RESUMEN
Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms. Ghrelin is secreted from the stomach in response to CR and regulates energy metabolism. We hypothesized that in CR ghrelin has a role in protecting aging-related diseases. We examined the physiological mechanisms underlying the ghrelin system during the aging process in three mouse strains with different genetic and biochemical backgrounds as animal models of accelerated or normal human aging. The elevated plasma ghrelin concentration was observed in both klotho-deficient and senescence-accelerated mouse prone/8 (SAMP8) mice. Ghrelin treatment failed to stimulate appetite and prolong survival in klotho-deficient mice, suggesting the existence of ghrelin resistance in the process of aging. However, ghrelin antagonist hastened death and ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases with decreased microglial activation in the brain and prolonged survival in klotho-deficient, SAMP8 and aged ICR mice. In vitro experiments, the elevated sirtuin1 (SIRT1) activity and protein expression through the cAMP-CREB pathway was observed after ghrelin and ghrelin potentiator treatment in ghrelin receptor 1a-expressing cells and human umbilical vein endothelial cells. Furthermore, rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression of the heart in the all three mouse models of aging. Pericarditis, myocardial calcification and atrophy of myocardial and muscle fiber were improved by treatment with rikkunshito. Ghrelin signaling may represent one of the mechanisms activated by CR, and potentiating ghrelin signaling may be useful to extend health and lifespan.
Asunto(s)
Ghrelina/metabolismo , Ghrelina/fisiología , Sirtuina 1/metabolismo , Envejecimiento/fisiología , Animales , Restricción Calórica , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/metabolismo , Medicamentos Herbarios Chinos/uso terapéutico , Hipotálamo , Ratones , Ratones Endogámicos ICR , Receptores de Ghrelina/genética , Transducción de Señal , Sirtuina 1/fisiologíaRESUMEN
A new fluorine-containing anthracycline derivative, ME2303, showed excellent antitumor activity against various experimental tumor models. The i.p. or i.v. administrations of ME2303 on Day 1 or on Days 1, 5, and 9 against i.p.-implanted L1210 leukemia cells rendered more than 50% of mice tumor free at wide ranges of nontoxic doses, whereas the incidence of cure obtained with Adriamycin (ADM) was less than that obtained with ME2303. ME2303 given i.p. or i.v. on Day 1 or Days 1, 5, and 9 was also effective against i.p.-implanted P388 leukemia cells, and higher incidences of cure were obtained than with ADM. ME2303 administered i.v. on Days 1, 8, 15, and 22 showed prominent antitumor activity against s.c.-implanted colon adenocarcinomas 26 and 38, Lewis lung carcinoma, B16 melanoma, and M5076 sarcoma. Against colon adenocarcinoma 26, ME2303 induced cure in 16 of 20 mice at doses of 35 to 71 mumol/kg, whereas no cure was observed with ADM. Significant growth inhibition of colon adenocarcinoma 38, Lewis lung carcinoma, B16 melanoma, and M5076 sarcoma cell lines was also observed at a dose of 18 to 106 mumol/kg. ME2303 was effective against human and murine multidrug-resistant cells in vitro. For example, human myelogenous leukemia K562 resistant to ADM (K562/ADM) was only 2.8-fold more resistant to ME2303, while the cells were 200-fold more resistant to ADM when the values for the concentration of drug required for 50% inhibition of cell growth were compared. ME2303 was also more effective than ADM against human leukemia CCRF-CEM resistant to vinblastine, human ovarian carcinoma A2780 resistant to ADM, human epidermoid carcinoma KB cells resistant to colchicine, and mouse leukemia P388 resistant to ADM and vincristine. Therapeutic effects were obtained in vivo against ADM- and, especially, vincristine-resistant P388 leukemia. ME2303 is one of the most interesting potential antitumor agents to be studied further.
