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.

Sister chromatid cohesion control and aneuploidy.

Apart from a personal tragedy, could Down syndrome, cancer and infertility possibly have something in common? Are there links between a syndrome with physical and mental problems, a tumor growing out of control and the incapability to reproduce? These questions can be answered if we look at the biological functions of a protein complex, named cohesin, which is the main protagonist in the regulation of sister chromatid cohesion during chromosome segregation in cell division. The establishment, maintenance and removal of sister chromatid cohesion is one of the most fascinating and dangerous processes in the life of a cell. Errors in the control of sister chromatid cohesion frequently lead to cell death or aneuploidy. Recent results showed that cohesins also have important functions in non-dividing cells, revealing new, unexplored roles for these proteins in human syndromes, currently known as cohesinopathies. In the last 10 years, we have improved our understanding of the molecular mechanisms of the cohesin and cohesin-interacting proteins regulating the different events of sister chromatid cohesion during cell division in mitosis and meiosis.

[Multi-organ failure as first clinical sign of macrophage activation syndrome in childhood Still's disease]. / Fracaso multiorgánico como forma de presentación del síndrome de activación macrofágica en la enfermedad de Still infantil.

Macrophage activation syndrome is a form of secondary haemophagocytic lymphohistiocytosis seen in the context of rheumatic diseases. It is seen most frequently in association with systemic onset juvenile arthritis or childhood Still's disease. Hemophagocytosis is part of a sepsis-like clinical syndrome caused by hypercytokinemia due to a highly stimulated but ineffective immune response. Coagulopathy and hemorrhages, decreased white cell count, elevated levels of aspartate aminotransferase, fever, rash, hepatosplenomegaly and central nervous system dysfunction are some of diagnostic criteria of macrophage activation syndrome, but it is very difficult to diagnose due to the lack of specific clinical signs. We report a 8-year-old child who was admitted to the ICU with lethargy, fever, acute respiratory failure, coagulopathy, metabolic acidosis and multiorgan failure. Septic shock was suspected, but he was diagnosed with macrophage activation syndrome and treated with corticosteroids and intravenous immunoglobulin and later discharged from the ICU.

Dynamics of cohesin proteins REC8, STAG3, SMC1 beta and SMC3 are consistent with a role in sister chromatid cohesion during meiosis in human oocytes.

BACKGROUND: Sister chromatid cohesion is essential for ordered chromosome segregation at mitosis and meiosis. This is carried out by cohesin complexes, comprising four proteins, which seem to form a ring-like complex. Data from animal models suggest that loss of sister chromatid cohesion may be involved in age-related non-disjunction in human oocytes. Here, we describe the distribution of cohesins throughout meiosis in human oocytes. METHODS: We used immunofluorescence in human oocytes at different meiotic stages to detect cohesin subunits REC8, STAG3, SMC1 beta and SMC3, [also synaptonemal complex (SC) protein 3 and shugoshin 1]. Samples from euploid fetuses and adult women were collected, and 51 metaphase I (MI) and 113 metaphase II (MII) oocytes analyzed. SMC1 beta transcript levels were quantified in 85 maturing germinal vesicle (GV) oocytes from 34 women aged 19-43 years by real-time PCR. RESULTS: At prophase I, cohesin subunits REC8, STAG3, SMC1 beta and SMC3 overlapped with the lateral element of the SC. Short cohesin fibers are observed in the oocyte nucleus during dictyate arrest. All four subunits are observed at centromeres and along chromosomal arms, except at chiasmata, at MI and are present at centromeric domains from anaphase I to MII. SMC1 beta transcripts were detected (with high inter-sample variability) in GV oocytes but no correlation between SMC1 beta mRNA levels and age was found. CONCLUSIONS: The dynamics of cohesins REC8, STAG3, SMC1 beta and SMC3 suggest their participation in sister chromatid cohesion throughout the whole meiotic process in human oocytes. Our data do not support the view that decreased levels of SMC1 beta gene expression in older women are involved in age-related non-disjunction.

Analysis of recombination along chromosome 21 during human female pachytene stage.

