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.

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.

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.

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.

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.

[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.

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.

[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.

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.

Reduction in plasma norepinephrine during fenfluramine treatment.

Plasma norepinephrine levels were reduced to 30% of placebo levels during the administration of fenfluramine, 120 mg/day, for 10 or more days. There was also a significant reduction in plasma dopamine beta-hydroxylase activity during fenfluramine treatment, suggesting that chronic administration may induce antiadrenergic effects which may be useful in the treatment of disorders affected by circulating catecholamines.

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.

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.

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.

Neonatally administered naltrexone affects several behavioral responses in adult rats of both genders.

The effects of a daily injection of the opioid antagonist naltrexone (NALTX, 1 mg/kg SC), from birth to weaning day, on several behavioral parameters were investigated in adult rats of both genders. This work uses three behavioral tests. The use of the open-field test in addition to the holeboard and plus-maze tests allows the measurement of complementary parameters, and provided further information on diverse components of behavior. The effect of repeated testing (habituation) over 3 consecutive days of open-field testing was also assessed. In accordance with previous reports, female rats showed higher locomotor activity and exploration and a lower level of emotionality than their male counterparts. The response of the animals to repeated testing seemed to be both sex and treatment dependent. In both males and females, NALTX treatment during the preweanling period did not affect locomotor activity, but increased the animals' levels of anxiety and emotionality and decreased exploratory behavior. Our results indicate that neonatal opioids may play an important role in the development of behavioral responses in the adult.

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.

Intermediate heterochromatic zones in Triturus (Amphibia: Caudata).

Employment of the C-banding procedure disclosed the presence of slightly stained bands in some chromosome regions of several Triturus species. These regions have been previously described in other organisms as 'grey C-bands'. Possible explanations for the presence of these bands in Triturus karyotypes are discussed.