RESUMEN
As we previously reported, N-methylpyrrolo[3,2-c]pyridine derivatives 1 (TAK-441) was discovered as a clinical candidate of hedgehog (Hh) signaling inhibitor by modification of the upper part. We next focused on modification of the lower part including core skeletons to discover new Hh signaling inhibitors with novel core rings. Efforts to find novel chemotypes by using X-ray single crystal structure analysis led to some potent Hh signaling inhibitors (2c, 2d, 2e, 2f) with novel core ring systems, which had benzamide moiety at the 5-position as a key component for potent activity. The suppression of Gli1 expression with these new Hh signaling inhibitors were weaker than that of compound 1 (TAK-441) because of low pharmacokinetic property. We recognized again TAK-441 is a good compound as clinical candidate with good structural and pharmacokinetic advantages.
Asunto(s)
Proteínas Hedgehog/antagonistas & inhibidores , Piridinas/química , Transducción de Señal , Animales , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Genes Reporteros , Semivida , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Conformación Molecular , Piridinas/síntesis química , Piridinas/farmacología , Pirroles/química , Pirroles/farmacología , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
A 45-year-old female went into cardiopulmonary arrest. She was in ventricular fibrillation (VF) and was defibrillated using an automated external defibrillator. After arrival at our hospital, electrocardiography monitoring showed QT prolongation. Serum potassium was low at 2.2 mEq/L, and hypokalemia-induced long QT syndrome was considered to be the cause of this patient's VF. An intravenous infusion of potassium and magnesium sulphate was started, which normalized her serum potassium and QTc interval, with no recurrence of ventricular arrhythmias. Endocrinological investigations showed a plasma renin activity of <0.1 ng/(mL h) and a plasma aldosterone concentration 258 pg/mL. Computed tomography scanning revealed a low signal area 16 mm × 20 mm in size of the right adrenal gland. From the above findings, this patient was diagnosed with a right adrenal tumor and primary aldosteronism. We concluded that the right adrenal tumor was excreting excess amounts of aldosterone from adrenal vein sampling, and performed laparascopic right adrenalectomy. Serum potassium levels rose immediately to normal levels postoperatively. We were able to withdraw her antihypertensive medication 3 months after adrenalectomy. We report a case of primary aldosteronism who experienced cardiopulmonary arrest, was resuscitated, and cured.
RESUMEN
Hedgehog (Hh) signaling is a highly conserved intercellular and intracellular communication mechanism that governs organogenesis and is dysregulated in cancers of numerous tissues, including prostate. Up-regulated expression of the Hh ligands, Sonic (Shh) and Desert (Dhh), has been reported in androgen-deprived and castration-resistant prostate cancer (CRPC). In a cohort of therapy naive, short- and long-term neoadjuvant hormone therapy-treated (NHT), and CRPC specimens, we observed elevated Dhh expression predominantly in long-term NHT specimens and elevated Shh expression predominantly in CRPC specimens. Together with previously demonstrated reciprocal signaling between Shh-producing prostate cancer (PCa) cells and urogenital mesenchymal fibroblasts, these results suggest that castration-induced Hh expression promotes CRPC progression through reciprocal paracrine signaling within the tumor microenvironment. We tested whether the orally available Smoothened (Smo) antagonist, TAK-441, could impair castration-resistant progression of LNCaP PCa xenografts by disrupting paracrine Hh signaling. Although TAK-441 or cyclopamine did not affect androgen withdrawal-induced Shh up-regulation or viability of LNCaP cells, castration-resistant progression of LNCaP xenografts was significantly delayed in animals treated with TAK-441. In TAK-441-treated xenografts, expression of murine orthologs of the Hh-activated genes, Gli1, Gli2 and Ptch1, was substantially suppressed, while expression of the corresponding human orthologs was unaffected. As androgen-deprived LNCaP cells up-regulate Shh expression, but are not sensitive to Smo antagonists, these studies indicate that TAK-441 leads to delayed castration-resistant progression of LNCaP xenografts by disrupting paracrine Hh signaling with the tumor stroma. Thus, paracrine Hh signaling may offer unique opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring of PCa progression.
