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1.
J Perinatol ; 37(3): 260-264, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-27929530

RESUMEN

OBJECTIVE: The objective of the study was to measure the effects of a 5-min delay (DCC) versus immediate cord clamping (ICC) on residual placental blood volume (RPBV) at birth, and hemoglobin and serum bilirubin at 24 to 48 h of age. STUDY DESIGN: In this prospective randomized controlled trial, 73 women with term (37 to 41 weeks) singleton fetuses were randomized to DCC (⩾5 min; n=37) or ICC (<20 s; n=36). RESULTS: Maternal and infant demographics were not different between the groups. Mean cord clamping time was 303±121 (DCC) versus 23±59 (ICC) s (P<0.001) with 10 protocol violations. Cord milking was the proxy for DCC (n=11) when the provider could not wait. Infants randomized to DCC compared with ICC had significantly less RPBV (20.0 versus 30.8 ml kg-1, P<0.001), higher hemoglobin levels (19.4 versus 17.8 g dl-1, P=0.002) at 24 to 48 h, with no difference in bilirubin levels. CONCLUSION: Term infants had early hematological advantage of DCC without increases in hyperbilirubinemia or symptomatic polycythemia.


Asunto(s)
Bilirrubina/sangre , Hemoglobinas/análisis , Circulación Placentaria , Nacimiento a Término/sangre , Cordón Umbilical/irrigación sanguínea , Adulto , Volumen Sanguíneo , Constricción , Parto Obstétrico/métodos , Femenino , Hematócrito , Humanos , Recién Nacido , Masculino , Placenta/irrigación sanguínea , Embarazo , Estudios Prospectivos , Rhode Island , Factores de Tiempo , Adulto Joven
2.
Neuroscience ; 324: 69-81, 2016 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-26964679

RESUMEN

Inter-alpha Inhibitor Proteins (IAIPs) are a family of related serine protease inhibitors. IAIPs are important components of the systemic innate immune system. We have identified endogenous IAIPs in the central nervous system (CNS) of sheep during development and shown that treatment with IAIPs reduces neuronal cell death and improves behavioral outcomes in neonatal rats after hypoxic-ischemic brain injury. The presence of IAIPs in CNS along with their exogenous neuroprotective properties suggests that endogenous IAIPs could be part of the innate immune system in CNS. The purpose of this study was to characterize expression and localization of IAIPs in CNS. We examined cellular expressions of IAIPs in vitro in cultured cortical mouse neurons, in cultured rat neurons, microglia, and astrocytes, and in vivo on brain sections by immunohistochemistry from embryonic (E) day 18 mice and postnatal (P) day 10 rats. Cultured cortical mouse neurons expressed the light chain gene Ambp and heavy chain genes Itih-1, 2, 3, 4, and 5 mRNA transcripts and IAIP proteins. IAIP proteins were detected by immunohistochemistry in cultured cells as well as brain sections from E18 mice and P10 rats. Immunoreactivity was found in neurons, microglia, astrocytes and oligodendroglia in multiple brain regions including cortex and hippocampus, as well as within both the ependyma and choroid plexus. Our findings suggest that IAIPs are endogenous proteins expressed in a wide variety of cell types and regions both in vitro and in vivo in rodent CNS. We speculate that endogenous IAIPs may represent endogenous neuroprotective immunomodulatory proteins within the CNS.


Asunto(s)
alfa-Globulinas/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/crecimiento & desarrollo , Células Cultivadas , Inmunohistoquímica , Ratones Endogámicos C57BL , Microglía/citología , Microglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/metabolismo , Ratas Wistar
3.
J Perinatol ; 33(2): 147-53, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22678140

