La respuesta a hemina in vitro es una caracteristica compartida por todos los miembros de la familia de las eIF2á quinasas

Four distinct eukaryotic initiation factor 2á (eIF2á) kinases phosphorylate eIF2á at Ser-51 and regulate protein synthesis in response to cellular stress conditions. This kinase family includes the hemin-regulated inhibitor (HRI); the doublestranded RNA-dependent kinase (PKR); the GCN2 protein kinase; and the endoplasmic reticulum-resident kinase (PERK). HRI mediates protein synthesis inhibition in heme-deficient reticulocyte lysates. Although HRI contains two putative heme regulatory motifs (HRMs) that are not present in other eIF2á kinases, the significance of these motifs in heme regulation is not clear. In fact, it had been characterized two novel eIF2á kinases from Schizosaccharomyces pombe that lacked any of the HRMs, but were sensitive to heme regulation in vitro. To investigate the importance of different regions in the regulation of HRI by heme, specific HRI mutants were generated, and kinase activities and heme responsiveness were analyzed in vitro. Mutational analysis indicated that the heme regulatory motifs were spread around some regions in the HRI catalytic domain, outside of the HRMs. In accordance with these results, both the autokinase and the eIF2á kinase activities of three distinct eIF2á kinases, including the human PKR, the mouse GCN2 and the Drosophila PERK were inhibited in vitro by hemin. Although the known regulatory mechanisms of these eIF2á kinases are very different, the data reported here indicate that all known eIF2á kinases are regulated in vitro by hemin. This finding provides evidence that hemin represents a regulatory mechanism unique to eIF2á kinases and underscores the role of hemin in the translational regulation of eukaryotic cells.Las cuatro eIF2ƒ¿ quinasas eucarioticas fosforilan el residuo Ser-51 de la subunidad alfa del factor de iniciacion 2 y regulan la sintesis de proteinas en respuesta a situaciones de estres celular. Esta familia de proteinas quinasas esta formada por el inhibidor regulado por hemina (HRI); la quinasa dependiente de RNA de doble cadena (PKR); la proteina quinasa GCN2 y la quinasa residente en el reticulo endoplasmico (PERK). El HRI inhibe la sintesis de proteinas en lisados de reticulocitos de conejo deficientes de hemina. Aunque el HRI contiene dos supuestos motivos reguladores de hemina (HRMs), que no estan presentes en las otras eIF2ƒ¿ quinasas, no esta claro aun el papel de estos motivos en la regulacion por hemina. De hecho, se han caracterizado dos nuevas eIF2ƒ¿ quinasas de Schizosaccharomyces pombe que carecen de dichos HRMs, pero son sensibles a la regulacion por hemina in vitro. Un analisis mutacional indico que los motivos reguladores de hemina estaban dispersos a lo largo del dominio catalitico, fuera de los HRMs. De acuerdo con estos resultados, las actividades autoquinasa y eIF2ƒ¿ quinasa de tres eIF2ƒ¿ quinasas distintas, la PKR humana, la GCN2 de raton y la PERK de Drosophila, se inhibian por hemina in vitro. Aunque los mecanismos de regulacion de todas estas eIF2ƒ¿ quinasas son muy diferentes, nuestros resultados indican que todas las eIF2ƒ¿ quinasas se regulan por hemina in vitro. Este descubrimiento soporta la evidencia de que la hemina representa un mecanismo de regulacion especifico de las eIF2ƒ¿ quinasas, y subraya su papel en la regulacion de la traduccion de celulas eucarioticas

La interdisciplinariedad y el itinerario curricular elegido por el estudiante, ejes fundamentales para desarrollar competencias curriculares en Química

Póster presentado en: V Jornadas de Innovación Docente de la UBU, Burgos, 21-22 de octubre de 2010, organizadas por el Instituto de Formación e Innovación Educativa-IFIE de la Universidad de Burgo

Clonaje y caracterización de nuevas e IF2[alfa] quinasas reguladas por hemina

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-12-200

Role of Mitogen-Activated Protein Kinase Sty1 in Regulation of Eukaryotic Initiation Factor 2α Kinases in Response to Environmental Stress in Schizosaccharomyces pombe▿

The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2α (eIF2α) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2α, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2α kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2α kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2α phosphorylation. Finally, the MAPK Sty1, but not the eIF2α kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2α kinase pathways for a particular range of stress responses

Low bone density with normal bone turnover in ovariectomized and streptozotocin-induced diabetic rats