Asunto(s)
Antineoplásicos/farmacología , Comunicación Paracrina/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Piridinas/farmacología , Pirroles/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Castración , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias de la Próstata/metabolismo , Receptor Smoothened , Microambiente Tumoral/efectos de los fármacos , Alcaloides de Veratrum/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human chymase, an angiotensin II-forming chymotrypsin-like serine proteinase, posses various biological actions mediating through local angiotensin II formation in the tissue level of many cardiovascular organs. Our previous experimental data have shown that chymase inhibitor increased a survival rate of the hamster post-myocardial infarction model with concomitant improvements of the cardiac function and hypertrophy, decreased hamster aortic atherosclerotic lesion induced by a high fat diet and improved hamster diabetic nephropathy decreasing the proteinuria and increased renal antiotensin II levels. Although chymase inhibitor has not yet been applied for clinical use, clinical cardiovascular diseases above mentioned appear to be the target of chymase inhibitor. The related basal and clinical circumstances are discussed in this review article for chymase inhibitor.
Asunto(s)
Enfermedades Cardiovasculares/prevención & control , Quimasas/antagonistas & inhibidores , Angiotensina II/metabolismo , Animales , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Quimasas/metabolismo , Humanos , Serina Proteasas/metabolismoRESUMEN
6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-3-(2,2,2-trifluoroethoxy)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (TAK-441) is a potent, selective hedgehog signaling pathway inhibitor that binds to Smo and is being developed for the treatment of cancer. The objectives of these studies were to explore the possibility of establishing of a link between the pharmacokinetics of TAK-441 and the responses of Gli1 mRNA in tumor-associated stromal or skin cells and the antitumor effect of hedgehog inhibition. To this end, we built pharmacokinetic and pharmacodynamic models that describe the relationship of the concentrations of TAK-441 plasma to the responses of Gli1 mRNA in the tumor (target) and skin (surrogate) and to tumor growth inhibition in mice bearing xenografts of human pancreatic tumors (PAN-04). The responses of Gli1 mRNA and tumor growth were described by an indirect response model and an exponential tumor growth model, respectively. The IC50 values for Gli1 mRNA inhibition in the tumor and skin by TAK-441 were estimated to be 0.0457 and 0.113 µg/ml, respectively. The IC90 value for tumor growth inhibition was estimated to be 0.68 µg/ml. These results suggest that a >83% inhibition of Gli1 mRNA expression in the skin or a >94% inhibition of Gli1 mRNA expression in the tumor would be required to sufficiently inhibit (>90%) hedgehog-related tumor growth in the xenografted model mice. We conclude that Gli1 mRNA expression in the tumor and skin could be a useful biomarker for predicting the antitumor effect of hedgehog inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Oncogénicas/genética , Piridinas/farmacología , Piridinas/farmacocinética , Pirroles/farmacología , Pirroles/farmacocinética , Transactivadores/genética , Animales , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Modelos Biológicos , Neoplasias/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Piel/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína con Dedos de Zinc GLI1RESUMEN
The Hedgehog (Hh) signaling pathway plays a significant role in the regulation of cell growth and differentiation during embryonic development. Since activation of the Hh signaling pathway is implicated in several types of human cancers, inhibitors of this pathway could be promising anticancer agents. Using high throughput screening, thieno[3,2-c]quinoline-4-one derivative 9a was identified as a compound of interest with potent in vitro activity but poor metabolic stability. Our efforts focused on enhancement of in vitro inhibitory activity and metabolic stability, including core ring conversion and side chain optimization. This led to the discovery of pyrrolo[3,2-c]quinoline-4-one derivative 12b, which has a structure distinct from previously reported Hh signaling inhibitors. Compound 12b suppressed stromal Gli1 mRNA expression in a murine model and demonstrated antitumor activity in a murine medulloblastoma allograft model.
