Transcriptional activity and splicing factors are preserved during physiological apoptosis.

Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism's life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the progression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome elements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.

Psychiatric rehabilitation in Latin America: challenges and opportunities.

Comprehensive psychiatric rehabilitation programs in Latin America have been designed across several countries in the region without yet achieving full implementation. Facing an increasing burden of disease due to mental disorders, including alcohol and substance use disorders, the region has responded unevenly to the challenge. Moreover, low priority for mental health in national policies and insufficient funding for mental health services are common barriers for the much-needed mental health services reforms. Reestablishing a primary care community-based model of care has been a shared aspiration for most countries during the last two decades. Comprehensive models of psychiatric rehabilitation developed predominantly in high-income countries need to be culturally adapted to local contexts, while strengthening health systems research will provide evidence on the efficiency of locally designed interventions and on the critical milestones to succeed in the scaling up strategies. Increasing participation of patients and their families in the mental health delivery system is another key factor in order to ensure comprehensive patient-centred psychosocial rehabilitation programs in Latin America.

Association between subclinical carotid atherosclerosis, hyperhomocysteinaemia and mild cognitive impairment.

OBJECTIVE: Evidence suggests that intima-media thickness (IMT) and plasma homocysteine (Hcy) levels are associated with one another, and both appear to be related to cognitive dysfunction. However, no connection between both factors taken together and mild cognitive impairment (MCI) has been established. This study analysed potential relationships between IMT, Hcy and MCI. METHODS: We included 105 patients with MCI and 76 controls with no history of vascular disease. All participants underwent laboratory analyses, a carotid ultrasound, and clinical and neuropsychological assessment. We used the Mantel-Haenszel test (MHT), ANCOVA and multiple linear regression models (MLRM) to examine any associations between IMT, Hcy and cognitive state. RESULTS: The MHT revealed a significant association between IMT and risk of MCI (z = 4.285, P < 0.0001). The OR for the upper quartile vs the lower quartile was 5.12 (95% CI: 2.12-12.36). MHT also showed a clear association between Hcy levels and risk of MCI (z = 3.01, P = 0.003). OR for the upper vs the lower quartile was 3.39 (95% CI: 1.41-8.12). Additionally, we found a correlation between IMT and Hcy (r = 0.162, P = 0.032). CONCLUSIONS: Our results suggest that there is a connection between IMT, Hcy levels and presence of amnestic MCI in a population with no history of clinically manifest atherosclerosis. Furthermore, there is also a connection between the IMT and Hcy levels themselves.

Immunohistochemical and ultrastructural study of the lamellae of oocytes in atretic follicles in relation to different processes of cell death.

Atresia is the process through which non-selectable oocytes are eliminated; it involves apoptosis and/or autophagy. This study used immunohistochemical and ultrastructural techniques to characterize the lamellae present in the cytoplasm of oocytes in follicles in the process of atresia in prepubertal and adult Wistar rats. The results indicate that the lamellae are positive to tubulin and myosin immunodetection under light and electron microscopy. Labeling is greater with anti-tubulin and lesser with anti-myosin. Our observations indicate that lamellae are present in oocytes at the initial antral stage in prepubertal rats; that is, from day 14 post-birth to adult age. We were able to determine that the increase in altered lamellae principally occurs in the apoptotic cells rather than in the autophagic cells.

Apoptotic and autophagic cell death induced by glucolaxogenin in cervical cancer cells.

