RESUMEN
Although the rate of preterm birth has increased in recent decades, a number of preterm infants have escaped death due to improvements in perinatal and neonatal care. Antenatal glucocorticoid (GC) therapy has significantly contributed to progression in lung maturation; however, its potential effects on other organs remain controversial. Furthermore, the effects of antenatal GC therapy on the fetal heart show both pros and cons. Translational research in animal models indicates that constant fetal exposure to antenatal GC administration is sufficient for lung maturation. We have established a premature fetal rat model to investigate immature cardiopulmonary functions in the lungs and heart, including the effects of antenatal GC administration. In this review, we explain the mechanisms of antenatal GC actions on the heart in the fetus compared to those in the neonate. Antenatal GCs may contribute to premature heart maturation by accelerating cardiomyocyte proliferation, angiogenesis, energy production, and sarcoplasmic reticulum function. Additionally, this review specifically focuses on fetal heart growth with antenatal GC administration in experimental animal models. Moreover, knowledge regarding antenatal GC administration in experimental animal models can be coupled with that from developmental biology, with the potential for the generation of functional cells and tissues that could be used for regenerative medical purposes in the future.
Asunto(s)
Glucocorticoides , Nacimiento Prematuro , Animales , Metabolismo Energético , Femenino , Corazón Fetal , Glucocorticoides/farmacología , Humanos , Recién Nacido , Recien Nacido Prematuro , Embarazo , RatasRESUMEN
It is difficult to observe the structure of the enzyme-substrate complex (ES complex) experimentally, since the complex changes to the enzyme and its product during observation. The molecular dynamics (MD) approach is ideal to observe the structural change of enzyme and of substrate in the ES complex. Analyses on the complex of L-Phe oxidase with L-Phe by MD showed 1) the distance between the α-hydrogen atom of L-Phe and the N5 atom of isoalloxazine ring of FAD to be 2.64 ± 0.19 Å, and 2) the angle CA-HA-N5 atoms to be 141.5 ± 10.7°. This result clearly showed that the α-hydrogen atom forms the hydrogen bond with the N5 atom of isoalloxazine ring of FAD in the enzyme-substrate complex. Thus, the complex is ready for the hydrogen transfer from substrate to FAD in the key step of the oxidation of substrate by the enzyme.
Asunto(s)
Flavina-Adenina Dinucleótido , Oxidorreductasas , Sitios de Unión , Cristalografía por Rayos X , Flavina-Adenina Dinucleótido/metabolismo , Hidrógeno , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Oxidación-Reducción , Oxidorreductasas/químicaRESUMEN
AIM: Jaundice is especially common in premature infant born before 35 weeks. Because the premature infant liver is not fully developed at birth it may be incomplete the bilirubin metabolism. The purpose was to evaluate the metabolism and the excretion of bilirubin in the premature infant rat liver following prenatal glucocorticoid (GC) administration. METHODS: Dexamethasone (DEX) was administered subcutaneously to pregnant Wistar rats for two consecutive days on gestational days 17 and 19. The fetus were delivered by cesarean section in gestational days 19 and 21. The mRNA levels and protein levels of bilirubin-metabolic enzymes and transporters in the fetal liver tissues were analyzed using RT-PCR immunohistochemistry staining and ELISA, respectively. We evaluated that the effect of bilirubin-metabolic enzymes in the primary fetal rat hepatocytes treated with DEX after pretreated with glucocorticoid receptor (GR, Nr3c1) and Pxr (Nr1i2) siRNA. RESULTS: Ugt1a1 and Bsep (Abcb11) mRNA levels were significantly increased in the fetuses by prenatal GC administration. The mRNA levels of nuclear transcription factors Nr1i2, Car (Nr1i3), and Rxrα (Nr2b1) were also significantly increased in the fetuses by prenatal GC administration. In addition, DEX increased Nr1i2, Ugt1a1, and Abcc2 (Mrp2) mRNA levels in the primary fetal hepatocytes. The Nr3c1 or Nr1i2 siRNA-mediated knockdown suppressed the increases of Ugt1a1, and Abcc2 mRNA levels induced by DEX, indicating that DEX are mediated by GC receptor and PXR in primary fetal hepatocytes. CONCLUSIONS: These results suggest that prenatal GC administration increases bilirubin-metabolic ability, in the premature liver, which may prevent jaundice in neonates.