Asunto(s)
4-Quinolonas/farmacología , Antineoplásicos/farmacología , Descubrimiento de Drogas , Proteínas Hedgehog/antagonistas & inhibidores , Meduloblastoma/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , 4-Quinolonas/síntesis química , 4-Quinolonas/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteínas Hedgehog/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Noqueados , Modelos Moleculares , Estructura Molecular , Células 3T3 NIH , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , Relación Estructura-Actividad , Trasplante Homólogo , Proteína con Dedos de Zinc GLI1RESUMEN
We recently reported the discovery of the novel pyrrolo[3,2-c]quinoline-4-one derivative 1 as a potent inhibitor of Hedgehog (Hh) pathway signaling. However, the PK evaluation of 1 at high dosage (100 mg/kg) revealed the C(max) value 3.63 µg/mL, likely due to poor solubility of this compound. Efforts to improve solubility by reducing the aromatic ring count of the core system led to N-methylpyrrolo[3,2-c]pyridine derivative 11. Further optimization of the 3-alkoxy group led to compound 11d with acceptable solubility and potent Hh inhibitory activity. Compound 11d suppressed transcription factor Gli1 mRNA expression in tumor-associated stromal tissue and inhibited tumor growth (treatment/control ratio, 3%) in a mouse medulloblastoma allograft model owing to the improved PK profile based on increased solubility. Compound 11d (TAK-441) is currently in clinical trials for the treatment of advanced solid tumors.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Proteínas Hedgehog/antagonistas & inhibidores , Meduloblastoma/tratamiento farmacológico , Piridinas/farmacología , Pirroles/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Células 3T3 NIH , Piridinas/administración & dosificación , Piridinas/síntesis química , Piridinas/química , Pirroles/administración & dosificación , Pirroles/síntesis química , Pirroles/química , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , Solubilidad , Relación Estructura-Actividad , Trasplante Homólogo , Proteína con Dedos de Zinc GLI1RESUMEN
Whey acidic protein (WAP) has been identified as a major whey protein in milk of a wide range of species and reportedly plays important roles in regulating the proliferation of mammary epithelial cells. However, in some species including humans, WAP is not synthesized in the mammary gland. The presence of WAP in carnivore species has not been reported. We searched the National Center for Biotechnology Information (NCBI) database for the dog WAP gene and tried biochemically to identify WAP in dog milk. The nucleotide sequence of the examined dog genomic DNA was completely identical to that in the NCBI database and showed that the dog WAP gene, like other known functional WAP genes, has four exons. Biochemical analysis of milk protein by reverse-phase HPLC and Western blotting demonstrated the presence of WAP in dog milk.
Asunto(s)
Perros/genética , Proteínas de la Leche/aislamiento & purificación , Leche/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting/veterinaria , Biología Computacional , Perros/metabolismo , Femenino , Leche/metabolismo , Proteínas de la Leche/química , Proteínas de la Leche/genética , Proteínas de la Leche/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , Alineación de Secuencia/veterinariaRESUMEN
Water-soluble black Chinese (Pu-Erh) tea extract (BTE), which contains high gallic acid content, has been demonstrated to elicit antiobese effects in animals. Because gallic acid is related with the reduction of visceral fat and cholesterol contents and improvement of obesity in animals, we investigated the effects of BTE intake on 36 preobese Japanese adults (body mass index [BMI], >25- <30 kg/m(2)) in a 12-week double-blind, randomized, placebo-controlled group comparison study using powdered barley tea with or without (placebo) BTE. A follow-up 4-week period after BTE intake termination was monitored to observe the withdrawal effect. All subjects ingested barley tea with or without BTE (333 mg) before each of the 3 daily meals. In the BTE-treated group, the mean pretreament values of body weight and BMI significantly decreased after intake and after BTE withdrawal. However, the corresponding values scored significant differences only from 8 weeks after intake (vs the placebo-treated group). The mean values of the waist circumference indicated a similar tendency. Furthermore, coronal navel section (same anatomical position) images of computed tomography of all BTE- and non-BTE-treated subjects revealed that the visceral fat areas (cm(2)) were significantly (P < .05) less in the former 12 weeks after BTE ingestion. Measured biochemical parameters did not indicate significant differences, and BTE-treated subjects did not complain of any adverse effects (abdominal distension, etc). Ingestion of BTE exhibited significant effects in reducing the mean waist circumference, BMI, and visceral fat values and might be useful for weight control and prevention of obesity development (or metabolic syndrome) in humans.