RESUMEN

OBJECTIVE: Hospital length of stay (LOS) is important to administrators and families of neonates admitted to the neonatal intensive care unit (NICU). A prediction model for NICU LOS was developed using predictors birth weight, gestational age and two severity of illness tools, the score for neonatal acute physiology, perinatal extension (SNAPPE) and the morbidity assessment index for newborns (MAIN). STUDY DESIGN: Consecutive admissions (n=293) to a New England regional level III NICU were retrospectively collected. Multiple predictive models were compared for complexity and goodness-of-fit, coefficient of determination (R (2)) and predictive error. The optimal model was validated prospectively with consecutive admissions (n=615). Observed and expected LOS was compared. RESULT: The MAIN models had best Akaike's information criterion, highest R (2) (0.786) and lowest predictive error. The best SNAPPE model underestimated LOS, with substantial variability, yet was fairly well calibrated by birthweight category. LOS was longer in the prospective cohort than the retrospective cohort, without differences in birth weight, gestational age, MAIN or SNAPPE. CONCLUSION: LOS prediction is improved by accounting for severity of illness in the first week of life, beyond factors known at birth. Prospective validation of both MAIN and SNAPPE models is warranted.


Asunto(s)
Enfermedades del Recién Nacido/diagnóstico , Unidades de Cuidado Intensivo Neonatal/estadística & datos numéricos , Tiempo de Internación/estadística & datos numéricos , Modelos Estadísticos , Benchmarking , Peso al Nacer , Estudios de Cohortes , Femenino , Edad Gestacional , Humanos , Recién Nacido , Enfermedades del Recién Nacido/terapia , Masculino , Análisis Multivariante , New England , Valor Predictivo de las Pruebas , Análisis de Regresión , Estudios Retrospectivos , Factores de Riesgo , Índice de Severidad de la Enfermedad
4.
J Comp Pathol ; 148(2-3): 252-8, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22819013

RESUMEN

Cytokines have gained increasing attention as therapeutic targets in inflammation-related disorders and inflammatory conditions have been investigated in sheep. Monoclonal antibodies (mAbs) specific for the ovine pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 could be used to study the effects of blocking pro-inflammatory cytokines in sheep. Ovine-specific IL-1ß and IL-6 proteins and mAbs specific for these molecules were produced and the ability of the mAbs to neutralize the proteins was tested in cultures of ovine splenic mononuclear cells. Expression of nuclear factor (NF)-κß and signal transducer and activator of transcription (STAT)-3 was evaluated by western blotting and densitometric quantification. Treatment with purified IL-1ß and IL-6 proteins increased NF-κß (P < 0.001) and STAT-3 (P < 0.01) expression, respectively, in cell culture. Treatment with these proteins that were pre-incubated with IL-1ß and IL-6 mAbs attenuated (P < 0.01) these effects. These results confirm the bioactivity of ovine IL-1ß and IL-6 proteins and the neutralizing capacity of anti-ovine-IL-1ß and -IL-6 mAbs in vitro. These mAbs could be used to investigate anti-inflammatory strategies for attenuation of the effects of these pro-inflammatory cytokines in sheep.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Citocinas/inmunología , Citocinas/metabolismo , Bazo/efectos de los fármacos , Bazo/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Células Cultivadas , Técnicas In Vitro , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/veterinaria , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Modelos Animales , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismo , Ovinos , Enfermedades de las Ovejas/tratamiento farmacológico , Enfermedades de las Ovejas/metabolismo , Bazo/citología
5.
J Dev Orig Health Dis ; 3(6): 395-408, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25084292

RESUMEN

Advances in understanding the molecular basis of behavior through epigenetic mechanisms could help explain the developmental origins of child mental health disorders. However, the application of epigenetic principles to the study of human behavior is a relatively new endeavor. In this paper we discuss the 'Developmental Origins of Health and Disease' including the role of fetal programming. We then review epigenetic principles related to fetal programming and the recent application of epigenetics to behavior. We focus on the neuroendocrine system and develop a simple heuristic stress-related model to illustrate how epigenetic changes in placental genes could predispose the infant to neurobehavioral profiles that interact with postnatal environmental factors potentially leading to mental health disorders. We then discuss from an 'Evo-Devo' perspective how some of these behaviors could also be adaptive. We suggest how elucidation of these mechanisms can help to better define risk and protective factors and populations at risk.


