Perceptions from nurses, surgeons, and anesthetists about the use and benefits of the surgical checklist in a teaching hospital.

OBJECTIVE: To assess attitudes and perceptions from nursing staff, surgeons and anesthetists about compliance, utility, and impact on patient's safety of the surgical checklist in a teaching hospital. We also aimed to identify improvement opportunities for strengthening the usefulness of the checklist in the operating theater. METHODS: We carried out a questionnaire-based an observational cross-sectional study. A questionnaire was distributed to operating room staff, including nursing staff, surgeons, and anesthetists. In addition to the information about surgical checklist, We also collected information regarding years of experience in the operating theater. Fisher's exact was used to compare proportions in each statement. Group discussion meetings with key professionals were held to jointly assess the results, propose improvement actions, and evaluate their feasibility. RESULTS: The overall response rate was 36.2% (131/362). Nursing staff was perceived as the most supportive group in the use of surgical checklist. A 64.3% of surgeons considered that using the checklist prevented adverse events vs 84.2% and 85.7% among anesthetists and nurses, respectively; p=0.028. Junior staff showed a supportive attitude toward the use of surgical checklist, considering it as a tool that gives them confidence. We ended up with a list of improvement actions aiming at strengthening the surgical checklist reliability and compliance. CONCLUSIONS: The perception of the surgical checklist usefulness as a tool to prevent adverse events was moderate among surgeons, but well appreciated by junior staff. Nursing staff were especially critical regarding compliance and support by other professionals. To reinforce the usefulness perception of the surgical checklist it is needed to increase the involvement of all professionals, especially senior staff and surgical leaders.

Dynamics of cohesin subunits in grasshopper meiotic divisions.

The cohesin complex plays a key role for the maintenance of sister chromatid cohesion and faithful chromosome segregation in both mitosis and meiosis. This complex is formed by two structural maintenance of chromosomes protein family (SMC) subunits and two non-SMC subunits: an α-kleisin subunit SCC1/RAD21/REC8 and an SCC3-like protein. Several studies carried out in different species have revealed that the distribution of the cohesin subunits along the chromosomes during meiotic prophase I is not regular and that some subunits are distinctly incorporated at different cell stages. However, the accurate distribution of the different cohesin subunits in condensed meiotic chromosomes is still controversial. Here, we describe the dynamics of the cohesin subunits SMC1α, SMC3, RAD21 and SA1 during both meiotic divisions in grasshoppers. Although these subunits show a similar patched labelling at the interchromatid domain of metaphase I bivalents, SMCs and non-SMCs subunits do not always colocalise. Indeed, SA1 is the only cohesin subunit accumulated at the centromeric region of all metaphase I chromosomes. Additionally, non-SMC subunits do not appear at the interchromatid domain in either single X or B chromosomes. These data suggest the existence of several cohesin complexes during metaphase I. The cohesin subunits analysed are released from chromosomes at the beginning of anaphase I, with the exception of SA1 which can be detected at the centromeres until telophase II. These observations indicate that the cohesin components may be differentially loaded and released from meiotic chromosomes during the first and second meiotic divisions. The roles of these cohesin complexes for the maintenance of chromosome structure and their involvement in homologous segregation at first meiotic division are proposed and discussed.

Diagnótico indirecto del abuso/dependencia al alcohol en población adulta peruana: validación de un encuesta / Indirect diagnose of alcohol of abuse/dependency in an adult peruvian population: validation of a survey

Objetivo: Realizar la validación de la encuesta para diagnóstico indirecto de abuso/dependencia al alcohol en la sierra peruana. Material y método: Se usó la base de datos del Estudio Epidemiológico de Salud Mental 2003 realizado en la sierra peruana que contenía módulos que evaluaban la situación de salud mental. Se utilizó el módulo de diagnóstico directo de abuso/dependencia al alcohol, aplicado al adulto y la encuesta diagnóstico indirecto de abuso/dependencia al alcohol, aplicado a la mujer unida. La muestra fue de 1 302 parejas mujer unida û adulto. Para hallar la confiabilidad se utilizó el coeficiente alfa de Cronbach. Se realizó la validez de criterio comparándola con el cuestionario directo y la de constructo utilizando el análisis factorial. Resultados: Se obtuvo una coherencia interna de 93,1 por ciento. La probabilidad estimada de que el criterio externo y el cuestionario coincidan en el diagnóstico es 77 por ciento. Siendo el punto de mejor sensibilidad (70,4 por ciento) y especificidad (74,9 por ciento) en 2 ítems. El análisis factorial indica que la mayoría de ítems satura a un factor predominante. Conclusiones: El cuestionario indirecto para diagnóstico de abuso/ dependencia de alcohol es parcialmente válido para poblaciones con características similares a las de la sierra peruana.