Asunto(s)
Índice de Masa Corporal , Peso Corporal/efectos de los fármacos , Camellia sinensis/química , Sobrepeso/tratamiento farmacológico , Extractos Vegetales/farmacología , Té/química , Pueblo Asiatico , Composición Corporal , Colesterol/sangre , Método Doble Ciego , Femenino , Humanos , Grasa Intraabdominal/efectos de los fármacos , Masculino , Persona de Mediana Edad , Tomógrafos Computarizados por Rayos X , Circunferencia de la Cintura/efectos de los fármacosRESUMEN
Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (ψlox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/ψlox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endogenous lox-like sequence termed pseudo-loxm5 (ψloxm5) in early mouse embryos. We characterized the Cre/ψloxm5 system in embryonic environment. Cre-expressing plasmid and a transgene (CMV/LacZ gene) flanked by ψloxm5 and ψloxcorem5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers, and the recombination products were investigated. The results show that the ψloxm5 site is an active substrate for Cre-mediated recombination in the mouse embryonic environment. The transgenesis efficiency was up to 27% (6/22). The site-specific integration of the transgene into the endogenous ψloxm5 site was found in 50 % of the transgenic pups. Our findings demonstrated that the Cre/ψloxm5 integrative recombination system is an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.
Asunto(s)
Embrión de Mamíferos/fisiología , Marcación de Gen , Integrasas/metabolismo , Recombinación Genética/genética , Transgenes/genética , Animales , Animales Modificados Genéticamente , Femenino , Operón Lac , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Plásmidos/genética , beta-Galactosidasa/metabolismoRESUMEN
We have previously reported the action of whey acidic protein (WAP) inhibiting the proliferation of mouse mammary epithelial cells in the experiments utilizing in vivo and in vitro systems. We report herein the bacteriostatic activity of WAP. Western blot analysis demonstrated successful isolation of WAP from whey fractions of rat milk by column chromatography. The WAP fraction inhibited the growth of Staphylococcus aureus JCM2413 in a dose-dependent manner, but did not inhibit the growth of Escherichia coli. The bacteriostatic activity of WAP was highest at pH 6.6 and was not affected by the presence of 150 mM NaCl. A scanning electron micrograph of bacteria treated with WAP exhibited the disruption of the bacterial cell walls.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Leche/farmacología , Animales , División Celular/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Ratones , Microscopía Electrónica de Rastreo , Leche/química , Proteínas de la Leche/aislamiento & purificación , Ratas , Proteínas Recombinantes/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/ultraestructuraRESUMEN
Whey acidic protein (WAP) is a major whey protein in milk that has structural similarity to the family of serine protease inhibitors with WAP motif domains characterized by a four-disulfide core. We previously reported that enforced expression of the mouse WAP transgene in mammary epithelial cells inhibits their proliferation in vitro and in vivo by means of suppressing cyclin D1 expression (Nukumi et al., 2004, Dev Biol 274: 31-44). This study was conducted in order to clarify the molecular mechanism of the inhibitory function of WAP in HC11 cells, a mammary epithelial cell line. The assembly of laminin, a component in the extracellular matrix, was much more prominent around WAP-clonal HC11 cells that stably expressed the WAP transgene than around mock-clonal HC11 cells, and the proliferation of WAP-clonal HC11 cells was particularly inhibited in the presence of laminin. A laminin degradation assay demonstrated that WAP inhibited the activity of the pancreatic elastase-mediated cleavage of laminin B1 and the phosphorylation of ERK1/2. ERK1/2 phosphorylation was blocked by an inhibitor of the epidermal growth factor (EGF) receptor AG1478. Treatment with pancreatic elastase was found to enhance the proliferation of mock-clonal HC11 cells, but had no effect on that of WAP-clonal HC11 cells. The proliferation of WAP-clonal HC11 cells was recovered by the addition of exogenous EGF. We concluded that WAP plays some role in regulating the proliferation of mammary epithelial cells by preventing elastase-type serine protease from carrying out laminin degradation and thereby suppressing the MAP kinase signal pathway.