Asunto(s)
Glucocorticoides , Receptores de Glucocorticoides , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/farmacología , Animales , Bilirrubina/metabolismo , Bilirrubina/farmacología , Cesárea , Dexametasona/metabolismo , Dexametasona/farmacología , Femenino , Feto/metabolismo , Expresión Génica , Glucocorticoides/metabolismo , Glucocorticoides/farmacología , Humanos , Hígado/metabolismo , Embarazo , Receptor X de Pregnano/genética , Receptor X de Pregnano/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Ratas , Ratas Wistar , Receptores de Glucocorticoides/genéticaRESUMEN
BACKGROUND: Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for the maturation of cardiopulmonary function. Preterm and low-birth-weight infants often experience liver dysfunction after birth because their livers are immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration on the maturation of liver hepatocytes in preterm rats. METHODS AND RESULTS: Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the mRNA levels of albumin, hepatocyte nuclear factor-4 alpha (HNF4α), hepatocyte growth factor (HGF), thymus cell antigen 1 (Thy-1), cyclin B, and Cyclin-dependent kinase 1 (CDK1) in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay were performed to examine protein production. The hepatocytes enlarged because of growth and prenatal DEX administration. Albumin, HNF4α, and HGF levels increased secondary to growth and prenatal DEX administration. The levels of the cell cycle markers cyclin B and CDK1 gradually decreased during growth and with DEX administration. CONCLUSIONS: The results suggest that prenatal GC administration leads to hepatocyte maturation via expression of HNF4α and HGF in preterm fetuses.
Asunto(s)
Glucocorticoides , Nacimiento Prematuro , Albúminas/metabolismo , Albúminas/farmacología , Animales , Cesárea , Ciclina B/metabolismo , Ciclina B/farmacología , Dexametasona , Femenino , Feto/metabolismo , Glucocorticoides/metabolismo , Hepatocitos , Hígado/metabolismo , Embarazo , Nacimiento Prematuro/metabolismo , Ratas , Ratas WistarRESUMEN
BACKGROUND: Cytokines play an important role in the immune response, angiogenesis, cell growth, and differentiation in hepatocellular carcinoma (HCC). OBJECTIVE: We performed a comprehensive study to identify tumor-related cytokines and pathways involved in HCC pathogenesis. METHODS: Cytokine production was evaluated in human HCC tissues and adjacent non-tumor tissues using an antibody-based protein array technique. We compared cytokine expression in HCC tissues with that of hepatic hemangioma (HH), liver metastasis of colorectal cancer, and noncancerous liver tissues from transplantation donors. The protein levels and localization of the candidate cytokines were analyzed by western blotting and immunohistochemistry. RESULTS: Increased expression of interleukin (IL)-1 receptor antagonist, macrophage migration inhibitory factor, and IL-16 was observed in HCC and paired adjacent non-tumor tissues compared with noncancerous livers. In addition, there were increased IL-16 levels in HCC tissues compared with HH. IL-16 treatment significantly increased cell proliferation in vitro. The expression of extracellular signal-regulated kinase (ERK)1/2 and cyclin D1 was markedly increased in cells from two HCC cell lines, Huh7 and HepG2, in a dose- and time-dependent manner. Phosphorylated to total ERK1/2 ratio was increased in Huh7 cells following IL-16 50âng/ml, but not HepG2 cells. ERK phosphorylation have occurred earlier than protein accumulation at 48âh. Pretreatment with the ERK inhibitor, FR18024, or an anti-IL-16 antibody reduced the increase in IL-16 production in HCC cells. CONCLUSIONS: These results suggest that cell proliferation induced by IL-16 is mediated through the ERK pathway, thus, we identified a new factor associated with HCC tumor growth.