Asunto(s)
Epigénesis Genética , Trastornos Mentales/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Niño , Desarrollo Fetal , Humanos , Trastornos Mentales/etiología , Sistemas Neurosecretores/fisiología , Polimorfismo de Nucleótido Simple
6.
Placenta ; 31(6): 528-34, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20421131

RESUMEN

LAT-1 (L-type amino acid transporter 1) is a system L, Na(+)-independent amino acid transporter responsible for transport of large neutral amino acids. Dysregulated expression of LAT-1 is characteristic of many primary human cancers and it's over expression is related to tumor invasion. LAT-1 is highly expressed in the trophoblast giant cells (TGCs) at the time of implantation. Since trophoblast giant cells are highly invasive during the process of endometrial implantation and placentation, LAT-1 may play a role in the invasive phenotype. Our objectives were to identify the effects of increased and decreased LAT-1 expression on mouse trophoblast invasion. We therefore examined the role of amino acid deprivation, pharmacologic blockade specific to leucine transport and gene silencing (siRNA) on LAT-1 expression and trophoblast cell invasion. We utilized mouse primary trophoblast stem (TS) cells. LAT-1 mRNA expression was quantified by real time qPCR, protein by Western blotting and cell invasion was measured in Transwell plates through Matrigel. Amino acid transport using uptake of tritiated leucine. Under limited leucine availability and/or pharmacologic blockage, LAT-1 gene expression was significantly increased, p<0.05. This was associated with a 3-fold increase in cell invasion, p<0.05. In contrast, following siRNA-mediated gene silencing decreased LAT-1 expression (both mRNA and protein) was associated with decreased cell invasion and decreased leucine uptake, p<0.05. Upregulation of LAT-1 gene expression via limited amino acid availability or following pharmacologic blockade of transport leads to an increase in mouse trophoblast stem cell invasiveness. Downregulation of LAT-1 expression via genetic silencing leads to inhibition of invasiveness. These results demonstrate that LAT-1 plays an important role in trophoblast invasion.


Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Trofoblastos/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+L , Análisis de Varianza , Animales , Western Blotting , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Expresión Génica , Leucina/farmacología , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre , Factores de Tiempo , Trofoblastos/efectos de los fármacos
7.
J Perinatol ; 30(1): 11-6, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19847185

RESUMEN

OBJECTIVE: The results from our previous trial revealed that infants with delayed cord clamping (DCC) had significantly lesser intraventricular hemorrhage (IVH) and late-onset sepsis (LOS) than infants with immediate cord clamping (ICC). A priori, we hypothesized that infants with DCC would have better motor function by 7 months corrected age. STUDY DESIGN: Infants between 24 and 31 weeks were randomized to ICC or DCC and follow-up evaluation was completed at 7 months corrected age. RESULT: We found no differences in the Bayley Scales of Infant Development (BSID) scores between the DCC and ICC groups. However, a regression model of effects of DCC on motor scores controlling for gestational age, IVH, bronchopulmonary dysplasia, sepsis and male gender suggested higher motor scores of male infants with DCC. CONCLUSION: DCC at birth seems to be protective of very low birth weight male infants against motor disability at 7 months corrected age.


Asunto(s)
Discapacidades del Desarrollo/prevención & control , Recién Nacido de muy Bajo Peso , Atención Perinatal , Cordón Umbilical/cirugía , Desarrollo Infantil , Constricción , Femenino , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , Masculino , Factores Sexuales
8.
Placenta ; 30(3): 270-6, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19193433