Diabetes and estrogen deficit are known causes of osteopenia, diabetes being associated with a low bone turnover and estrogen deficit with a high bone turnover. In the present work, we studied the effect of combined ovariectomy and diabetes on bone mineral content (BMC) and bone mineral density (BMD) and several bone markers in the rat. Four groups of rats were studied: control (C), ovariectomized (O), diabetic (D), and ovariectomized and diabetic (DO). Twelve weeks after starting the experiments, BMC and BMD of the first six lumbar vertebrae were measured; a bone formation marker (BGP) and a bone resorption marker (free collagen cross-links, PYD) were also analyzed. Diabetic rats showed diminished gain in bone mass, BMC (D: 0.417 +/- 0.028 g, DO: 0.422 +/- 0.020 g) and BMDs (D: 0.171 +/- 0.006 g/cm2, DO: 0.174 +/- 0.006 g/cm2) both being significantly (P < 0.001) lower than those of control (C: BMC 0.727 +/- 0.024 g and BMD 0.258 +/- 0.004 g/cm2) and ovariectomized (O: BMC 0.640 +/- 0.044 g and BMD 0.240 +/- 0.009 g/cm2) groups. Moreover, the BMC and BMD of the C group were significantly (P < 0.05) higher than that of the O group. BGP and PYD levels were significantly (P < 0.01) higher in the O group (BGP: 138.2 +/- 16.8 ng/ml, PYD: 270.2 +/- 17.8 nM/mM) than those found in the control rats (BGP: 44.7 +/- 4.8 ng/ml, PYD: 165.6 +/- 12.5 nM/mM); the D group showed significantly (P < 0.01) lower values (BGP: 27.4 +/- 14.6 ng/ml, PYD: 55.0 +/- 7.4 nM/mM) than those of the control group. The DO group showed similar levels (BGP: 43.4 +/- 5.1 ng/ml, PYD: 146.7 +/- 14.6 nM/mM) to those found in the C group. Although bone marker levels in the O and D groups were in accordance with those expected in these situations, in the DO group the corresponding levels are apparently "normal." Also, the decrease of gain in bone mass observed after combining estrogen deficit and diabetes (DO group) did not seem to be more marked than that caused by diabetes alone.This study was partially supported by a grant from the Comisión Interministerial de Ciencia y Tecnología, Spain (SAL 91-0802). The densitometer was generously donated by Rhône-Poulenc-Rorer S.A.Peer reviewe

Heterogeneous decrease of bone mineral density in the vertebral column of ovariectomized rats

The long-term effect of ovariectomy on the loss of bone mineral density (BMD) was evaluated in rats with and without estrogen treatment; BMD was studied in the lumbar and caudal vertebrae, measured by DXA, to find how the losses of BMD occur in the axial skeleton. Seventy female Wistar rats of 3 months of age were divided into four groups as follows: group 1: control animals; group 2: ovariectomized animals; group 3: ovariectomized animals undergoing treatment with estrogen (0.25 mg/kg per week of 17-beta estradiol); group 4: ovariectomized rats undergoing estrogen treatment only during the last 3 months of the experimental period. No significant differences were found among the groups in regard to the BMD values of the caudal vertebrae at either 3 or 6 months. Likewise, in the lumbar vertebrae there were no significant differences among the groups after 3 months. However, at 6 months, a decrease in the BMDs of the ovariectomized animals with respect to the remaining groups was found: 226 +/- 11 mg/cm2 in the ovariectomized group; 262 +/- 14 mg/cm2 in the controls; 255 +/- 4 mg/cm2 in the rats receiving estrogen treatment for 6 months; and 259 +/- 5 mg/cm2 in the animals receiving estrogen for 3 months. The study also reveals the absence of differences in the bone mineral density between the ovariectomized and control rats when the former received estrogen treatment. According to the changes in bone remodeling, the ovariectomized rats showed an increase in the hydroxyproline/creatinine and Ca++/creatinine ratios; this increase is accompanied by an increase in serum alkaline phosphatase and osteocalcin levels. In summary, measurement of the BMD in rats by the DXA method permits detection of loss of bone mineral density occurring in certain zones of the axial skeleton at 6 months after ovariectomy.This study was partially supported by a grant from the Comisión Interministerial de Ciencia y Tecnologia, Spain (SAL 91-0802). The densitometer was generously donated by Rhône-Poulenc Rorer S,A.Peer reviewe

Vida y aventuras de Robinsón Crusoé,

"Madrid: Imprenta de Jaime Ratés ..."Publisher's advertisements: [10] p. at end.Contains pt. 1 only.Mode of access: Internet.SPEC: Copy 2: [4], [7]-335, [13] p. Illustrated bookplate on front pastedown: William S. Lloyd. Bookplate on front pastedown: Philip H. and A.S.W. Rosenbach Foundation Museum