Asunto(s)
Carcinoma Hepatocelular/genética , Proteína Antagonista del Receptor de Interleucina 1/genética , Interleucina-16/genética , Neoplasias Hepáticas/genética , Hígado/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Hemangioma/tratamiento farmacológico , Hemangioma/genética , Hemangioma/patología , Células Hep G2 , Humanos , Interleucina-16/antagonistas & inhibidores , Interleucina-16/biosíntesis , Interleucina-16/farmacología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Metástasis de la Neoplasia , ProteómicaRESUMEN
Biased agonism is a paradigm that may explain the selective activation of a signaling pathway via a GPCR that activates multiple signals. The autoantibody-induced inactivation of the calcium-sensing receptor (CaSR) causes acquired hypocalciuric hypercalcemia (AHH). Here, we describe an instructive case of AHH in which severe hypercalcemia was accompanied by an increased CaSR antibody titer. These autoantibodies operated as biased allosteric modulators of CaSR by targeting its Venus flytrap domain near the Ca2+-binding site. A positive allosteric modulator of CaSR, cinacalcet, which targets its transmembrane domain, overcame this autoantibody effect and successfully corrected the hypercalcemia in this patient. Hence, this is the first study to our knowledge that identifies the interaction site of a disease-causing GPCR autoantibody working as its biased allosteric modulator and demonstrates that cinacalcet can correct the AHH autoantibody effects both in vitro and in our AHH patient. Our observations provide potentially new insights into how biased agonism works and how to design a biased allosteric modulator of a GPCR. Our observations also indicate that the diagnosis of AHH is important because the severity of hypercalcemia may become fatal if the autoantibody titer increases. Calcimimetics may serve as good treatment options for some patients with severe AHH.
Asunto(s)
Autoanticuerpos/metabolismo , Hormonas y Agentes Reguladores de Calcio/administración & dosificación , Cinacalcet/administración & dosificación , Hipercalcemia/tratamiento farmacológico , Receptores Sensibles al Calcio/metabolismo , Anciano de 80 o más Años , Regulación Alostérica/efectos de los fármacos , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Sitios de Unión/efectos de los fármacos , Calcio/sangre , Calcio/metabolismo , Células HEK293 , Humanos , Hipercalcemia/sangre , Hipercalcemia/diagnóstico , Hipercalcemia/inmunología , Masculino , Receptores Sensibles al Calcio/inmunología , Receptores Acoplados a Proteínas G/inmunología , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: ATP synthesis and cardiac contraction-related protein production are accelerated in the immature fetal heart by antenatal glucocorticoids (GC). This study investigated the structural maturity of the myocardium and underlying signal pathway associated with cardiac growth in fetal rats that received antenatal GC. METHODS AND RESULTS: Dexamethasone (DEX) was given to pregnant rats for 2 days from day 17 or day 19 of gestation, and the hearts of 19 and 21 day fetuses and 1-day-old neonates were analyzed. Although irregular myofibril orientation was observed morphologically in 19 day fetal hearts, the myofibril components were organized in fetuses after DEX. The cross-sectional area of the myocardium and Ki-67-positive cells were significantly increased in fetal DEX groups, suggesting that cardiac enlargement resulted from myocyte proliferation. Glycogen synthase kinase-3ß (GSK-3ß) protein was significantly decreased in fetal DEX groups. ß-Catenin and vascular endothelial growth factor protein were also significantly increased. Furthermore, increased cardiomyocyte proliferation appeared to be mediated by GC receptors after culture with DEX in vitro. CONCLUSIONS: Antenatal DEX induces structural maturity accompanying cardiomyocyte proliferation in the premature fetal rat heart, and GSK-3ß and ß-catenin are thought to contribute to cardiac growth.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Dexametasona/farmacología , Glucocorticoides/farmacología , Corazón/efectos de los fármacos , Animales , Western Blotting , Células Cultivadas , Femenino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Corazón/crecimiento & desarrollo , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Ratas , Transducción de Señal , beta Catenina/metabolismoRESUMEN