Objective: Validation of a survey for making an indirect diagnosis of abuse/dependence of alcohol survey in the Peruvian Andes. Material and method: We used the database of The Mental Health Epidemiological Study conducted in the Peruvian Andes in 2003, which includes surveys assessing mental health. We used the direct diagnosis of alcohol abuse/dependence module in adult males, and indirect diagnosis of alcohol abuse/dependence survey in their spouses. The sample size was 1,302 adult couples. CronbachÆs Alpha coefficient was used in order to verify reliability of the tests. Validity of criteria was obtained by comparing them with the direct questionnaire and the validity of the construct was determined by using a factorial analysis. Results: The internal consistency of the questionnaire was 93.1 percent. The estimated probability of matching between the indirect survey and the external criteria is 77 percent. Best sensitivity (70.4 percent) and specificity (74.9 percent) values were located in 2 positive items. The factorial analysis indicated that most of the items saturate one predominant factor. Conclusions: The questionnaire for indirect diagnosis of alcohol abuse/ dependence is partially valid in populations with similar characteristics to those from the Peruvian Andes.

Incomplete synapsis and chiasma localization: the chicken or the egg?

In the present study, and as a sincere tribute from the Cytogenetics teams from Madrid to Professor Máximo Drets on his 80th birthday, we have analyzed and compared 3 different grasshopper species with different synaptic patterns, a standard pattern, a second pattern with synapsis restricted to the proximal regions, and a third pattern with synapsis restricted to the distal regions. In the 3 species we have thoroughly analyzed the relationships among cohesin axis morphogenesis, formation of double strand breaks (DSBs) and recombination initiation. Our results demonstrate that in every case recombination initiation precedes synapsis, and that there is a direct relationship between the absence of meiotic recombination and the existence of particular unsynapsed chromosomal regions during prophase I. Based on our results we propose and discuss the mechanisms underlying the existence of incomplete synapsis and the localization of chiasma in wild species.

[Photodynamic therapy with verteporfin in choroidal neovascularization after refractive surgery]. / Terapia fotodinámica con verteporfina en neovascularización coroidea después de cirugía refractiva.

PURPOSE: To analyze the results obtained with Photodynamic Therapy (PDT) to treat subfoveal and juxtafoveal Choroidal Neovascularization (CNV) in patients with high myopia corrected by Laser-Assisted in situ Keratomileusis (LASIK) or by implanting a Phakic Intraocular Lens (PIOL). METHODS: We analyzed the results from 14 highly myopic eyes corrected by LASIK (seven cases) or by PIOL implantation (seven cases), which later developed CNV and were treated by PDT with verteporfin. RESULTS: Mean Best Corrected Visual Acuity (BCVA) after refractive surgery was 0.45 SD 0.17 (range, 0.2 to 0.8), with residual spherical equivalent (RSE) -0.5 SD 1.8 D (range, 1 to 5.5 D). After CNV appearance, BCVA was 0.10 SD 0.19 (range, 0.025 to 0.7). CNV was treated in all cases by PDT (mean, 2.0 SD 0.8 treatments). After CNV closure, the mean BCVA improved up to 0.22 SD 0.18 (range, 0.1 to 0.63) (RSE -1.4 SD 1.4 D, range, 0.5 to -4 D). Differences in RSE after refractive surgery and after PDT, and differences between BCVA after CNV appearance and final were not statistically significant (p=0.82 and p=0.06, respectively, Student's t test paired data). CONCLUSION: We consider that PDT is effective in achieving closure of CNV in myopic patients after refractive surgery without inducing changes in spherical equivalent.

Incomplete posterior hyaloid detachment after intravitreal pegaptanib injection in diabetic macular edema.