RESUMEN

OBJECTIVES: LAT-1 (L-type amino acid transporter 1) is a system L, Na(+)-independent amino acid transporter responsible for transport of large neutral amino acids. Dysregulated expression of LAT-1 is characteristic of many primary human cancers and is related to tumor invasion. Primary rat hepatocytes in culture increase LAT-1 mRNA in response to amino acid depletion. Transformed hepatic cell lines demonstrate constitutive expression of LAT-1. These observations suggest that LAT-1 expression confers a growth and survival advantage under limited amino acid availability. LAT-1 is highly expressed in the placenta. It has been shown previously that amino acids are fundamental regulators of cell function and energy metabolism in pre-implantation embryos. Our objectives were to analyze qualitatively and quantitatively LAT-1 expression in pre-implantation stages of mouse embryo development and to identify cell types expressing LAT-1 in post-implantation stages. METHODS: LAT-1 was quantified by real-time qPCR. Localization of expression was by laser capture microdissection, in situ hybridization and immunohistochemistry. RESULTS: Our results show increasing mRNA levels of LAT-1 as the embryo develops from zygote to blastocyst with highest levels at hatching blastocyst. Expression studies of LAT-1 on microdissected samples from developing mouse placenta show highest levels of LAT-1 mRNA in trophoblast giant cells (TGCs) at the time of implantation (E7.5), followed by maternal decidua, ectoplacental cone and epiblast. At later stages of development (E9.5 and E11.5) no differential expression of LAT-1 was observed. In situ hybridization and immunohistochemistry also showed differential expression of LAT-1 mRNA and protein, respectively, with darkest staining in TGCs at E7.5. By E9.5 and E11.5 mRNA expression was no longer preferentially localized to TGCs, hybridization was equal across the different cell types and regions. LAT-1 protein expression, however, still showed highest intensity of staining in TGCs at E9.5 and E11.5. CONCLUSIONS: Since trophoblast giant cells are invasive cells that displace and phagocytose the uterine epithelial cells, these data suggest that LAT-1 may play a role in the invasive phenotype. The mechanism of LAT-1 regulation during placentation, therefore, might provide valuable clues to its role in tumor progression and invasion.


Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Implantación del Embrión , Embrión de Mamíferos/metabolismo , Placenta/metabolismo , Sistema de Transporte de Aminoácidos y+L , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Ratones , Microdisección , Placentación , Reacción en Cadena de la Polimerasa , Embarazo
9.
Am J Physiol Regul Integr Comp Physiol ; 281(6): R1966-74, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11705783

RESUMEN

The placenta expresses high levels of 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) that converts cortisol into inactive 11-keto metabolites and effectively protects the developing fetus from maternal cortisol during pregnancy. Impairment of this glucocorticoid barrier has adverse effects on fetal outcomes. A similar spectrum of adverse fetal effects is induced by antenatal stress during pregnancy. To examine the hypothesis that physiological stress may regulate placental 11betaHSD2 gene expression, we examined the effects of the catecholamines norepinephrine (NE) and epinephrine (E) on 11betaHSD2 expression in human trophoblastic cells. With the use of Northern blotting and semiquantitative RT-PCR, we determined that NE and E rapidly downregulate 11betaHSD2 steady-state mRNA levels in early- and late-gestation human trophoblasts and BeWo trophoblastic cells. Experiments using different adrenoceptor subtype-selective agonists and antagonists demonstrated that this catecholamine suppression of 11betaHSD2 mRNA expression is mediated via both alpha(1)- and alpha(2)-adrenoceptors and is independent of beta-adrenergic stimulation. To examine transcriptional regulation, BeWo cells were transiently transfected with a reporter construct in which an 11betaHSD2 human promoter sequence was inserted upstream of the luciferase gene. Treatment with 10(-7) M NE decreased luciferase activity by ~60% (n = 3, P < 0.01). These results suggest the NE/E-mediated decrease in placental 11betaHSD2 gene expression is an instance of alpha-adrenoceptor-specific rapid transcriptional inhibition of an adrenergic target gene. This molecular mechanism may be involved in the deleterious effects of antenatal physiological stress on fetoplacental growth and development.