The antiproliferative and cytotoxic activity of glucolaxogenin and its ability to induce apoptosis and autophagy in cervical cancer cells are reported. We ascertained that glucolaxogenin exerts an inhibitory effect on the proliferation of HeLa, CaSki and ViBo cells in a dose-dependent manner. Analysis of DNA distribution in the cell-cycle phase of tumor cells treated with glucolaxogenin suggests that the anti-proliferative activity of this steroid is not always dependent on the cell cycle. Cytotoxic activity was evaluated by detection of the lactate dehydrogenase enzyme in supernatants from tumor cell cultures treated with the steroid. Glucolaxogenin exhibited null cytotoxic activity. With respect to the apoptotic activity, the generation of apoptotic bodies, the presence of active caspase-3 and annexin-V, as well as the DNA fragmentation observed in all tumor lines after treatment with glucolaxogenin suggests that this compound does indeed induce cell death by apoptosis. Also, a significantly increased presence of the LC3-II, LC3 and Lamp-1 proteins was evidenced with the ultrastructural existence of autophagic vacuoles in cells treated with this steroidal glycoside, indicating that glucolaxogenin also induces autophagic cell death. It is important to note that this compound showed no cytotoxic effect and did not affect the proliferative capacity of mononuclear cells obtained from normal human peripheral blood activated by phytohaemagglutinin. Thus, glucolaxogenin is a compound with anti-proliferative properties that induces programmed cell death in cancer cell lines, though it is selective with respect to normal lymphocytic cells. These findings indicate that this glycoside could have a selective action on tumor cells and, therefore, be worthy of consideration as a therapeutic candidate with anti-tumor potential.

[The impact of neurological and medical complications on the mortality and functional situation of acute stroke patients]. / Impacto de las complicaciones neurologicas y medicas sobre la mortalidad y situacion funcional de pacientes con ictus agudo.

INTRODUCTION: Stroke patients have a high risk of presenting complications, the appearance of which can condition the prognosis of the stroke. We studied the frequency and impact of the onset of several different complications on the early and mid-term prognosis of these patients. PATIENTS AND METHODS: We conducted an observation-based study of the patients admitted to a stroke unit. The complications that occurred while hospitalised were recorded, a distinction being drawn between neurological and medical complications. The study examined their influence, according to the subtype of stroke, on intra-hospital mortality and that at 90 days, as well as on the functional situation at 90 days, by analysing the clinical factors that are predictive for the appearance of complications. RESULTS: The sample consisted of 847 patients. Altogether, 29.5% of the patients presented complications, which were more frequent in haemorrhagic stroke (50.5% versus 26.6%; p < 0.0001). The most usual complications were of a neurological nature (21%). For both subtypes, the presence of complications was associated with a higher rate of mortality both in hospital (2.1% versus 12.6%; p < 0.0001) and at 90 days (5.7% versus 29.6%; p < 0.0001), and a lower probability of independence at 90 days (72.9% versus 30.4%; p < 0.0001). The severity of the stroke on admission revealed itself as the most powerful predictor of the onset of any type of complication. CONCLUSIONS: The appearance of complications during the acute phase of the stroke has an adverse influence on mortality and on the functional prognosis. The identification of predictive factors could reduce the impact upon the progress of acute stroke patients.

TITLE: Impacto de las complicaciones neurologicas y medicas sobre la mortalidad y situacion funcional de pacientes con ictus agudo.Introduccion. Los pacientes con ictus presentan un elevado riesgo de presentar complicaciones. Su aparicion puede condicionar el pronostico del ictus. Estudiamos la frecuencia y el impacto de la aparicion de diversas complicaciones en el pronostico precoz y a medio plazo en estos pacientes. Pacientes y metodos. Estudio observacional de los pacientes ingresados en una unidad de ictus. Se registraron las complicaciones durante su estancia, distinguiendose entre complicaciones neurologicas y medicas. Se estudio la influencia de estas segun subtipo de ictus en la mortalidad intrahospitalaria y a los 90 dias, y en la situacion funcional a los 90 dias, analizandose los factores clinicos predictores para la aparicion de complicaciones. Resultados. Muestra de 847 pacientes. Un 29,5% de los pacientes presento complicaciones, que fueron mas frecuentes en el ictus hemorragico (50,5% frente a 26,6%; p < 0,0001). Las complicaciones mas habituales fueron las neurologicas (21%). Para ambos subtipos, la presencia de complicaciones se asocio a mayor mortalidad intrahospitalaria (2,1% frente a 12,6%; p < 0,0001) y a 90 dias (5,7% frente a 29,6%; p < 0,0001), y menor probabilidad de independencia a 90 dias (72,9% frente a 30,4%; p < 0,0001). La gravedad del ictus al ingreso se mostro como el predictor mas potente en la aparicion de cualquier tipo de complicacion. Conclusiones. La aparicion de complicaciones durante la fase aguda del ictus influye de forma adversa en la mortalidad y en el pronostico funcional. La identificacion de factores predictores podria disminuir el impacto sobre la evolucion del paciente con un ictus agudo.