PURPOSE: Posterior hyaloid adherences play a role in the pathogenesis of diabetic macular edema (DME). Intravitreal antivascular endothelial growth factor (VEGF) drugs are presently being used to treat DME. The authors report one case of incomplete posterior hyaloid detachment (PHD) following intravitreal pegaptanib to treat DME. This case shows a combined mechanism of DME resolution by anti VEGF and PHD. METHODS: Prospective, interventional, single case report. One male patient with bilateral DME was treated by intravitreal pegaptanib in his right eye every 6 weeks for 6 months (five injections) and followed for 42 months. RESULTS: Central macular thickness decreased from 511 to 376 microm at month 4 in the treated eye and remained within 10% of this value during follow-up. The posterior hyaloid became taut and partially detached after the third injection and was almost completely detached 1 year later. Visual acuity remained unchanged in both eyes during follow-up. CONCLUSIONS: PHD may play an important role in cases where macular thickness is successfully reduced or better acuity is achieved after intravitreal injections.

Intravitreal bevacizumab for adult-onset vitelliform dystrophy: a case report.

PURPOSE: Adult-onset foveomacular vitelliform dystrophy (AFVD) is often misdiagnosed as occult choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). The authors report the anatomic and functional outcome of intravitreal bevacizumab in a case of AFVD associated with a suspected occult CNV. METHODS: Prospective, interventional, single case report. One female patient with decreased visual acuity (VA) and metamorphopsia secondary to AFVD received one single intravitreal injection of bevacizumab 1.25 mg. RESULTS: The patient reported unchanged VA and decreased metamorphopsia 6 weeks after the injection. Fluorescein angiography (FA) and optical coherence tomography (OCT) showed progressive decrease of subretinal fluid until complete disappearance. VA, OCT, and FA remained unchanged during 10 months follow-up. CONCLUSIONS: Intravitreal bevacizumab showed a morphologic improvement and stable VA in a patient with AFVD. Further case series are required to confirm this observation.

Therapeutic window for melanoma treatment provided by selective effects of the proteasome on Bcl-2 proteins.

Melanoma cells depend on sustained proteasomal function for survival. However, bortezomib, the first proteasome inhibitor in clinical use, is not sufficient to improve the poor prognosis of metastatic melanoma patients. Since the proteasome is also expressed in all normal cell compartments, it is unclear how to enhance the efficacy of bortezomib without exacerbating secondary toxicities. Here, we present pharmacological and genetic analyses of mechanisms of resistance to proteasome inhibition. We focused on Bcl-2, Bcl-x(L) and Mcl-1 as main antiapoptotic factors associated with melanoma progression. Despite an efficient blockage of the proteasome, bortezomib could not counteract the intrinsically high levels of Bcl-2 and Bcl-x(L) in melanoma cells. Moreover, Mcl-1 was only downregulated at late time points after treatment. Based on these results, a combination treatment including (-)-gossypol, an inhibitor of Mcl-1/Bcl-2/Bcl-x(L), was designed and proven effective in vivo. Using a specific RNA interference approach, the survival of bortezomib-treated melanoma cells was found to rely primarily on Mcl-1, and to a lesser extent on Bcl-x(L) (but not on Bcl-2). Importantly, neither Mcl-1 nor Bcl-x(L) inactivation affected the viability of normal melanocytes. This hierarchical requirement of Bcl-2 family members for the maintenance of normal and malignant cells offers a therapeutic window to overcome melanoma chemoresistance in a tumor cell-selective manner.

Réplica. Receptores de glutamato de tipo kainato y epilepsia / Reply. Kainate-type glutamate receptors and epilepsy

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[The neurochemistry of epilepsy, inhibitory neurotransmission and experimental models: new perspectives]. / Neuroquímica de la epilepsia, neurotransmisión inhibitoria y modelos experimentales: nuevas perspectivas.

AIMS: The biological mechanisms of epilepsy allow pathophysiological patterns to be established which are essential for the selection of new therapeutic targets. The identification of these mechanisms also provides us with knowledge about the dynamics of neuronal arrangement, synaptogenesis, synaptic transmission and the receptors involved, and even the development of the brain. DEVELOPMENT: Recent advances in neurobiology regarding the GABAergic system point to it as playing a leading role in the pathophysiology of epilepsy. We evaluate the different functional formats of the ionotropic (GABAA) and metabotropic (GABAB) gamma-aminobutyric (GABA) receptors. Although the main function posited is inhibitory, owing to the variability of their location, subunits and neuronal physiology/maturation they can even end up expressing excitatory functions. We discuss the anomalies in the GABAergic system identified in animal models with epilepsy and in brain tissue samples from patients submitted to surgery due to epilepsy. The mechanism inhibiting the activation of GABA receptors is performed by hyperpolarisation achieved by entry of Cl- into the neuron--a process mediated by the cotransporter KCC2, typically expressed in the neuron. Mutations in the KCC2 gene produce mice that are susceptible to seizures. In some animal models it has been found that loop diuretics (furosemide) suppress seizures. Mutations in genes that code for ion channels have been identified in numerous epileptic syndromes and this pushes epilepsy ever further inside the broad group of disorders known as channelopathies. The origin could be polygenetic in many cases. CONCLUSIONS: The GABAergic system seems to situate itself as the main system implicated in the pathophysiology of epilepsy, although conditions that have been considered to be idiopathic up till now could have a polygenic nature.