Asunto(s)
Antagonistas Adrenérgicos alfa/farmacología , Azepinas/farmacología , Hidroxiesteroide Deshidrogenasas/genética , Placenta/enzimología , Receptores Adrenérgicos alfa/fisiología , Trofoblastos/enzimología , Línea Celular , Células Cultivadas , Cesárea , Vellosidades Coriónicas/enzimología , Cartilla de ADN , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Edad Gestacional , Humanos , Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Norepinefrina/farmacología , Reacción en Cadena de la Polimerasa , Embarazo , Primer Trimestre del Embarazo , Propanolaminas/farmacología , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transcripción Genética
10.
Pediatr Res ; 50(5): 575-80, 2001 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11641450

RESUMEN

The classical model of gene regulation by hormones involves a hormone-bound receptor interacting with a DNA response element to increase or decrease gene transcription. Steroid hormone regulation more commonly involves atypical cis-elements, co-receptors, accessory proteins, and unique modes of interaction on different genes. The thyroid hormone and retinoic acid receptors belong to the super family of steroid nuclear receptors and may modify gene expression even in the absence of ligand binding. In these studies, we characterized thyroid receptor- and retinoic acid receptor-mediated regulation of beta1 adrenergic receptor (beta1AR) gene expression. Using cloned fragments of the ovine beta1AR in a luciferase reporter vector, we examined the effects of thyroid receptor and retinoic acid receptor, alone and in combination with T3 or retinoic acid on beta1AR expression. We examined expression in SK-N-SH neuroblastoma cells, CV-1 fibroblasts, and, in neonatal rat, primary cardiomyocytes. We demonstrated that even in the absence of ligand binding, thyroid receptor and retinoic acid receptor can significantly increase beta1AR transcription activity. This effect is important in the developmental transition in beta1AR expression during fetal and postnatal life.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Receptores Adrenérgicos beta 1/genética , Receptores de Ácido Retinoico/fisiología , Receptores de Hormona Tiroidea/fisiología , Transcripción Genética/fisiología , Animales , Línea Celular , Humanos , Ligandos , Ratas , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/metabolismo
11.
FASEB J ; 15(11): 1921-6, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11532972

RESUMEN

Cardiomyocyte development switches from hyperplasmic to hypertrophic growth between postnatal days 3 and 4 in rats. The mechanisms responsible for this transition have been controversial. beta-Adrenergic receptor (betaAR) activation of mitogenic responses in vitro has been reported. We hypothesized that tonic activation of the betaAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling kinases known to be important in cell cycle regulation. The purpose of the current study was to elucidate the roles of betaAR in rat cardiomyocyte growth in vivo. We demonstrated that betaAR blockade induced a significant reduction in cardiomyocyte proliferation as measured by the BrdU labeling index. Blockade of betaAR did not affect p38 or p44/42 MAPK activities. We further demonstrated that betaAR blockade induced a prompt deactivation of the p70 ribosomal protein S6 kinase (p70 S6K). To confirm these results, we measured p70 S6K activity directly. Basal activity of p70 S6K in neonatal cardiomyocytes was fourfold higher than that of insulin-treated adult rat liver. The activity of p70 S6K was reduced by 60% within 1 min after betaAR blockade. We conclude that the betaAR are involved in regulation of neonatal cardiomyocyte proliferation and that this mitogenic control may be mediated via the p70 S6K pathway.


Asunto(s)
Miocardio/citología , Receptores Adrenérgicos beta/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Antagonistas Adrenérgicos beta/farmacología , Animales , Animales Recién Nacidos , División Celular , Femenino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Propranolol/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos
12.
Am J Obstet Gynecol ; 185(1): 190-7, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11483927

RESUMEN

OBJECTIVE: Previously we have shown that neonatal lung function in sheep after preterm birth is profoundly enhanced by intra-amniotic injection of endotoxin, with a magnitude at least equal to that induced by maternal betamethasone administration. This study investigated the effects of betamethasone on lung maturation and growth in the presence of inflammation by treating sheep with both maternal intramuscular betamethasone and intra-amniotic endotoxin injections. STUDY DESIGN: Time-mated pregnant ewes at 118 days' gestation were allocated at random to receive maternal intramuscular or intra-amniotic saline solution injection (n = 10), maternal intramuscular betamethasone injection (0.5 mg/kg; n = 7), intra-amniotic endotoxin injection (20 mg Escherichia coli B055;B5; n = 11) by ultrasonographic guidance, or both betamethasone and endotoxin injections (n = 7). The lambs were delivered abdominally at 125 days' gestation (term is 150 days' gestation), and the neonates were ventilated for 40 minutes before postmortem examination. RESULTS: Combined treatment with betamethasone and endotoxin resulted in significantly greater improvements in neonatal lung function than occurred after treatment with either agent alone, and this effect was not accompanied by a further increase in surfactant levels. The reduction in birth weight that is seen after maternal betamethasone treatment was not seen when this treatment was combined with endotoxin. Endotoxin treatment resulted in inflammatory responses in cord blood and alveolar wash, and these responses were not inhibited by betamethasone treatment. There were no pregnancy losses. CONCLUSION: Both intra-amniotic endotoxin injection and maternal intramuscular betamethasone injection promoted fetal lung maturation. When these treatments were combined, there were additive effects on short-term postnatal lung function but not on surfactant levels. Endotoxin negated the growth restriction in sheep caused by maternal betamethasone treatment. These findings provide evidence that the lung maturation induced by glucocorticoids and that induced by endotoxin are mediated by different mechanisms.