Asunto(s)
Doxorrubicina/análogos & derivados , Neoplasias Experimentales/tratamiento farmacológico , Animales , Doxorrubicina/uso terapéutico , Resistencia a Medicamentos , Técnicas In Vitro , Leucemia L1210/tratamiento farmacológico , Leucemia P388/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma Experimental/tratamiento farmacológico , Ratones , Sarcoma Experimental/tratamiento farmacológico , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
A 19-year-old man was punched on the back, and anterior chest pain appeared about 3 hours after injury. The patient was consulted a physician complaining of anterior chest pain. On chest X-ray, mediastinal emphysema was suspected, and transferred to our hospital. Chest computed tomography (CT) revealed mediastinal emphysema. On esophageal radiography and bronchofiberscopy, no abnormal findings were detected. Conservative therapy was conducted, and symptoms had gradually improved. On the 8th hospital day, mediastinal emphysema was improved on chest CT. The patient was discharged on the 10th hospital day. The most frequent cause of mediastinal emphysema after trauma is traffic or downfall accident, and no report on this condition after the punch on the back was found.
Asunto(s)
Traumatismos de la Espalda/complicaciones , Enfisema Mediastínico/etiología , Heridas no Penetrantes/complicaciones , Adulto , Dolor en el Pecho/etiología , Humanos , Masculino , Enfisema Mediastínico/diagnóstico por imagen , Enfisema Mediastínico/terapia , Tomografía Computarizada por Rayos XRESUMEN
Activation of P2X7 receptor (P2X7R), a purinergic receptor, expressed by neurons is well-known to induce their death, but whether or not their sensitivity to ATP depends on its expression levels remains unclear. Here, we examined the effect of the expression level of P2X7Rs on cell viability using pure neuron cultures, co-cultures with astrocytes derived from SJL- and ddY-strain mice, and mouse P2X7R-expressing HEK293T cell systems. Treatment of pure neuron cultures with 5mM ATP for 2h, followed by 3-h incubation in fresh medium, resulted in death of both types of neurons, and their death was prevented by administration of P2X7R-specific antagonists. In both SJL- and ddY-neurons, ATP-induced neuronal death was inhibited by a mitochondrial permeability transition pore inhibitor cyclosporine A, mitochondrial dysfunction being involved in their death. The ATP-induced neuronal death was greater for SJL-neurons than for ddY-ones, this being correlated with the expression level of P2X7R in them, and the same results were obtained for the HEK293T cell systems. Co-culture of neurons with astrocytes increased the ATP-induced neuronal death compared to the case of pure neuron cultures. Overall, we reveal that neuronal vulnerability to ATP depends on the expression level of P2X7R, and co-existence of astrocytes exacerbates ATP-induced neuronal death.
Asunto(s)
Adenosina Trifosfato/farmacología , Muerte Celular/fisiología , Neuronas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animales , Astrocitos/metabolismo , Western Blotting , Muerte Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Células HEK293 , Humanos , Inmunohistoquímica , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Neuronas/efectos de los fármacos , Reacción en Cadena de la PolimerasaRESUMEN
Phoborhodopsin (pR; also called sensory rhodopsin II, sRII) is a receptor of negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. These receptors contain retinal as a chromophore which binds to a lysine residue via Schiff base. This Schiff base can be cleaved with hydroxylamine to loose their color (bleaching). In dark, the bleaching rate of ppR was very slow whereas illumination accelerated considerably the bleaching rate. Addition of azide accelerated the decay of the M-intermediate while its formation (decay of the L-intermediate) is not affected. The bleaching rate of ppR under illumination was decreased by addition of azide. Essentially no reactivity with hydroxylamine under illumination was observed in the case of D75N mutant which lacks the M-intermediate in its photocycle. Moreover, we provided illumination by flashes to ppR in the presence of varying concentrations of azide to measure the bleaching rate per one flash. A good correlation was obtained between the rate and the mean residence time, MRT, which was calculated from flash photolysis data of the M-decay. These findings reveal that water-soluble hydroxylamine reacts selectively with the M-intermediate and its implication was discussed.