Asunto(s)
Proliferación Celular , Células Epiteliales/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Laminina/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Proteínas de la Leche/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular , Células Clonales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Inmunohistoquímica , Laminina/análisis , Glándulas Mamarias Animales/citología , Ratones , Proteínas de la Leche/genética , Elastasa Pancreática/análisis , Plásmidos , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , TransfecciónRESUMEN
Whey acidic protein (WAP) is a major component of whey, which has two or three WAP motif domains characterized by a four-disulfide core (4-DSC) structure similar to the serine protease inhibitor. We have previously found that WAP inhibits the proliferation of mammary epithelial cells in vitro and in vivo [N. Nukumi, K. Ikeda, M. Osawa, T. Iwamori, K. Naito, H. Tojo, Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro, Dev. Biol. 274 (2004) 31-44]. We report herein that WAP also reduces the progression of human breast cancer cells (MCF-7 and MDA-MB-453 cells). We have demonstrated that the forced expression of WAP in MCF-7 cells reduces the proliferation in either the presence or absence of estrogen. The tumor progression of WAP-expressing MCF-7 cells in nude mice is significantly suppressed more than that of mock-MCF-7 cells following the reduced expression of angiopoietin-2 gene. We have confirmed that the invasive activity of breast cancer cells is reduced to approximately 30% of that of mock cells by the forced expression of exogenous WAP through its inhibition of degradation of laminin. These data suggest that WAP has a protease-inhibitory function on the progression of breast cancer cells. It is therefore possible to utilize WAP as therapeutic protein against tumorigenesis of breast cancer.
Asunto(s)
Neoplasias de la Mama/prevención & control , Transformación Celular Neoplásica/metabolismo , Proteínas de la Leche/metabolismo , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Proteínas de la Leche/genética , Péptido Hidrolasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pilsicainide is a class IC antiarrhythmic drug, which has a pure sodium channel blocking action with slow recovery pharmacokinetics. In experimental studies, pilsicainide has a depressant effect on intra-atrial conduction and a prolonging effect on the atrial effective refractory period (ERP). In patients with paroxysmal atrial fibrillation (AF), pilsicainide significantly prolonged the ERP of the distal pulmonary vein (PV), PV-left atrium (LA) junction and LA, and the conduction time from the distal PV to the PV-LA junction. In some patients, PV-LA conduction block has been observed just before pilsicainide-induced termination of AF; this isolation of the PV may provide a new insight into the mechanism of pharmacological conversion of AF. Hybrid therapy with pilsicainide and PV isolation (by radiofrequency catheter ablation) appears to be an effective therapeutic approach for AF. The pharmacological PV isolation by pilsicainide and its suppression of focal discharges from atrial tissue may prevent the development of AF after unsuccessful ablation.
Asunto(s)
Antiarrítmicos/uso terapéutico , Fibrilación Atrial/tratamiento farmacológico , Lidocaína/análogos & derivados , Bloqueadores de los Canales de Sodio/uso terapéutico , Animales , Fibrilación Atrial/cirugía , Ablación por Catéter , Terapia Combinada , Técnicas Electrofisiológicas Cardíacas , Humanos , Lidocaína/uso terapéutico , Venas Pulmonares/efectos de los fármacos , Venas Pulmonares/fisiologíaRESUMEN
Sequential activation of muscle-specific transcription factors is the critical basis for myogenic differentiation. However, the complexity of this process does not exclude the possibility that other molecules and systems are regulatory as well. We observed that myogenic differentiation proceeded through three distinct stages of proliferation, elongation and fusion, which are distinguishable by their cellular morphologies and gene expression patterns of proliferation- and differentiation-specific markers. Treatment of the differentiating myoblasts with inhibitors of matrix metalloproteinases (MMPs) revealed that MMP activity at the elongation stage is a critical prerequisite to complete the successive myoblast cell fusion. The MMP regulated the myogenic differentiation independently from the genetic program that governs expression of the myogenic genes. Membrane-type 1 matrix metalloproteinase (MT1-MMP) was identified as a major contributor to this checkpoint for morphological differentiation and degraded fibronectin, a possible inhibitory factor for myogenic cell fusion. A MT1-MMP deficiency caused similar myogenic impediments forming smaller myofibers in situ. Additionally, the mutant mice demonstrated some central nucleation of the myofibers typically found in muscular dystrophy and MT1-MMP was found to cleave laminin-2/4 in the basement membrane. Thus, MT1-MMP is a new multilateral regulator for muscle differentiation and maintenance through processing of stage-specific distinct ECM substrates.