Asunto(s)
Endotoxinas/farmacología , Glucocorticoides/farmacología , Pulmón/fisiología , Reacción de Fase Aguda , Animales , Animales Recién Nacidos/fisiología , Betametasona/administración & dosificación , Betametasona/farmacología , Peso al Nacer , Dióxido de Carbono/sangre , Interacciones Farmacológicas , Endotoxinas/administración & dosificación , Escherichia coli , Femenino , Sangre Fetal/química , Edad Gestacional , Concentración de Iones de Hidrógeno , Recuento de Leucocitos , Pulmón/efectos de los fármacos , Pulmón/embriología , Linfocitos , Neutrófilos , Oxígeno/sangre , Embarazo , Ovinos
13.
Mol Cell Endocrinol ; 181(1-2): 165-78, 2001 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-11476950

RESUMEN

The effects of glucocorticoids on expression of the beta1-adrenergic receptor (beta1AR) gene have been varied. To study the mechanism underling hormonal regulation of the beta1AR, transient transfection of progressively deleted ovine beta1AR promoter fragments was used to identify a 43-bp region (-1274 to -1232 from the translation start site) that contains a novel glucocorticoid regulatory unit (GRU) and confers glucocorticoid responsiveness. Using DNase I footprinting and electrophoretic mobility shift assays (EMSA), we demonstrated the GRU was composed of a palindrome, 5'-TAATTA-3', which is a core binding motif for the homeodomain proteins, an E-box (5'-CACGTG-3'), binding site for the Myc/Max family proteins, and an overlapping glucocorticoid response element (GRE) half-site (5'-TGTTCT-3'). EMSA demonstrated that the GRE half-site is critical for GRU-protein interactions, which also require binding of proteins to the E-box and the homeodomain region. Co-transfection of a plasmid expressing a c-myc antisense construct significantly reduced glucocorticoid responsiveness of the ovine beta1AR promoter. Furthermore, expression of proteins binding to the GRU was shown to be developmentally regulated, being high in embryonic, reduced in newborn and not detectable in adult heart. We conclude that the ovine beta1AR promoter contains a novel, functional GRU and that glucocorticoid receptor (GR) and the Myc/Max family proteins are involved in the cell-specific nuclear factor binding and transactivation via this element. The results suggest an alternative pathway through which glucocorticoids may exert their effects on genes lacking a full consensus GRE.


Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Receptores Adrenérgicos beta 1/genética , Elementos de Respuesta/genética , Ovinos/genética , Factores de Transcripción , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Línea Celular , ADN/genética , ADN/metabolismo , Huella de ADN , Sondas de ADN/genética , Sondas de ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ratas
14.
Biochim Biophys Acta ; 1518(3): 267-70, 2001 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-11311938

RESUMEN

The rat LAT-1 (L-amino acid transporter-1) gene is a CD98 light chain highly expressed in cancer and development. As an initial study of the molecular basis underlying regulation of its expression, we cloned 2 kb of the LAT-1 5' flanking region. Inverse RACE and primer extension methods were used to define the transcription initiation site at 80 bp upstream from the translational start site. Functional studies carried out in normal hepatic cells using constructs containing progressive 5' deletion from region -1958 to -185 showed 3-5-fold beta-galactosidase activities over control. The presence of an activator site(s) between -52 and -185 was indicated by low activities conferred by the construct spanning this region.