Neuroquímica de la epilepsia, neurotransmisión inhibitoria y modelos experimentales: nuevas perspectivas / The neurochemistry of epilepsy, inhibitory neurotransmission and experimental models: new perspectives

Objetivo. Los mecanismos biológicos de la epilepsia permiten establecer patrones fisiopatológicos clave para la selección de nuevas dianas terapéuticas. La identificación de estos mecanismos aporta además conocimiento sobre la dinámica de ordenación neuronal, la sinaptogénesis, la transmisión sináptica y los receptores implicados e incluso el desarrollo cerebral. Desarrollo. Los recientes avances neurobiológicos sobre el sistema gabérgico identifican a éste como un agente principal implicado en la fisiopatología de la epilepsia. Evaluamos los distintos formatos funcionales de los receptores g-aminobutíricos ionotrópicos (GABAA) y metabotrópicos (GABAB): aunque se postula una función inhibitoria principalmente, con la variabilidad en su localización, las subunidades y la maduración/fisiología neuronal pueden acabar expresando funciones incluso excitadoras. Se discuten las anomalías en el sistema gabérgico identificadas en modelos animales con epilepsia y muestras cerebrales de pacientes sometidos a cirugía a causa de la epilepsia. El mecanismo inhibitorio de la activación de receptores GABA se lleva a cabo por la hiperpolarización obtenida mediante la entrada de Cl– a la neurona, mediada por el cotransportador KCC2, de expresión típicamente neuronal. Mutaciones en el gen de KCC2 producen ratones susceptibles a crisis. En algunos modelos animales se ha comprobado una supresión de las convulsiones con diuréticos de asa (furosemida). La identificación en múltiples síndromes epilépticos de mutaciones en genes que codifican canales iónicos sitúan a la epilepsia dentro del cada vez más amplio grupo de trastornos conocidos como canalopatías. El origen podría ser poligenético en numerosos casos. Conclusión. El sistema gabérgico parece postularse como el principal sistema implicado en la fisiopatología de la epilepsia, aunque los cuadros hasta ahora considerados idiopáticos podrían tener un carácter poligénico (AU)

Aims. The biological mechanisms of epilepsy allow pathophysiological patterns to be established which are essential for the selection of new therapeutic targets. The identification of these mechanisms also provides us with knowledge about the dynamics of neuronal arrangement, synaptogenesis, synaptic transmission and the receptors involved, and even the development of the brain. Development. Recent advances in neurobiology regarding the GABAergic system point to it as playing a leading role in the pathophysiology of epilepsy. We evaluate the different functional formats of the ionotropic (GABAA) and metabotropic (GABAB) gamma-aminobutyric (GABA) receptors. Although the main function posited is inhibitory, owing to the variability of their location, subunits and neuronal physiology/maturation they can even end up expressing excitatory functions. We discuss the anomalies in the GABAergic system identified in animal models with epilepsy and in brain tissue samples from patients submitted to surgery due to epilepsy. The mechanism inhibiting the activation of GABA receptors is performed by hyperpolarisation achieved by entry of Cl¨C into the neuron ¨Ca process mediated by the cotransporter KCC2, typically expressed in the neuron. Mutations in the KCC2 gene produce mice that are susceptible to seizures. In some animal models it has been found that loop diuretics (furosemide) suppress seizures. Mutations in genes that code for ion channels have been identified in numerous epileptic syndromes and this pushes epilepsy ever further inside the broad group of disorders known as channelopathies. The origin could be polygenetic in many cases. Conclusions. The GABAergic system seems to situate it self as the main system implicated in the pathophysiology of epilepsy, although conditions that have been considered to beidiopathic up till now could have a polygenic nature (AU)

Spatio-temporal dynamics of a neutralized B chromosome in the grasshopper Eyprepocnemis plorans.