Vasoactive intestinal peptide (VIP) is a modulator of inflammatory responses. VIP receptors are expressed in several tumor types, such as colorectal carcinoma. The study described herein was conducted to confirm the presence of VIP and its receptors (VPAC1 and VPAC2) in surgically resected hepatocellular carcinoma (HCC) tissues and in the HCC cell line Huh7. The mechanism responsible for apoptosis of HCC cells was then examined because VIP treatment (10-10 M) significantly suppressed proliferation of Huh7 cells. In examining apoptosis-related proteins, we found caspase-3 to be significantly increased and Bcl-xL and cyclic AMP (cAMP) response element-binding protein (CREB) to be significantly decreased in Huh7 cells cultured with VIP. Furthermore, the CREB level and phosphorylation were reduced. These effects were reversed by the addition of VIP receptor antagonist or cAMP antagonist Rp-cAMPS. Pretreatment with cAMP analogue blocked the increased apoptosis, suggesting that VIP induces apoptosis via a PKA-independent signaling mechanism. Our data indicate that VIP prevents the progression of HCC by apoptosis through the cAMP/Bcl-xL pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/metabolismo , AMP Cíclico/metabolismo , Neoplasias Hepáticas/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Proteína bcl-X/metabolismo , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Fosforilación/efectos de los fármacos , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Transducción de Señal/efectos de los fármacos , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Adipose tissue contains multipotent cells known as adipose-derived stem/stromal cells (ASCs), which have therapeutic potential for various diseases. Although the demand for adipose tissue for research use remains high, no adipose tissue bank exists. In this study, we attempted to isolate ASCs from cryopreserved adipose tissue with the aim of developing a banking system. ASCs were isolated from fresh and cryopreserved adipose tissue of rats and compared for proliferation (doubling time), differentiation capability (adipocytes), and cytokine (hepatocyte growth factor and vascular endothelial growth factor) secretion. Finally, ASCs (2.5 × 106) were intravenously infused into rats with spinal cord injury, after which hindlimb motor function was evaluated. Isolation and culture of ASCs from cryopreserved adipose tissue were possible, and their characteristics were not significantly different from those of fresh tissue. Transplantation of ASCs derived from cryopreserved tissue significantly promoted restoration of hindlimb movement function in injured model rats. These results indicate that cryopreservation of adipose tissue may be an option for clinical application.
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Tejido Adiposo/citología , Criopreservación , Traumatismos de la Médula Espinal/cirugía , Células del Estroma/trasplante , Animales , Diferenciación Celular , Proliferación Celular , Separación Celular , Modelos Animales de Enfermedad , Femenino , Factor de Crecimiento de Hepatocito/metabolismo , Miembro Posterior , Humanos , Laminectomía , Locomoción , Ratas , Ratas Sprague-Dawley , Medicina Regenerativa , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND AIMS: Adipose tissue has therapeutic potential for spinal cord injury (SCI) because it contains multipotent cells known as adipose-derived stem/stromal cells (ASCs). In this study, we attempted intravenous ASC transplantation in rats with SCI to examine the effect on functional recovery. METHODS: ASCs (2.5 × 106) were intravenously infused into SCI rats, after which hindlimb motor function was evaluated. Distribution of transplanted ASCs was investigated and growth factor/cytokine levels were determined. RESULTS: Intravenous transplantation of ASCs promoted the functional recovery in SCI rats and reduced the area of spinal cord cavitation. A distribution study revealed that ASCs gradually accumulated at the site of injury, but long-term survival of these cells was not achieved. Levels of growth factors increased only slightly in the spinal cord after ASC transplantation. Unexpectedly, cytokine-induced neutrophil chemoattractant (CINC)-1 showed a transient but substantial increase in the spinal cord tissue and blood of the ASC group. CINC-1 was secreted by ASCs in vitro, and the sponge implantation assay showed that CINC-1 and ASCs induced angiogenesis. CINC-1 promoted functional recovery in SCI rats, which was similar to the ASCs. Expression of glial cell line-derived neurotrophic factor was greater in the ASC group than in the CINC-1 group, although both promoted extracellular signal-regulated kinase (ERK)1/2 phosphorylation; Akt phosphorylation was enhanced in the spinal cord after ASC transplantation. CONCLUSIONS: Our findings indicated that intravenously transplanted ASCs gradually accumulated in the injured spinal cord, where cytokines such as CINC-1 activated ERK1/2 and Akt, leading to functional recovery.