Asunto(s)
Metaloproteinasas de la Matriz Asociadas a la Membrana/metabolismo , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Animales , Diferenciación Celular/fisiología , Fusión Celular , Células Cultivadas , Fibronectinas/metabolismo , Regulación de la Expresión Génica , Laminina/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasas de la Matriz Asociadas a la Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/fisiología , Mioblastos Esqueléticos/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regeneración , Inhibidor Tisular de Metaloproteinasa-2/genéticaRESUMEN
The acetylation of nuclear core histone has been suggested to work as an epigenetic mark for transmitting gene expression patterns to daughter cells. Global histone deacetylations, presumably involved in the reprogramming of the gene expression, have been observed after germinal vesicle breakdown (GVBD) in a cell cycle-dependent manner during meiotic maturation of mouse and porcine oocytes, although the regulation mechanism of histone deacetylation has not been studied well. In the present study, we examined the involvement of a crucial cell-cycle-regulator, maturation-promoting factor (MPF), and a meiosis-related kinase, mitogen-activated protein kinase (MAPK), in the global histone deacetylation during porcine oocyte maturation. In order to know whether the activities of MPF and MAPK were required, or the breakdown of GV membrane was sufficient, for the global histone deacetylation observed after GVBD, we artificially destroyed the GV membrane of the porcine immature oocytes. The artificial GV destruction (AGVD) induced histone deacetylation without the activation of MPF and MAPK. This deacetylation after AGVD was not affected by an MPF inhibitor, roscovitine, or an inhibitor of protein synthesis, cycloheximide, but was completely prevented by an inhibitor of histone deactylases (HDACs), trichostatine A. HDAC1 was present in the GV of the immature oocytes and localized on chromosomes after GVBD and AGVD. These results suggest that the MPF and MAPK activities were dispensable and the breakdown of the GV membrane was sufficient for the global histone deacetylation, which was catalyzed by HDAC activity.
Asunto(s)
Histonas/metabolismo , Factor Promotor de Maduración/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Oocitos/fisiología , Acetilación , Animales , Cromatina/ultraestructura , Cicloheximida/farmacología , Activación Enzimática , Femenino , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/farmacología , Immunoblotting/métodos , Inmunohistoquímica/métodos , Factor Promotor de Maduración/antagonistas & inhibidores , Meiosis , Mesotelina , Ratones , Inhibidores de la Síntesis de la Proteína/farmacología , Purinas/farmacología , RoscovitinaRESUMEN
We investigated gender difference in the effects of chronic exposure to human growth hormone (hGH) on cardiac risk biomarkers using transgenic mice with non-pulsatile circulating hGH. Blood plasma was obtained from transgenic and control mice at 8, 12, and 16 weeks of age, and was used for the measurement of hGH and the following cardiac risk biomarkers: total cholesterol (CHO), triglyceride (TG), HDL cholesterol (HDL), LDL cholesterol (LDL), non esterified free fatty acids (NEFA), and lipid peroxides (LPO). The hearts and the livers of transgenic mice were weighed and histopathologically examined, and the results were compared with those of control mice. Transgenic males exhibited higher levels of LDL at 8 and 12 weeks of age and higher levels of LPO at every week of age examined, as compared to those of the control males, while transgenic females exhibited somewhat lower levels of LDL and LPO from 8 to 16 weeks of age, as compared to the control females. The relative heart weight in males increased with aging and was significantly higher in the 16-week-old transgenic males compared to those of the control mice. The present results demonstrate that transgenic males had cardiac risk potential caused by chronic-exposure to hGH as compared to females. The results also show that the present transgenic mouse line is a useful model for the study of gender difference in cardiac disorders caused by hGH.