Functional outcome of stroke and the cumulative experience of a stroke unit.

OBJECTIVE: Patients with acute stroke are more likely to survive and achieve independence if they are treated in a stroke unit. Available information in our setting is scarce. We analyse the outcomes of our patients on the basis of cumulative experience in a stroke unit. PATIENTS AND METHODS: A retrospective cohort study of patients admitted to a stroke unit. We differentiate between two groups according to the year of admission: group A (July 2007-December 2009) and group B (January 2010-December 2011), analysing early outcome based on the score on the National Institute of Health stroke scale and mortality at discharge, and medium-term outcome in terms of mortality and functional status according to the modified Rankin scale at three months. RESULTS: A total 1070 patients were included. There were no differences between groups with respect to favourable outcome (68.3% vs 63.9), hospital mortality (5.1% vs 6.6%), or 90-day mortality (12.8% vs 13.1%). The percentage of patients who were independent at 90 days was greater in group B (56.3% vs 65.5%, P=.03). In the multivariate analysis adjusted for stroke subtype and fibrinolytic therapy, the association between patient independence and admission period remained present. CONCLUSIONS: The probability of functional independence in our patients increased alongside accumulated experience in our stroke unit with no differences in mortality.

Plasticity and metaplasticity of adult rat hippocampal mossy fibers induced by neurotrophin-3.

Changes in synaptic efficacy and morphology are considered as the downstream mechanisms of consolidation of memories and other adaptive behaviors. In the last decade, neurotrophin-3 (NT-3) has emerged as one potent mediator of synaptic plasticity. In the adult brain, expression of NT-3 is largely confined to the hippocampal dentate gyrus (DG). Our previous studies show that application of high-frequency stimulation (HFS) sufficient to elicit long-term potentiation (LTP) at the DG-CA3 pathway as well as acute intrahippocampal microinfusion of brain-derived neurotrophin factor produce mossy fiber (MF) structural reorganization. Here, we show that intrahippocampal microinfusion of NT-3 induces a long-lasting potentiation of synaptic efficacy in the DG-CA3 projection accompanied by an MF structural reorganization of adult rats in vivo. It is considered that the capacity of synapses to express plastic changes is itself subject to variation depending on previous experience; taking into consideration the effects of NT-3 on MF synaptic plasticity, we thus used intrahippocampal microinfusion of NT-3 to analyse its effects on functional and structural plasticity induced by subsequent MF-HFS sufficient to induce LTP in adult rats, in vivo. Our results show that NT-3 modifies the ability of the MF pathway to present subsequent LTP by HFS, and modifies the structural reorganization pattern. The modifications in synaptic efficacy and morphology elicited by NT-3 at the MF-CA3 pathway were blocked by the presence of a Trk receptor inhibitor (K252a). These findings support the idea that NT-3 actions modify subsequent synaptic plasticity, a homeostatic mechanism thought to be essential for maintaining synapses in the adult mammalian brain.

Analysis of different cell death processes of prepubertal rat oocytes in vitro.

The processes of cell death were studied in vitro in populations of oocytes isolated from prepubertal rats. In order to identify apoptosis, the externalized phosphatidylserine was recognized with Annexin-V coupled to FITC and the fragmentation of DNA was demonstrated by means of electrophoresis. Oocytes were tested for autophagy by means of the incorporation of monodansylcadaverine and monitoring Lc3-I/Lc3-II by western blot. The expression of mRNA marker genes of autophagy and of apoptosis was studied by means of RT-PCR in pure populations of oocytes. Some oocytes expressed at least one of the following markers: caspase-3, lamp1 and Lc3. Some oocytes were positive to Annexin-V or to monodansylcadaverine. However, most of them were simultaneously positive to both markers. The relative frequency of oocytes simultaneously positive to markers of apoptosis and autophagy did not change in the different ages studied. The transformation of Lc3-I in Lc3-II was present in all populations of oocytes studied. The mRNAs for caspase-3, lamp1 and Lc3 were present in all populations of oocytes analyzed. Our results demonstrate that oocytes of rats from new born to prepubertal age are eliminated by means of three different cell death processes: apoptosis, autophagy and a mixed event in which both routes to cell death participate in the same cell.

