive to apoptosis and to self-renewal inhibition induced by RA or T3. The behaviour of a non-transforming mutant of v-erbA suggests that this v-erbA-induced protection is related to its transforming potential.
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Células Madre Hematopoyéticas/citología , Receptores de Ácido Retinoico/fisiología , Receptores de Hormona Tiroidea/fisiología , Proteínas Oncogénicas de Retroviridae/fisiología , Animales , Apoptosis/genética , Secuencia de Bases , Diferenciación Celular/genética , Pollos , Cartilla de ADN , Eritrocitos/citología , Datos de Secuencia Molecular , Proteínas Oncogénicas v-erbA , ARN Mensajero/metabolismo , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/genética , Receptores de Hormona Tiroidea/antagonistas & inhibidores , Receptores de Hormona Tiroidea/genética , Proteínas Oncogénicas de Retroviridae/genética , Tretinoina/farmacología , Triyodotironina/farmacología , Células Tumorales CultivadasRESUMEN
The biological activity of various natural retinoids and the time "window" when vitamin A activity is required for normal cardiovascular development were examined in vitamin A-deprived Japanese quail embryos. The administration of 1 microgram of retinol at the beginning of incubation resulted in normal cardiovascular development in 97% of embryos; retinoic acid was toxic at this dose level. Treatment of embryos with 0.1 microgram of all-trans-retinol or 13-cis-retinoic acid at the beginning of incubation resulted in normal cardiovascular development in 47 and 12% of embryos, respectively; administration of these retinoids at other time points attenuated the percentage of embryos with normal cardiovascular development. Didehydroretinol, 0.1 microgram, and 9-cis-retinoic acid, 0.1 microgram, were inactive at all time points examined; 9-cis-retinoic acid did not enhance the biological activity of all-trans-retinoic acid. All-trans-retinoic acid, 0.1 microgram, administered during 22-28 hr of incubation induced normal cardiovascular development in 20-34% of embryos; biological activity was optimal when it was administered at 24 hr. All retinoids tested were inactive in establishing normal cardiovascular development when administered at 36 hr of incubation or later. The studies suggest that all-trans-retinoic acid is the biologically active form of vitamin A required for normal cardiovascular development in the avian embryo. There is a critical time point within the first 22-28 hr of quail embryogenesis when all-trans-retinoic acid initiates events that lead to normal cardiovascular development.
Asunto(s)
Sistema Cardiovascular/embriología , Coturnix/embriología , Retinoides/farmacología , Animales , Embrión no Mamífero/fisiología , Desarrollo Embrionario , Inducción Embrionaria/fisiología , Femenino , Deficiencia de Vitamina A/embriologíaRESUMEN
Retinoyl beta-D-glucuronide is a biologically active metabolite of retinoic acid. The kinetics of UDP-glucuronosyltransferase-catalyzed biosynthesis of retinoyl beta-D-glucuronide was examined in rat liver and intestinal native microsomes incubated with [3H retinoic acid incorporated into liposomes. The product was identified by cochromatography with authentic all-trans retinoyl beta-D-glucuronide, by hydrolysis with beta-D-glucuronidase, and by mass spectrometry. In vitamin A-sufficient rats the apparent Km values for all-trans-retinoic acid were 173 microM and 125 microM, and the apparent Vmax, 62 and 41 pmol/min per mg, for small intestinal and liver microsomes, respectively. In vitamin A-deficient rats repleted with all-trans-retinyl acetate, the apparent Km (91 microM) and Vmax (53 pmol/min per mg) for intestinal microsomes were in range of those of vitamin A-sufficient rats. The similarities in the kinetic parameters for UDP-glucuronosyltransferase in small intestinal mucosa and liver suggest that the reactions are catalyzed by the same enzyme. In vitamin A-deficient rats given a large amount all-trans-retinoic acid (1.2 mmol/day for 3 days) the apparent Km was 105 microM and Vmax, 127 pmol/min per mg of intestinal microsomal protein. We conclude that the kinetics of intestinal retinoic acid glucuronidation are not characteristic of simple detoxification reactions. Retinoyl glucuronide may be important in mediating retinoic acid metabolism and function.