Spatial and temporal patterns of frequency variation for a neutralized B chromosome in the grasshopper Eyprepocnemis plorans were analyzed along six transects in the east of Spain to explore possible factors affecting the population dynamics of this polymorphism. Three parameters were employed to quantify B frequency: prevalence, load and mean frequency. Of them, load seemed to be the less sensitive parameter, probably due to its small range of variation. Prevalence, however, shows ample variation, but the mean frequency of B chromosomes per individual is the best parameter to characterize B frequency. Only river transects revealed significant differences among populations, and the use of two geographic explicit approaches (Mantel test and distograms) revealed significant isolation by distance (IBD), especially at the Segura River mouth, presumably due to low gene flow and drift. No temporal trend was found in the Segura River transects, which is consistent with the slow changes in B frequency expected during the random walk for neutralized B chromosomes. But these transects showed a clear spatial pattern, with B1 showing lower frequency in the upper course of this river. The present results provide the first empirical evidence of IBD in the evolution of a neutralized B chromosome, and support the notion that B dynamics at this evolutionary stage is best explained by a metapopulation approach.

Telomeric and interstitial telomeric-like DNA sequences in Orthoptera genomes.

A (TTAGG)n-specific telomeric DNA probe was hybridized to 11 orthopteroid insect genomes by fluorescence in situ hybridization. Nine different genera, mainly distributed within two evolutionary branches with male chromosome numbers 2n = 23 and 2n = 17 were included in the analysis. Telomere sequences yielded positive signals in every telomere and there was a considerable number of interstitial telomeric-like sequences, mainly located at the distal end of some, but not all, subterminal chromosome regions. One of the species, Pyrgomorpha conica, showed massive hybridization signals associated with constitutive heterochromatin. The results are discussed along two lines: (i) the chromosomal evolutionary trends within this group of insects and (ii) the putative role that ITs may play in a genome when they are considered telomere-derived, but not telomere-functional, DNA sequences.

Surgical removal of subfoveal choroidal neovascularisation in highly myopic patients.

AIM: To analyse the visual results obtained in the treatment of subretinal choroidal neovascularisation (CNV) in patients with high myopia by vitrectomy and extraction of the neovascular membrane. METHODS: 22 eyes of 22 patients with high myopia (>-6 dioptres and/or axial length >26 mm) with subfoveal CNV treated by extraction of the CNV were analysed retrospectively. The patients' mean age was 60.27 (SD 16.41) years (range 32-83 years). The mean follow up was 29.3 (9.9) months (range 12-42 months). RESULTS: Best corrected visual acuity (BCVA) preoperatively was 0.09 (0.07) (range 0.01-0.3). After treatment BCVA was 0.12 (0.10) (range 0.01-0.4), a difference without statistical significance (p=0.03, Student's t test paired data). In four cases the CNV recurred, in three cases cataract developed that required extraction, and in one case retinal detachment occurred in the early postoperative period. Topical treatment was necessary in two cases to lower the intraocular pressure. CONCLUSIONS: The treatment of subfoveal CNV in highly myopic patients by surgical removal by vitrectomy does not achieve any significant improvement of the BCVA.

Possible mechanisms involved in the down-regulation of translation during transient global ischaemia in the rat brain.

The striking correlation between neuronal vulnerability and down-regulation of translation suggests that this cellular process plays a critical part in the cascade of pathogenetic events leading to ischaemic cell death. There is compelling evidence supporting the idea that inhibition of translation is exerted at the polypeptide chain initiation step, and the present study explores the possible mechanism/s implicated. Incomplete forebrain ischaemia (30 min) was induced in rats by using the four-vessel occlusion model. Eukaryotic initiation factor (eIF)2, eIF4E and eIF4E-binding protein (4E-BP1) phosphorylation levels, eIF4F complex formation, as well as eIF2B and ribosomal protein S6 kinase (p70(S6K)) activities, were determined in different subcellular fractions from the cortex and the hippocampus [the CA1-subfield and the remaining hippocampus (RH)], at several post-ischaemic times. Increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) and eIF2B inhibition paralleled the inhibition of translation in the hippocampus, but they normalized to control values, including the CA1-subfield, after 4--6 h of reperfusion. eIF4E and 4E-BP1 were significantly dephosphorylated during ischaemia and total eIF4E levels decreased during reperfusion both in the cortex and hippocampus, with values normalizing after 4 h of reperfusion only in the cortex. Conversely, p70(S6K) activity, which was inhibited in both regions during ischaemia, recovered to control values earlier in the hippocampus than in the cortex. eIF4F complex formation diminished both in the cortex and the hippocampus during ischaemia and reperfusion, and it was lower in the CA1-subfield than in the RH, roughly paralleling the observed decrease in eIF4E and eIF4G levels. Our findings are consistent with a potential role for eIF4E, 4E-BP1 and eIF4G in the down-regulation of translation during ischaemia. eIF2 alpha, eIF2B, eIF4G and p70(S6K) are positively implicated in the translational inhibition induced at early reperfusion, whereas eIF4F complex formation is likely to contribute to the persistent inhibition of translation observed at longer reperfusion times.