Asunto(s)
Antígenos CD/genética , Proteínas Portadoras/genética , Regiones Promotoras Genéticas , Animales , Antígenos CD/química , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/química , Línea Celular , Clonación Molecular , Proteína-1 Reguladora de Fusión , Regulación de la Expresión Génica , Biblioteca Genómica , Ratones , Datos de Secuencia Molecular , Ratas , Transfección
15.
Placenta ; 22(2-3): 162-70, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11170820

RESUMEN

We developed a model of maternal hyperglycaemia with secondary hyperinsulinaemia and hypoaminoacidaemia in pregnant sheep (H) to determine the effect of these conditions on uterine, uteroplacental and fetal amino-acid uptake rates and fetal amino-acid concentrations [AA]. Results were compared with normal pregnant ewes (C). Plasma glucose concentrations were greater in H versus C animals: 7.7+/-0.3 versus 3.9+/-0.1 mmol/l maternal, P< 0.005; 2.6+/-0.1 versus 1.1+/-0.1 mmol/l fetal, P< 0.005. Maternal insulin concentrations [I] were greater in the H group (132+/-30 H versus 31+/-5 C microU/ml, P< 0.005); fetal [I] were not different (15+/-2 H versus 16+/-2 C microU/mL). Maternal [AA] were lower in H than C groups except for SER (P=ns) and GLY (approx twofold higher, P< 0.01). Uterine, uteroplacental and fetal uptake rates of several AA, particularly the branch chain AA, were lower in H than C animals, producing lower total fetal nitrogen uptake rates (270+/-64 mg N/kg fetus/day H, 696+/-75 mg N/kg fetus/day C, P=0.001) and lower fetal plasma concentrations for the branch chain AA. Most fetal [AA], however, remained at control values, which could occur by relative increase in fetal amino-acid production and/or decrease in utilization, but not by increased uteroplacental transport rates.


Asunto(s)
Aminoácidos/deficiencia , Aminoácidos/metabolismo , Placenta/metabolismo , Aminoácidos/sangre , Animales , Transporte Biológico , Femenino , Feto/metabolismo , Edad Gestacional , Hiperglucemia/metabolismo , Hiperinsulinismo/metabolismo , Nitrógeno/metabolismo , Embarazo , Ovinos , Factores de Tiempo
16.
Brain Res Mol Brain Res ; 83(1-2): 128-32, 2000 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-11072103

RESUMEN

We isolated a 2.5-kb fragment of the promoter for the rat norepinephrine transporter (NET) gene. The transcription start site was identified approximately 200 base pairs upstream from the translation start site. Several potential regulatory elements were identified by sequence analysis. The structure of the rat NET promoter was compared to mouse and human. Expression studies in placental and neuroblastoma cells suggested the presence of a 'repressor' element more than 500 base pairs upstream from the transcription start site. These studies provide the basis for examination of transcriptional regulation of this gene and for understanding its temporal and tissue-specific modes of regulation.


Asunto(s)
Proteínas Portadoras/genética , Regiones Promotoras Genéticas/genética , Simportadores , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Clonación Molecular , Colforsina/farmacología , AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Intrones , Luciferasas/genética , Datos de Secuencia Molecular , Neuroblastoma , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática , Ratas , Análisis de Secuencia de ADN , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Células Tumorales Cultivadas
17.
Am J Respir Crit Care Med ; 162(5): 1656-61, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11069792