Asunto(s)
Biomarcadores/sangre , Cardiopatías/sangre , Hormona de Crecimiento Humana/sangre , Animales , Cardiomegalia/sangre , Cardiomegalia/genética , Cardiomegalia/patología , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Femenino , Cardiopatías/genética , Cardiopatías/patología , Hormona de Crecimiento Humana/genética , Peróxidos Lipídicos/sangre , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocitos Cardíacos/patología , Tamaño de los Órganos , Factores de Riesgo , Factores SexualesRESUMEN
Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células de la Granulosa/citología , Proteína de la Región Y Determinante del Sexo/genética , Proteína de la Región Y Determinante del Sexo/metabolismo , Animales , Linaje de la Célula/genética , Células Cultivadas , Femenino , Células de la Granulosa/enzimología , Células de la Granulosa/metabolismo , Integrasas/genética , Integrasas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Ovario/citología , Ovario/metabolismo , Regiones Promotoras Genéticas , Conejos , Recombinación Genética , Células de Sertoli/citología , Células de Sertoli/enzimología , Células de Sertoli/metabolismo , Testículo/citología , Testículo/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
Although whey acidic protein (WAP) has been identified in the milk of a range of species, it has been predicted that WAP is not secreted into human milk as a result of critical point mutations within the coding region. In the present study, we first investigated computationally the promoter region of mutated human WAP genes by comparing with those of other known WAP genes. Computational database analyses showed that the human WAP promoter region was highly conserved, as in other species with milk WAP. Next, we evaluated the activity of the human WAP promoter (2.6 kb) using a reporter gene assay. MCF-7 cells were stably transfected with the hWAP/hGH (human growth hormone) fusion gene, cultured on Matrigel, and treated with lactogenic hormones. Radioimmunoassay detected hGH in the culture medium, indicating that the human WAP promoter was responsible for the lactogenic hormones. The human WAP promoter was significantly more active in MCF-7 cells than the mouse WAP promoter (2.4 kb). The present results provide us with important information on the molecular evolution of milk protein genes.
Asunto(s)
Regiones Promotoras Genéticas , Línea Celular Tumoral , Colágeno/metabolismo , Colágeno/farmacología , Biología Computacional , Secuencia Conservada , Bases de Datos Genéticas , Combinación de Medicamentos , Evolución Molecular , Humanos , Laminina/metabolismo , Laminina/farmacología , Proteínas de la Leche/genética , Leche Humana , Modelos Genéticos , Modelos Estadísticos , Mutación Puntual , Proteoglicanos/metabolismo , Proteoglicanos/farmacología , Radioinmunoensayo , Análisis de Secuencia de ADN , TransfecciónRESUMEN
Mammalian immature oocytes contain large nuclei referred to as germinal vesicles (GVs). The translocation of maturation/M-phase promoting factor (MPF) into GVs just before the activation of MPF has been reported in several species. To examine whether the GV is required for MPF activation in mammalian oocytes, porcine immature oocytes were enucleated and their MPF activity and CCNB (also known as cyclin B) levels were investigated. The activation of MPF at the start of maturation was detected at normal levels in enucleated oocytes, whereas reactivation to induce the second meiosis was not observed. Although protein synthesis was found to be normal both qualitatively and quantitatively, even in the absence of the nucleus, CCNB1 did not sufficiently accumulate in the enucleated oocytes. The defects in the enucleated oocytes were reversed by the injection of GV material into the enucleated oocytes. Furthermore, the inhibition of CCNB1 degradation revealed drastic accumulation of CCNB1, indicating active synthesis of CCNB1 in enucleated oocytes. The mitogen-activated protein kinase cascade remained unaffected by enucleation. These results indicate that GV is not required for the activation of MPF during the first meiosis, but that it is required for the second meiosis because of its promotion of CCNB1 accumulation.