Ischemia-induced inhibition of the initiation factor 2alpha phosphatase activity in the rat brain.

Rats were subjected to the standard four-vessel occlusion model of brain transient ischemia for 30 min. Following different recirculation periods, the level of phosphorylation of the initiation factor 2 subunit alpha (eIF2alpha) and the eIF2alpha kinase/s and phosphatase/s activity were determined. eIF2alpha phosphorylation significantly increased very early during reperfusion (10-30 min), recovering at 4 h of reperfusion. Activation of any eIF2alpha kinases studied during ischemia or reperfusion was not noted. Conversely, eIF2alpha phosphatase activity significantly decreased at 10-15 min of reperfusion, reaching values even higher than in controls at 2-4 h of reperfusion. Our results support the hypothesis that the reperfusion-induced phosphorylated eIF2alpha changes are at least a result of the transiently eIF2alpha phosphatase inhibition.

Lauryl gallate inhibits the activity of protein tyrosine kinase c-Src purified from human platelets.

The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu,Na,Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.

Resistance of initiation factor 2 (eIF-2alpha) kinases to staurosporine: an approach for assaying the kinases in crude extracts.

We studied the effect of staurosporine on two well characterised mammalian eIF-2alpha kinases, the heme-regulated translational inhibitor (HRI), and interferon-inducible double-stranded RNA-activated protein kinase (PKR). Both pure eIF-2 and a synthetic peptide used to measure the activity of purified or immunoprecipitated enzymes (sequence ILLSELSRRRIRAI) were phosphorylated with purified enzymes and crude preparations of tissues or cells in the presence of the inhibitor. In the presence of 0.25 microM staurosporine (a concentration which completely inhibits a wide range of Ser/Thr protein kinases), the phosphorylation of eIF-2alpha by HRI and PKR was not inhibited. The lack of response of eIF-2alpha kinases to staurosporine allowed us to measure PKR activity in salt washed postmicrosomal supernatants without previous purification of the enzyme. In the presence of poly(I):poly(C), the PKR activator, we detected both an increased phosphorylation of eIF-2alpha and an increment in the autophosphorylation of PKR. We also confirmed an induction of PKR in cultured neuronal cells after treatment with interferon. The results obtained following phosphorylation of the synthetic peptide with crude extracts are less conclusive. Although its phosphorylation is specific because it displaces eIF-2 phosphorylation, and the presence of staurosporine prevents its phosphorylation by other serine/threonine kinases, it is a rather poor substrate for PKR.

Calcium mobilization by ryanodine promotes the phosphorylation of initiation factor 2alpha subunit and inhibits protein synthesis in cultured neurons.

Protein synthesis plays an important role in the viability and function of the cell. There is evidence indicating that Ca2+ may be a physiological regulator of the translational process. In the present study, the effect of agents that increase intracellular calcium levels by different mechanisms, as well as repercussion on the rate of protein synthesis, including phosphorylation of initiation factor 2alpha subunit, and double-stranded RNA-dependent eIF-2alpha kinase (PKR) activity were analyzed. Glutamate (100 microM) and K+ (60 mM), which increase intracellular calcium levels (the former mostly by the influx of extracellular calcium via voltage-sensitive calcium channels, and the latter by receptor-operated calcium channels), and carbachol (1 mM), as well as glutamate, which mobilizes intracellular calcium from the endoplasmic reticulum via activation of inositol 1,4,5-trisphosphate receptor, did not modify any of the analyzed parameters. Nevertheless, 100 nM ryanodine, which increases intracellular calcium concentration by activating the ryanodine receptor, promoted a significant decrease in the rate of protein synthesis and increased both initiation factor 2alpha subunit phosphorylation and PKR activity. From our results, we can conclude that inhibition of protein synthesis is dependent on the mobilization of intracellular calcium from internal stores. Moreover, they strongly suggest that this inhibition is only promoted when calcium is increased via ryanodine receptor, and possibly by activation of PKR activity.