RESUMEN

Antenatal exposure to glucocorticoids, amnionitis, intraamniotic interleukin (IL)-1alpha, or endotoxin can improve postnatal lung function after preterm delivery. The relationship between early lung maturation and the dose and duration of a proinflammatory stimulus has not been evaluated. The effects of proinflammatory stimuli on fetal plasma cortisol also have not been evaluated. We hypothesized that intraamniotic endotoxin would induce early lung maturation in fetal sheep without increasing fetal cortisol. Intraamniotic injections of 1, 4, 20, or 100 mg of Escherichia coli 055:beta5 endotoxin caused 2-fold increases in compliance, 4- to 5-fold increases in lung gas volumes, and 20-fold increases in alveolar saturated phosphatidylcholine (Sat PC) when given 7 d before preterm delivery at 125 d gestation. Animals treated with 20 mg endotoxin for treatment to delivery intervals of 5 h to 15 d had no significant elevations in cord plasma cortisol levels. Increases in Sat PC in lung tissue and alveolar washes were detected 2 d after endotoxin treatment and lung function improved 4 d after endotoxin treatment. Two doses of endotoxin given 3 and 7 d or 7 and 15 d before treatment resulted in lung maturation responses equivalent to single dose comparison groups without elevations in cortisol. Early lung maturation induced by intraamniotic endotoxin in fetal sheep occurred without an increase in fetal plasma cortisol, indicating that endotoxin promoted lung maturation by a mechanism independent of cortisol.


Asunto(s)
Endotoxinas/farmacología , Madurez de los Órganos Fetales/efectos de los fármacos , Hidrocortisona/fisiología , Pulmón/embriología , Amnios , Animales , Relación Dosis-Respuesta a Droga , Endotoxinas/administración & dosificación , Femenino , Sangre Fetal/química , Madurez de los Órganos Fetales/fisiología , Edad Gestacional , Hidrocortisona/sangre , Inyecciones , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Fosfatidilcolinas/metabolismo , Ovinos
18.
Am J Physiol Regul Integr Comp Physiol ; 279(3): R830-8, 2000 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10956240

RESUMEN

Glucocorticoids are administered for preterm labor to improve postnatal adaptation. We assessed the effect of antenatal betamethasone (Beta) treatment on preterm newborn lamb neuroendocrine [catecholamine, arginine vasopressin (AVP)] and endocrine [triiodothyronine (T(3)), ANG II, and atrial natriuretic factor (ANF)] adaptive responses following delivery and a hypoxic challenge. Beta treatment included direct fetal injection at 0.2 (F(0.2); n = 8) or 0.5 (F(0.5); n = 7) mg/kg estimated fetal body weight or maternal injection with 0.2 (n = 8) or 0.5 mg/kg (M(0.5); n = 8). Control animals received fetal and maternal intramuscular injections of saline (n = 8). After 24 h, lambs were delivered by cesarean section, surfactant treated, and ventilated for 4 h. Relative to the control lambs, 3 h after delivery, there was a marked suppression of plasma cortisol, epinephrine, norepinephrine, and ANG II levels and elevated plasma T(3) and ANF levels, systolic blood pressure, and left ventricular contractility (dP/dt; F(0.5) and M(0.5)) values in F(0.5) and both maternal Beta-treated groups. However, Beta treatment augmented the cardiac output, cortisol, norepinephrine, AVP, and ANF responses to 20 min of hypoxia (PO(2) = 25-30 mmHg). We concluded that short-term (24 h) antenatal glucocorticoid exposure 1) alters preterm newborn postnatal blood pressure regulation in the face of marked depression of plasma cortisol, catecholamine, and ANG II levels and 2) augments the postnatal neuroendocrine and endocrine responses to a hypoxic challenge.


Asunto(s)
Betametasona/farmacología , Glucocorticoides/farmacología , Hipoxia/fisiopatología , Sistemas Neurosecretores/efectos de los fármacos , Sistemas Neurosecretores/fisiología , Triyodotironina/sangre , Angiotensina II/sangre , Animales , Animales Recién Nacidos , Arginina Vasopresina/sangre , Factor Natriurético Atrial/sangre , Análisis de los Gases de la Sangre , Presión Sanguínea/efectos de los fármacos , Epinefrina/sangre , Femenino , Edad Gestacional , Frecuencia Cardíaca/efectos de los fármacos , Hidrocortisona/sangre , Concentración de Iones de Hidrógeno , Hipoxia/sangre , Norepinefrina/sangre , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ovinos , Arterias Umbilicales
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