Asunto(s)
Proteínas Arqueales , Bacteriorodopsinas/química , Carotenoides , Halorrodopsinas , Hidroxilamina/química , Luz , Rodopsinas Sensoriales , Sitios de Unión , Fotoquímica , Relación Estructura-ActividadRESUMEN
Phoborhodopsin (pR or sensory rhodopsin II, sRII) and pharaonis phoborhodopsin (ppR or pharaonis sRII, psRII) have a unique absorption maximum (lambda(max)) compared with three other archaeal rhodopsins: lambda(max) of pR and ppR is approx. 500 nm and of others (e.g. bacteriorhodopsin, bR) is 560-590 nm. To determine the residue contributing to the opsin shift from ppR to bR, we constructed various ppR mutants, in which a single residue was substituted for a residue corresponding to that of bR. The residues mutated were those which differ from that of bR and locate within 5 A from the conjugated polyene chain of the chromophore or any methyl group of the polyene chain. The shifts of lambda(max) of all mutants were small, however. We constructed a mutant in which all residues which differ from those of bR in the retinal binding site were simultaneously substituted for those of bR, but the shift was only from 499 to 509 nm. Next, we constructed a mutant in which 10 residues located within 5 A from the polyene as described above were simultaneously substituted. Only 44% of the opsin shift (lambda(max) of 524 nm) from ppR to bR was obtained even when all amino acids around the chromophore were replaced by the same residues as bR. We therefore conclude that the structural factor is more important in accounting for the difference of lambda(max) between ppR and bR rather than amino acid substitutions. The possible structural factors are discussed.
Asunto(s)
Proteínas Arqueales/química , Carotenoides/química , Halorrodopsinas , Retinaldehído/química , Rodopsinas Sensoriales , Proteínas Arqueales/genética , Sitios de Unión , Carotenoides/genética , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Modelos Moleculares , Mutación , EspectrofotometríaRESUMEN
Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.
Asunto(s)
Melaninas/farmacología , Fenilalanina Hidroxilasa/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Glándulas Suprarrenales/enzimología , Animales , Bovinos , Núcleo Caudado/enzimología , Cinética , Hígado/enzimología , Especificidad de Órganos , Ratas , Especificidad de la EspecieRESUMEN
Blood flow in bypass grafts and recipient left anterior descending coronary arteries was evaluated with combined two-dimensional and Doppler echocardiography in 15 patients with an internal mammary artery graft and in 24 patients with a saphenous vein graft. Comparative studies of coronary hemodynamics were also performed regarding these two different grafting techniques. The graft vessel was detected in 11 (79%) of 14 patients with an internal mammary artery graft and in 20 (87%) of 23 with a saphenous vein graft. The recipient left anterior descending coronary artery was detected in 10 (67%) of the former group and 17 (71%) of the latter. The blood flow patterns obtained were generally biphasic, consisting of systolic and diastolic phases with higher velocity during diastole. The maximal diastolic flow velocity in internal mammary artery grafts was much higher than that in saphenous vein grafts. In patients with an internal mammary artery graft, the flow pattern characteristics within the recipient coronary artery were quite similar to those within the arterial graft, and flow velocities within the recipient coronary artery and the arterial graft were quantitatively almost identical. This outcome may contribute to the long-term patency seen in internal mammary artery grafts. On the other hand, the flow velocity in saphenous vein grafts was fairly low throughout the cardiac cycle. Flow velocity in the recipient coronary artery in patients with a saphenous vein graft was accelerated only in early diastole. As a result, the recipient coronary artery flow pattern and velocity differed substantially from those in the saphenous vein graft.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Puente de Arteria Coronaria , Circulación Coronaria/fisiología , Ecocardiografía Doppler , Ecocardiografía , Anastomosis Interna Mamario-Coronaria , Vena Safena/trasplante , Velocidad del Flujo Sanguíneo , Enfermedad Coronaria/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Contracción Miocárdica/fisiología , Grado de Desobstrucción VascularRESUMEN
Our previous study showed that PG490-88 effectively ameliorated both functional and histological changes of chronic rejection in the rat. In this experiment, we investigated the intragraft gene expression profiles of PG490-88 under successful prevention of chronic rejection in rat kidney allografts. Kidneys of F344 rats were transplanted into bilaterally nephrectomized LEW recipients. Recipients with a brief course of low-dose FK506 (1 mg/kg per day for 10 days) were dosed with PG490-88 0.5 mg/kg per day, which was predetermined and defined as the effective dose of preventing chronic allograft rejection in this model, for 90 days after grafting. Kidney grafts were harvested on day 90 after transplantation and subjected to gene expression analysis by real-time RT-PCR. Overall, the expression levels of all genes tested were upregulated in the brief course of low-dose FK506 control. PG490-88 treatment exhibited significant inhibition of intragraft m RNA levels of iNOS, IL-6, and perforin and marginal downregulation of IL-2, IFNgamma, IRF-1, TNFalpha, and TGFbeta. There was no change in IL-10, granzyme B, and PDGFalpha, when compared to the brief course of low-dose FK506 control. These results suggested that downregulation of multiple intragraft gene expression by mainly suppression of iNOS, IL-6, and perforin might be responsible for successful prevention of chronic kidney allograft nephropathy by PG490-88 in rats.
Asunto(s)
Citocinas/genética , Diterpenos/farmacología , Perfilación de la Expresión Génica , Rechazo de Injerto/prevención & control , Inmunosupresores/farmacología , Trasplante de Riñón/fisiología , Trasplante Homólogo/inmunología , Animales , Trasplante de Riñón/inmunología , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tacrolimus/farmacologíaRESUMEN
PG490-88 is a semisynthetic derivative of the novel compound PG490 (triptolide) purified from a Chinese herb. It has been shown to prolong acute allograft survival in multiple experimental organ transplant models. However, the effect of PG490-88 on prevention of acute and chronic renal allograft rejection has not been determined. Kidneys of ACI or F344 rats were transplanted into bilaterally nephrectomized LEW recipients as the acute or chronic allograft rejection models, respectively. Treatment of LEW recipients with PG490-88 significantly prolonged ACI kidney graft survival in a dose-dependent manner when compared with the untreated allograft controls. LEW recipients of F344 kidney grafts who received PG490-88 for 90 days with a brief course of low-dose FK506 showed normal serum creatinine levels and markedly reduced histological changes of chronic rejection at day 90 after transplantation. These results suggest that PG490-88 significantly prolongs kidney allograft survival in an acute rejection model and prevents chronic allograft rejection in rats.
Asunto(s)
Diterpenos/uso terapéutico , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/inmunología , Inmunosupresores/uso terapéutico , Trasplante de Riñón/inmunología , Enfermedad Aguda , Animales , Enfermedad Crónica , Rechazo de Injerto/patología , Supervivencia de Injerto/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas ACI , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Factores de Tiempo , Trasplante Homólogo/inmunologíaRESUMEN
OBJECTIVE: One of the mechanisms for mobilization of hematopoietic stem cells and progenitor cells is alternation of adhesion molecules. We investigated the mobilization of hematopoietic progenitor cells in blood by administration of anti-vascular cell adhesion molecule (VCAM)-1 antibody (Ab) in mice. MATERIALS AND METHODS: Twelve- to 14-week old C57BL/6J mice were injected intravenously with anti-VCAM-1 Ab and anti-very late antigen (VLA)-4 Ab at a dose of 5 mg/kg for 2 days. RESULTS: The number of colony-forming cells (CFCs) in blood was increased 11.4-fold after anti-VCAM-1 Ab treatment, but the number of CFCs was not increased after treatment with anti-VLA-4 Ab. The number of colony-forming unit spleen (CFU-S) also was increased 21.6-fold in the peripheral blood by administration of anti-VCAM-1 Ab. The number of CFCs and CFU-S in the bone marrow of mice treated with anti-VCAM-1 Ab was decreased and that in the spleen also was decreased. On administration of recombinant human granulocyte colony-stimulating factor (125 microg/kg twice daily) with anti-VCAM-1 Ab, the numbers of CFCs and CFU-S were increased 141.8-fold and 439-fold, respectively. CONCLUSIONS: These observations demonstrated that administration of anti-VCAM-1 Ab induced mobilization of hematopoietic progenitor cells into blood from bone marrow and spleen and that granulocyte colony-stimulating factor has synergistic effects on anti-VCAM-1 Ab-induced mobilization.
Asunto(s)
Anticuerpos/farmacología , Factor Estimulante de Colonias de Granulocitos/farmacología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/patología , Molécula 1 de Adhesión Celular Vascular/inmunología , Animales , Anticuerpos/inmunología , Sinergismo Farmacológico , Factor Estimulante de Colonias de Granulocitos/inmunología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , RatonesRESUMEN
Although the administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhances myeloid engraftment and reduces infectious morbidity after autologous and allogeneic bone marrow transplantations, the effect of rhG-CSF on neutrophil recovery in autologous blood stem cell transplantation (ABSCT) is controversial. We previously demonstrated that a low dose, delivered subcutaneously, of rhG-CSF (50 micrograms/m2) accelerates neutrophil recovery in ABSCT, but the optimal dosage of rhG-CSF is not known. To elucidate the effect of rhG-CSF on neutrophil recovery, we determined serum levels of endogenous and exogenously administered G-CSF in 24 patients receiving ABSCT. Of these, five received bolus subcutaneous injection of 50 micrograms/m2 rhG-CSF, 10 received 150 micrograms/m2, and nine received no rhG-CSF. Endogenous G-CSF levels rose immediately after ABSCT, and an inverse correlation was found between the serum level of G-CSF and the absolute neutrophil count (r = -0.73, p < 0.01). The pre-dose level in patients receiving rhG-CSF rose gradually, reaching a maximum between days 3 and 6. The level gradually decreased as the neutrophil count began to rise, even through administration of the same dose of rhG-CSF continued. Pharmacokinetic data showed that the half-life of elimination of G-CSF (t1/2) exceeded 15 hours during severe neutropenia but decreased during the recovery of neutrophils. These observations suggest that neutrophils provide a negative feedback mechanism for clearing G-CSF from the circulation. Pre-dose levels of G-CSF in patients receiving 50 micrograms/m2 rhG-CSF reached 10 ng/mL, equivalent to the concentrations used in clonogenic assay in vitro to stimulate myeloid progenitor cells.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/sangre , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Adolescente , Adulto , Femenino , Factor Estimulante de Colonias de Granulocitos/farmacocinética , Humanos , Japón , Cinética , Leucemia/terapia , Recuento de Leucocitos , Linfoma/terapia , Masculino , Persona de Mediana Edad , Mieloma Múltiple/terapia , Neoplasias/terapia , Neutrófilos , Proteínas Recombinantes/uso terapéutico , Trasplante AutólogoRESUMEN
Effects of the cardiotonic agent FK664, 6-(3, 4-dimethoxy-phenyl)-1-ethyl-4-mesitylimino-3-methyl-3,4-dihydro-2 (1H)-pyrimidone, on isolated guinea pig ventricular muscles and rabbit sinus node pacemaker cells were studied using micro-electrode techniques. In ventricular muscles driven at 0.5-1.0 Hz, FK664 above 3 mumol.litre-1 caused an increase in contractile force and a shortening of time to peak tension. This positive inotropic effect of FK664 was accompanied by a slight elevation of the early plateau phase of the action potential, while other action potential variables were unaffected. The change in contractile force induced by FK664 was abolished in a low Ca2+ medium (0.12 mmol.litre-1) or by treatment with ryanodine (2 mumol.litre-1), whereas it was relatively well preserved in the preparations pretreated with nefedipine (1 mumol.litre-1). The slow action potentials induced by isoprenaline (0.3 mumol.litre-1) in high K+ medium (30 mmol.litre-1) and the slow inward current measured by single sucrose gap voltage clamp at a holding potential of -40 mV were unaffected by FK664. In sinus node pacemaker cells, FK664 (1-10 mumol.litre-1) caused a dose dependent acceleration of phase 4 depolarisation and a shortening of spontaneous firing cycle length. This positive chronotropic effect of FK664 was markedly inhibited in a low Ca2+ medium (0.3 mmol.litre-1). These findings suggest that FK664 has positive inotropic and chronotropic effects on the heart, due to an enhancement of transsarcolemmal calcium influx through the low threshold, dihydropyridine insensitive Ca2+ channel population.
Asunto(s)
Cardiotónicos/farmacología , Corazón/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Pirimidinonas/farmacología , Nodo Sinoatrial/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Calcio/farmacocinética , Canales de Calcio/metabolismo , Cobayas , Ventrículos Cardíacos/efectos de los fármacos , Miocardio/metabolismo , Nifedipino/farmacología , Músculos Papilares/efectos de los fármacos , Conejos , Rianodina/farmacología , Sarcolema/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
A novel platform for transient photodetector component screening has been developed whereby an optical fiber tip serves as the counter electrode when placed in a variety of dielectric media, connected to a photoresponsive working electrode. The soft processing conditions allow for ubiquitous photodetection for organic and biological systems.
Asunto(s)
Complejos de Coordinación/química , Electrónica/instrumentación , Dispositivos Ópticos , Proteínas Bacterianas/efectos de la radiación , Bacteroidetes , Electrodos , Diseño de Equipo , Escherichia coli , Imidazoles/química , Líquidos Iónicos/química , Fibras Ópticas , Rodopsinas Sensoriales/efectos de la radiaciónRESUMEN
The rat S14 gene is synergistically regulated by thyroid hormone and carbohydrates. The 5'-flanking region of this gene contains both carbohydrate and thyroid hormone response elements (TREs). We recently located the carbohydrate response element (CHORE) between -1583 and -1069 bases from the start site of transcription that is required for glucose induction of this gene. We have now studied the relationship between the effects of CHORE, TREs, and thyroid hormone receptor on the carbohydrate regulation of transcription. We used a transient cotransfection assay with a plasmid containing 5 kilobases (kb) of the up-stream region from the S14 gene ligated to a luciferase reporter and a thyroid hormone receptor expression vector. Unliganded thyroid hormone receptor reduced the response of the reporter to glucose. Ligand-bound thyroid hormone receptor significantly enhanced the glucose response from the 5-kb promoter. While deletion of the CHORE (5-kb delta CHORE) from the 5-kb S14 promoter eliminated the glucose response without cotransfection of the thyroid receptor, deletion of the CHORE did not affect the glucose response when both thyroid hormone receptor was cotransfected and thyroid hormone was present. However, deletion of the TREs (-3261 to -2110) from the 5-kb delta CHORE construct abolished the effect. These data suggest that the thyroid hormone receptor functions as or in association with a trans-acting glucose response factor. The site of interaction between thyroid hormone and glucose is localized to an area of the gene that is different from the previously described CHORE.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Receptores de Hormona Tiroidea/fisiología , Animales , Secuencia de Bases , Carbohidratos/farmacología , Hígado/metabolismo , Masculino , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Tiroidea/genética , Hormonas Tiroideas/farmacología , Transcripción Genética/efectos de los fármacos , Transfección , Triyodotironina/farmacologíaRESUMEN
S14 is a nuclear protein that is rapidly and synergistically induced by glucose and thyroid hormone, and the level of it's messenger RNA correlates with hepatocyte and adipocyte lipogenesis. We previously reported that the calcium ionophore A23187 markedly inhibits the carbohydrate response of the S14 gene without inhibiting glucose metabolism. Because the calcium ionophore not only increased intracellular cytosolic free calcium but also depletes intracellular calcium stores, we examined which of these two possibilities accounts for the regulation of S14 gene transcription. We found that increasing cytosolic calcium with arginine vasopressin is insufficient to inhibit S14 gene transcription. Furthermore, reduction of intracellular calcium by addition of EGTA to medium containing A23187 leads to further inhibition of S14 transcription. Measurement of intracellular free calcium in indo-1-loaded hepatocytes showed no significant changes induced by high glucose. These results suggested that depletion of an intracellular pool of calcium by A23187 causes the inhibition of S14 transcription. Addition of thapsigargin, which depletes intracellular calcium pools by inhibition of endoplasmic reticulum Ca2+-ATPase, led to significant inhibition of glucose-regulated S14 transcription. Lastly, continuous incubation in 5.5 mM glucose depletes the thapsigargin-sensitive calcium pool. These studies imply that the ability of glucose to induce S14 transcription is related to a thapsigargin-sensitive calcium pool, and depletion of this pool by lowering glucose inhibits S14 transcription.
Asunto(s)
Calcio/metabolismo , Glucosa/farmacología , Hígado/metabolismo , Biosíntesis de Proteínas , Proteínas , Tapsigargina/farmacología , Transcripción Genética , Adipocitos/metabolismo , Animales , Calcimicina/farmacología , Células Cultivadas , Clorpromazina/farmacología , Ácido Egtácico/farmacología , Genes Reporteros , Glucosa/metabolismo , Ionomicina/farmacología , Cinética , Hígado/efectos de los fármacos , Luciferasas/biosíntesis , Masculino , Proteínas Nucleares , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacología , Timidina Quinasa/biosíntesis , Factores de Transcripción , Transcripción Genética/efectos de los fármacos , Transfección , Vasodilatadores/farmacología , Vasopresinas/farmacologíaRESUMEN
Transcription of the rat S14 gene is induced in response to increased carbohydrate metabolism in the liver. Because carbohydrate-induced changes in lipogenesis are mediated in part by changes in phosphorylation of multiple proteins, we investigated the role of protein phosphorylation on transcriptional regulation of the two carbohydrate response elements, a thyroid hormone receptor-independent carbohydrate response element and a thyroid receptor-dependent glucose response element located up-stream of the S14 gene. S14 reporter constructs were transiently transfected into rat primary hepatocytes and incubated with the protein or phosphatase inhibitor okadaic acid calyculin-A, or one of several protein kinase activators. Low dose okadaic acid blocked glucose induction from both elements without inhibiting glucose metabolism. Calyculin-A, a preferential phosphatase-1 inhibitor, only blocked the glucose response when glucose metabolism was inhibited. The protein kinase-C activator, 12-myristate 13-acetate, did not change the glucose responses, whereas the protein kinase-A activator, 8-(4-chlorophenylthio)cAMP, inhibited S14 transcription by inhibiting glucose metabolism. In contrast, the calcium ionophore A23187, a calmodulin kinase activator, mimicked the effect of low dose okadaic acid, but had no effect on glucose metabolism. We conclude that protein phosphatase-2A and calmodulin kinases may be involved in the glucose signaling pathway of the S14 gene. A similar phosphorylation step may be involved in the two distinct glucose response pathways.
Asunto(s)
Glucosa/metabolismo , Hígado/metabolismo , Fosfoproteínas/metabolismo , Proteínas/genética , Transducción de Señal , Transcripción Genética , Animales , Células Cultivadas , Éteres Cíclicos/farmacología , Masculino , Virus del Tumor Mamario del Ratón/genética , Toxinas Marinas , Proteínas Nucleares , Ácido Ocadaico , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Regiones Promotoras Genéticas , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Tiroidea/genética , Timidina Quinasa/genética , Factores de Transcripción , TransfecciónRESUMEN
The rat S14 gene provides an excellent model to examine the DNA sequences associated with carbohydrate regulation of hepatic gene transcription. We constructed internal deletions within 5 kilobases of the 5'-up-stream region and ligated these to a luciferase reporter gene. The constructs were transfected into primary hepatocytes and pancreatic HIT cells. In hepatocytes, an increase in the medium glucose concentration led to a parallel increase in endogenous mRNA S14 content and transfected luciferase reporter activity driven by 5 kilobases of the S14 promoter. Internal deletions of several sequences from -2706 to -285 each led to a decrease in glucose-stimulated activity, suggesting that multiple elements are necessary for the transcriptional response to glucose. Deletion from -1583 to -1069 nearly abolished the glucose effect in both cell types and delineated the carbohydrate response element (CHORE). The CHORE deletion was specific for glucose, because it did not alter the response to thyroid hormone, another known regulator of this gene. Although the CHORE sequence did not confer glucose activation to either a heterologous promoter or the basal S14 promoter (bases -285 to +19), a 5-fold enhanced response was observed when two copies of the CHORE were ligated to the first 2110 basepairs of the S14 promoter. The results suggest that the CHORE contains a carbohydrate regulatory element and operates as an enhancer in concert with other sequences within the S14 gene.
Asunto(s)
Glucosa/farmacología , Hígado/metabolismo , Regiones Promotoras Genéticas , Proteínas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Línea Celular , Línea Celular Transformada , Cricetinae , Elementos de Facilitación Genéticos , Eliminación de Gen , Luciferasas/genética , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión , Mapeo Restrictivo , Factores de Transcripción , TransfecciónRESUMEN
BACKGROUND: Neuroimaging studies have suggested the possible role of the cerebellum in the pathophysiology of schizophrenia. However, no study has investigated the detailed structures of the cerebellum in patients without a history of neuroleptic medication. The objective of this study is to examine the volume of detailed structures of the cerebellum in neuroleptic-naive schizophrenic patients and to examine the relationship between cerebellar morphology and clinical symptoms. METHODS: Magnetic resonance imaging scans were acquired from 20 male neuroleptic-naive schizophrenic patients and 20 healthy control subjects. We measured the volumes of the cerebrum, cerebellar hemisphere, cerebellar gray and white matter, and vermis. Symptoms were assessed with the Brief Psychiatric Rating Scale. Total Brief Psychiatric Rating Scale scores and subscale scores were used for analysis. RESULTS: The volume of the vermis was significantly reduced in the schizophrenic group relative to the control group, whereas no significant differences were found in the volumes of other cerebellar structures and the cerebrum. Reduction in the vermal volume correlated with the total Brief Psychiatric Rating Scale Depression subscore and Paranoia subscore. CONCLUSIONS: This study indicates that the volume of the vermis is reduced in patients with schizophrenia, and reduction in vermal volume is suggested to be related to the pathophysiology of the disease.
Asunto(s)
Núcleos Cerebelosos/patología , Esquizofrenia/patología , Adulto , Femenino , Lateralidad Funcional , Humanos , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Masculino , Escalas de Valoración Psiquiátrica , Psicología del EsquizofrénicoRESUMEN
OBJECTIVES: To investigate the function of the muscarinic cholinergic receptor (mAchR) in narcolepsy and the effects of pharmacotherapy on mAchRs. BACKGROUND: Muscarinic neural transmission serves as the main executive system in REM sleep. Studies in canine narcolepsy reported an increase in mAchRs in the pons. METHODS: The mAchRs of 11 drug naive/free patients with narcolepsy and 21 normal controls were investigated using PET with [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB). Measurements were done in the pons, thalamus, striatum, and cerebral cortex. Seven of the 11 patients also underwent additional PET scans after the alleviation of symptoms by pharmacotherapy. RESULTS: There were no differences in [11C]NMPB binding between the control and drug naive/free patients in all areas analyzed. At the time of on-medication PET scan, [11C]NMPB binding in the thalamus was decreased, but only to a small degree compared with that by anticholinergic drugs. CONCLUSION: The present results do not support the notion that the mAchR is the main site of action of pharmacotherapy in the marked clinical improvement of human cataplexy.