Combined apoptosis and autophagy, the process that eliminates the oocytes of atretic follicles in immature rats.

We studied the alterations of dying oocytes in 1-28 days old rats using TUNEL method, immunolocalizations of active caspase 3, lamp1, localization of acid phosphatase, and DAPI staining. All procedures were performed in adjacent sections of each oocyte. In most dying oocytes exist simultaneously features of apoptosis as active caspase 3 and DNA breaks, and a large increase of lamp1 and acid phosphatase characteristic of autophagy. Large clumps of compact chromatin and membrane blebbing were absent. Electron microscope observations demonstrated the presence of small clear vesicles and autophagolysosomes. All these features indicate that a large number of oocytes are eliminated by a process sharing features of apoptosis and autophagy. In dying oocytes of new born rats the markers of apoptosis predominate over those of autophagy. However, fragmentation and apoptotic bodies were not found. These features suggest that in different cytophysiological conditions the processes of cell death may be differently modulated.

Fine structural and cytochemical analysis of the processes of cell death of oocytes in atretic follicles in new born and prepubertal rats.

The process of cell death of oocytes was studied in atretic ovarian follicles of rats aged from 1 to 28 days using light and electron microscope and cytochemical methods. These methods were TUNEL procedure for DNA breaks, active caspase-3 and lysosome-associated membrane protein 1 (LAMP-1) immunolocalizations. The structural features of the process of oocyte death are mainly characterized by the presence of abundant clear vacuoles and autophagosomes, as well as by the absence of large clumps of compact chromatin associated to the nuclear envelope and apoptotic bodies. These features are common to oocytes in all types of follicles studied. Cytochemical features consisting in positive reactions to TUNEL method, active caspase-3 and LAMP-1 immunolocalizations, are common to the cell death of oocytes in all types of follicles. Particular features of the process of cell death of oocytes are found in different types of follicles. Two morphological patterns of cell death occur in pre-follicular oocytes of the new born and in primordial follicles in 1 to 5 days old rats. One is distinguished by clear nucleoli and moderate compaction of chromatin in clumps frequently resembling meiotic bivalents. The second pattern is characterized by nucleolar condensation and by the absence of compact chromatin. The process of cell death of oocytes in antral follicles is characterized by ribonucleoprotein ribbon-like cytoplasmic structures, pseudo-segmentation, and loss of contact with granulosa cells.

Spatial long-term memory is related to mossy fiber synaptogenesis.

Structural synaptic changes have been suggested to underlie long-term memory formation. In this work, we investigate if hippocampal mossy fiber synaptogenesis induced by water maze overtraining can be related with long-term spatial memory performance. Rats were trained in a Morris water maze for one to five identical daily sessions and tested for memory retrieval 1 week and 1 month after training. After the last test session, the rat brains were obtained and processed for Timm's staining to analyze mossy fiber projection. The behavioral results showed that with more training, animals showed a better performance in the memory tests, and this performance positively correlates with Timm's staining in the stratum oriens. Furthermore, with the use of the NMDA antagonist MK801 before, but not after acquisition, water maze spatial memory was impaired. Increased Timm's staining in the stratum oriens was observed in the animals treated with MK801 after acquisition but not in those treated before. Finally, we observed that mossy fiber synaptogenesis occurs mainly in the septal region of the dorsal hippocampus, supporting the idea that this anterior region is important for spatial memory. Altogether, these results suggest that mossy fiber synaptogenesis can be related with spatial long-term memory formation.

Long-term potentiation in the insular cortex enhances conditioned taste aversion retention.

Long-lasting changes in synaptic strength, such as long-term potentiation (LTP), are thought to underlie memory formation. Recent studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and retention of conditioned taste aversion (CTA), have demonstrated that tetanic stimulation of the basolateral nucleus of the amygdala (Bla) induce LTP in the IC of adult rats in vivo, as well as, that blockade of N-methyl-D-aspartate (NMDA) receptors disrupts CTA and IC-LTP induction in vivo. Here, we present experimental data showing that induction of LTP in the Bla-IC projection previous to CTA training enhances the retention of this task. These findings are of particular interest since they provide support for the view that the neural mechanisms underlying neocortical LTP may contribute to memory related functions performed by the IC.

Synaptogenesis of mossy fibers induced by spatial water maze overtraining.

Synaptic plasticity has been proposed as a mechanism underlying learning and memory. Synaptic reorganization of hippocampal mossy fibers has been observed after experimentally induced epilepsy, and after brief high-frequency activation inducing long-term potentiation. Furthermore, it has been suggested that synaptic changes in the hippocampus may occur after spatial learning. In this study, by using a zinc-detecting histologic technique (Timm), we demonstrate a significant increase of mossy fiber terminals in the CA3 stratum oriens region induced by training rats during 3 days in a spatial Morris water maze. In contrast, animals trained for only 1 day and animals that were just allowed to swim or were overtrained in a stress-motivated inhibitory avoidance task did not show increments of mossy fiber terminals in the stratum oriens. Electron microscopy confirmed that synaptic density of mossy fiber terminals in the stratum oriens increases significantly in water maze overtrained animals compared with the swimming control animals. Taken together, these results suggest that overtraining in a spatial learning task induces mossy fiber synaptogenesis that could be involved in the mechanisms underlying long-term memory storage. Hippocampus 1999;9:631-636.

[Onychomycosis due to Malassezia.]. / Onicomicosis por Malassezia.

The records of fifty patients presenting Malassezia spp. associated onychomycosis were compiled from two different mycology laboratories from Medellín, Colombia. Malassezia spp. was isolated by culture as the only etiological agent in 32% of the cases and associated to a yeast of the genus Candida in 30% of the cases. In 22% of the cases although Malassezia spp. was observed by direct examination, it was no isolated but others species were obtained. No etiological agent was isolated by culture in 16% of the cases. We found evidence of the Malassezia spp.- Candida relationship in 48% of the cases by either direct examination or by culture isolation. The level of detection of Malassezia spp. by culture isolation was of 62% as compared to the direct examination. Results showed similar patterns of distribution of epidemilogical factors for both entities: onychomycosis by Candida albicans and onychomycosis by Malassezia spp.

The NMDA receptor antagonist CPP impairs conditioned taste aversion and insular cortex long-term potentiation in vivo.

It has been proposed that long-term potentiation (LTP) a form of activity-dependent modification of synaptic efficacy, may be a synaptic mechanism for certain types of learning. Recent studies on the insular cortex (IC) a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that tetanic stimulation of the basolateral nucleus of the amygdala (Bla) induce an N-methyl-d-aspartate (NMDA) dependent LTP in the IC of adult rats in vivo. Here we present experimental data showing that intracortical administration of the NMDA receptor competitive antagonist CPP (-3(-2 carboxipiperazin-4-yl)-propyl-1-phosphonic acid) disrupts the acquisition of conditioned taste aversion, as well as, the IC-LTP induction in vivo. These findings are of particular interest since they provide support for the view that the neural mechanisms underlying NMDA dependent neocortical LTP, constitute a possible mechanism for the learning related functions performed by the IC.

[Prescriptions, access, and expenses on drugs among users of health services in Mexico]. / Prescripción, acceso y gasto en medicamentos entre usuarios de servicios de salud en México.

OBJECTIVE: To analyze the medical prescription, drug access and drug expenditure by patients based on the National Health Survey in Mexico, 1994. MATERIAL AND METHODS: A descriptive analysis of drug access and expenditure was undertaken and predictive factors for medical prescription were identified by logistic regression for 3,324 patients. RESULTS: 78% of the patients received drug prescriptions. 92% of the Social Security patients and 35% of the Ministry of Health patients received drugs free of charge (p = 0.000). The region with the highest poverty index received the least amount of drugs free of charge. Regarding drug expenditure of patients who purchased drugs, median expenditure was 40.00 pesos (12.50 USD). Private health service patients spent significantly more than public health service patients. CONCLUSIONS: Drug access and drug expenditure are linked to socioeconomic factors and to the institutions attended by patients. The Mexican health system faces, among others, the challenge of increasing the equity of access to medical drugs.