It is accepted that recombination errors during human female meiotic prophase have some influence on the origin of trisomy 21. A total of 335 oocytes from four euploid fetuses were analysed by immunofluorescence and fluorescence in-situ hybridization in order to assess the recombination nodules along chromosome 21. Results based on the analysis of recombination points on the bivalent 21 during human female meiosis showed that both number [none (3.70%), one (79.01%) and two (17.29%)1 and distribution (always positioned interstitially on the q-arm) are different in males, ensuring that the two homologues more efficiently remain together until anaphase 1.Therefore, the mainly maternal origin of trisomy 21 appears not be linked to the first stages of oocyte development during fetal life, and this leads to the suggestion that the influence of environmental factors on the segregation of chromosome 21 homologues in later meiotic stages could have a significant role in the predominant maternal origin of trisomy 21.

Cohesin complexes and sister chromatid cohesion in mammalian meiosis.

Maintenance and precise regulation of sister chromatid cohesion is essential to ensure correct attachment of chromosomes to the spindle, thus preserving genome integrity by correct chromosome segregation. Errors in these processes often lead to aneuploidy, frequently implicated in cell death and/or tumor development. The so-called cohesin complexes are essential in sister chromatid cohesion during both mitosis and meiosis; they are responsible for maintaining sister chromatids together physically until their segregation during the metaphase/anaphase transition. The recent identification of new molecules involved in the control of sister chromatid cohesion, and the characterization of mouse loss-of-function models, have improved our understanding of the variety of cohesin complexes and their chromatin binding and removal regulation. This review will focus basically on the distribution and function of cohesin complexes during mammalian meiosis.

Fibrilación ventricular recidivante en intoxicación por fluxilicato de magnesio / Recurrent ventricular fibrillation in magnesium fluosilicate poisoning

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[Training in the care of the critical and multiple-injured patient: role of clinical simulation]. / Formación en la asistencia al paciente crítico y politraumatizado: papel de la simulación clínica.

Clinical simulation is suggested as a new educational instrument to learn and train in different medical skills. It is conceived as a new method that integrates scientific knowledge and human factors. Experience with these systems has been limited up to now, but it is now being widely accepted since it seems to accelerate acquisition of skills and knowledge in a safe setting, that is, without risk for the patient. However, its effect on clinical performance has not been validated yet. They are two types of simulators for intensive cares: screen based and human patient simulator (HPS). These systems make it possible to simulate different situations that require the application of action protocols or the management of new drugs in the clinical practice, promoting the rational use of resources in urgent care of the critical and multiple-injured patients. The limiting factors that prevent the expansion of the simulation for clinical training are its high cost, human resources needed, and the difficulties to assess the effectiveness of the training in real situations.

Intoxicación por metadona en la edad pediátrica / Pediatric methadone poisoning

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Evaluation of the Stag3 gene and the synaptonemal complex in a rat model (as/as) for male infertility.

Affected males (as/as) from the mutant TT rat strain are sterile due to spermatogenesis impairment with meiotic arrest at the pachytene stage. The as locus is on rat chromosome 12, in a region that shows conserved synteny to cM 74-94 on mouse chromosome 5. Stag3, a new member of the stromalin protein family, is expressed specifically in testis and associates to the synaptonemal complex. Mouse Stag3 gene has been assigned to cM 78 on chromosome 5. In this study, we have characterized the rat Stag3 gene and examined it as a candidate for male infertility in as/as rats. The rat Stag3 cDNA is 4181 nucleotides long, contains a highly polymorphic hexanucleotide repeat in the coding region, and encodes a 1256 amino acid protein with 93 and 77% sequence identity to mouse and human Stag3 proteins, respectively. No mutations or differences in size or abundance of Stag3 mRNA were detected between as/as and control rats, suggesting that Stag3 is not responsible for the aspermic phenotype. In addition, immunohistochemistry with antibodies against SCP1 and SPC3 proteins suggest that the synaptonemal complex structures are not primarily affected in these rats.

Mammalian STAG3 is a cohesin specific to sister chromatid arms in meiosis I.

Cohesins, which have been characterized in budding yeast and Xenopus, are multisubunit protein complexes involved in sister chromatid cohesion. Regulation of the interactions among different cohesin subunits and the assembly/disassembly of the cohesin complex to chromatin are key steps in chromosome segregation. We previously characterized the mammalian STAG3 protein as a component of the synaptonemal complex that is specifically expressed in germinal cells, although its function in meiosis remains unknown. Here we show that STAG3 has a role in sister chromatid arm cohesion during mammalian meiosis I. Immunofluorescence results in prophase I cells suggest that STAG3 is a component of the axial/lateral element of the synaptonemal complex. In metaphase I, STAG3 is located at the interchromatid domain and is absent from the chiasma region. In late anaphase I and the later stages of meiosis, STAG3 is not detected. STAG3 interacts with the structural maintenance chromosome proteins SMC1 and SMC3, which have been reported to be subunits of the mitotic cohesin complex. We propose that STAG3 is a sister chromatid arm cohesin that is specific to mammalian meiosis I.

A meiotic chromosomal core consisting of cohesin complex proteins recruits DNA recombination proteins and promotes synapsis in the absence of an axial element in mammalian meiotic cells.

The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.

STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3-related genes flanking the Williams-Beuren syndrome deletion.

Chromatin rearrangements in the meiotic prophase are characterized by the assembly and disassembly of synaptonemal complexes (SC), a protein structure that stabilizes the pairing of homologous chromosomes in prophase. We report the identification of human and mouse cDNA coding for stromalin 3 (STAG3), a new mammalian stromalin member of the synaptonemal complex. The stromalins are a group of highly conserved proteins, represented in several organisms from yeast to humans. Stromalins are characterized by the stromalin conservative domain (SCD), a specific motif found in all proteins of the family described to date. STAG3 is expressed specifically in testis, and immunolocalization experiments show that STAG3 is associated to the synaptonemal complex. As the protein encoded by the homologous gene (Scc3p) in Saccharomyces cerevisiae was found to be a subunit of a cohesin complex that binds chromosomes until the onset of anaphase, our data suggest that STAG3 is involved in chromosome pairing and maintenance of synaptonemal complex structure during the pachytene phase of meiosis in a cohesin-like manner. We have mapped the human STAG3 gene to the 7q22 region of chromosome 7; six human STAG3-related genes have also been mapped: two at 7q22 near the functional gene, one at 7q11.22, and three at 7q11.23, two of them flanking the breakpoints commonly associated with the Williams-Beuren syndrome (WBS) deletion. Since the WBS deletion occurs as a consequence of unequal meiotic crossing over, we suggest that STAG3 duplications predispose to germline chromosomal rearrangement within this region.

Drosophila grim induces apoptosis in mammalian cells.

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.

Molecular cloning and expression of stromalin protein from Drosophila melanogaster: homologous to mammalian stromalin family of nuclear proteins.

We report the cloning of a new cDNA from Drosophila melanogaster that encodes an open reading frame of 1116 amino acid residues. It is the insect homolog of the previously reported stromalin (SA) family of nuclear proteins in mammals (Carramolino et al. [1997]. Gene 195, 151-159). Taking into account the identical domain present in all the SA family members characterized to date, we have carried out polymerase chain reaction (PCR) using degenerate oligonucleotides from the 5' and 3' ends of one of those regions of the molecule and cDNA from D. melanogaster embryos. We isolated the homologous domain of the putative Drosophila SA molecule (DSA). This cDNA fragment was used as a radiolabeled probe for screening a cDNA library from Drosophila embryos, and we have cloned a full-length cDNA for the SA homolog from an insect. The protein shows a good degree of identity with the mammalian stromalins SA-1 and SA-2, with the N and C ends being the most divergent regions of the molecule. The mRNA coding for this protein shows a molecular size of about 3.7 kb by Northern blot analysis and is essentially expressed in embryonic stage. The in situ hybridization experiments indicate that the DSA messenger is expressed mainly in neurogenic territories in the embryonic development of Drosophila. The DSA protein has been cloned and expressed in a baculovirus system, and polyclonal antibodies were generated against the recombinant molecule. Western blot analysis using these antibodies detected a main band corresponding to about 120 kDa, principally in embryos.

[Acute respiratory infections in pediatric intensive care units. A multicenter prospective study]. / Infecciones respiratorias agudas en unidades de cuidados intensivos pediátricos. