RESUMEN
Akt, also known as protein kinase B, has a central role in various signaling pathways that regulate cellular processes such as metabolism, proliferation and survival. On stimulation, phosphorylation of the activation loop (A-loop) and hydrophobic motif (HM) of Akt by the kinase phosphoinositide-dependent kinase 1 (PDK1) and the mammalian target of rapamycin complex 2 (mTORC2), respectively, results in Akt activation. A well-conserved threonine in the turn motif (TM) is also constitutively phosphorylated by mTORC2 and contributes to the stability of Akt. The role of TM phosphorylation in HM and A-loop phosphorylation has not been sufficiently evaluated. Using starfish oocytes as a model system, this study provides the first evidence that TM phosphorylation has a negative role in A-loop phosphorylation. In this system, the maturation-inducing hormone, 1-methyladenine, stimulates Akt to reinitiate meiosis through activation of cyclin B-Cdc2. The phosphorylation status of Akt was monitored via introduction of exogenous human Akt (hAkt) in starfish oocytes. TM and HM phosphorylation was inhibited by microinjection of an anti-starfish TOR antibody, but not by rapamycin treatment, suggesting that both phosphorylation events depend on TORC2, as reported in mammalian cells. A single or double alanine substitution at each of three phosphorylation residues revealed that TM phosphorylation renders Akt susceptible to dephosphorylation on the A-loop. When A-loop phosphatase was inhibited by okadaic acid (OA), TM phosphorylation still reduced A-loop phosphorylation, suggesting that the effect is caused at least partially through reduction of sensitivity to PDK1. Negative regulation by TM phosphorylation was also observed in constitutively active Akt and was functionally reflected in meiosis resumption. By contrast, HM phosphorylation enhanced A-loop phosphorylation and achieved full activation of Akt via a mechanism at least partially independent of TM phosphorylation. These observations provide new insight into the mechanism controlling Akt phosphorylation in the cell.
Asunto(s)
Secuencias de Aminoácidos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Activación Enzimática , Humanos , Meiosis , Ácido Ocadaico/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/química , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Sirolimus , Estrellas de Mar , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Transthoracic two-dimensional echocardiography (TTE) has been an accepted noninvasive procedure used to diagnose infective endocarditis by demonstrating the presence of vegetations and other complications such as ring abcess, mycotic lesions or sinus of valsalva aneurysm. Moreover, complementary Doppler and Color Flow imaging are very useful in detecting early valvular regurgitation and in evaluating the severity of such regurgitant lesions. Occasionally, TTE fails to provide an adequate quality of imaging because of the patient's obesity, chest deformity or emphysema. Transesophageal echocardiography (TEE) on the other hand, a relatively new technique, allows ultrasonic imaging of the heart through the esophagus and provides a clear visualization of all cardiac structures without any interference from the lungs, chest wall or rib cage. We present a case of aortic valve endocarditis diagnosed by TEE.
Asunto(s)
Válvula Aórtica/diagnóstico por imagen , Ecocardiografía/métodos , Endocarditis Bacteriana/diagnóstico por imagen , Anciano , Ecocardiografía Doppler , Esófago , Enfermedades de las Válvulas Cardíacas/diagnóstico por imagen , Humanos , MasculinoRESUMEN
It has been generally accepted for 20 years that the growth of Shionogi carcinoma 115 (SC115) is stimulated only by androgen in vivo and in cell culture. However, we recently found that the growth of SC115 is also stimulated by pharmacological, but not physiological, doses of glucocorticoid both in vivo and in cell culture and by pharmacological doses of estrogen only in vivo. In the present study, therefore, we investigated the effect of dexamethasone on androgen-induced growth of SC115 cells in vivo and in cell culture. In a serum-free medium [Ham's F-12:Eagle's minimum essential medium (1:1, v/v) containing 0.1% bovine serum albumin], the proliferation of SC-3 cells (a cloned cell line from SC115 cells) estimated by cell number and DNA synthesis reached a plateau at 10(-8) M testosterone (up to 93-fold) or 10(-6) M dexamethasone (up to 7.2-fold); high stimulation induced by higher than 10(-8) M testosterone was inhibited by the addition of 10(-5)-10(-8) M dexamethasone in a concentration-dependent manner, whereas low stimulation induced by lower than 10(-10) M testosterone was significantly enhanced by the addition of dexamethasone. The presence of typical glucocorticoid and androgen receptors in SC-3 cells was also demonstrated; dexamethasone did not bind to androgen receptor and testosterone did not bind to glucocorticoid receptor. In castrated mice, the concomitant administration of dexamethasone again significantly inhibited the high growth of SC115 tumors induced by high doses of androgen but significantly enhanced the low growth induced by low doses of androgen. The present results demonstrate both inhibitory and stimulatory effects of glucocorticoid on androgen-induced proliferation of SC115 cells in cell culture and probably in vivo.
Asunto(s)
Dexametasona/farmacología , Testosterona/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Línea Celular , Masculino , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Orquiectomía , Receptores Androgénicos/análisis , Receptores de Glucocorticoides/análisisRESUMEN
It has been generally accepted for 20 years that the growth of Shionogi carcinoma 115 (SC115) is stimulated only by androgen. However, we recently found that the growth of SC115 cells is also stimulated by pharmacological doses of estrogen in vivo but not in cell culture. In the present study, the growth-stimulatory effect of glucocorticoid on SC115 cells was examined. In castrated mice, daily injections of high doses of dexamethasone (100 micrograms/mouse) markedly stimulated the tumor growth, and the growth approached that found in normal males. However, daily injections of physiological doses of dexamethasone (4 micrograms/mouse) or high doses of epitestosterone, progesterone, or cholesterol (200-5000 micrograms/mouse) did not enhance the tumor growth in castrated mice. The androgen dependency, growth speed, steroid receptors, and histological type of the tumors grown by pharmacological doses of glucocorticoid were not significantly different from those of the original SC115 tumors grown by androgen. In a serum-free medium [Ham's F-12:Eagle's minimum essential medium (1:1, v/v) containing 0.1% bovine serum albumin], the proliferation of SC-3 cells (a cloned cell line from SC115 cells) was markedly (by up to 25-fold) stimulated by 10(-10)-10(-8) M testosterone, whereas the proliferation was only slightly but significantly (by up to 3.3-fold) stimulated by 10(-8)-10(-5) M dexamethasone. The present findings demonstrate that the growth of SC115 cells in vivo and in cell culture is significantly stimulated by physiological doses of androgen or pharmacological doses of glucocorticoid.