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Tejido Adiposo/citología , Traumatismos de la Médula Espinal/terapia , Trasplante de Células Madre/métodos , Animales , Quimiocina CXCL1/metabolismo , Femenino , Miembro Posterior/fisiopatología , Infusiones Intravenosas , Células Madre Multipotentes/trasplante , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Recuperación de la Función , Médula Espinal/metabolismo , Células del Estroma/trasplanteRESUMEN
Disease-causing mutations in G protein-coupled receptor (GPCR) genes, including the V2 vasopressin receptor (V2R) gene, often cause misfolded receptors, leading to a defect in plasma membrane trafficking. A novel V2R mutation, T273M, identified in a boy with partial nephrogenic diabetes insipidus (NDI), shows intracellular localization and partial defects similar to the two mutants we described previously (10). Although non-peptide V2R antagonists have been shown to rescue the membrane localization of V2R mutants, their level of functional rescue is weak. Interestingly, it has been reported that a non-peptide agonist, OPC51803, activates misfolded V2R mutants intracellularly without degradation, thus potentially serving as a therapeutic agent against NDI (14). In our current experiments, however, a peptide antagonist blocked arginine vasopressin (AVP)- or OPC51803-stimulated cAMP accumulation both in COS-7 and MDCK cells, suggesting that OPC51803 mainly stimulates cell surface V2R mutants. In addition, our analyses revealed that OPC51803 works not only as a non-peptide agonist that causes activation/ß-arrestin-dependent desensitization of V2R mutants expressed at the plasma membrane but also as a pharmacochaperone that promotes the endoplasmic reticulum-retained mutant maturation and trafficking to the plasma membrane. The ratio of the pharmacochaperone effect to the desensitization effect likely correlates negatively with the residual function of the tested mutants, suggesting that OPC5 has a more favorable effect on the V2R mutants with a less residual function. We speculated that the canceling of the desensitization effect of OPC51803 by the pharmacochaperone effect after long-term treatment may produce sustainable signaling, and thus pharmacochaperone agonists such as OPC51803 may serve as promising therapeutics for NDI caused by misfolded V2R mutants.
Asunto(s)
Benzazepinas/farmacología , Diabetes Insípida Nefrogénica , Mutación , Pirrolidinas/farmacología , Receptores de Vasopresinas , Animales , Células COS , Chlorocebus aethiops , Diabetes Insípida Nefrogénica/dietoterapia , Diabetes Insípida Nefrogénica/genética , Diabetes Insípida Nefrogénica/metabolismo , Perros , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Masculino , Receptores de Vasopresinas/agonistas , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismoRESUMEN
In the title compound, C10H19N3O4, the N- and C-termini are protonated and ionized, respectively, and the mol-ecule forms a zwitterion. The main chain is in a folded form. In the crystal, the N-terminal -NH3 (+) group hydrogen bonds to three C-terminal -COO groups and one carbonyl O atom, forming a three-dimensional network. In addition, an N-Hâ¯O hydrogen bond between the amide groups of the middle glycine residue and a C-Hâ¯O inter-action continue along the a-axis direction. The side chains of the leucyl residues form a hydro-phobic region along the a axis.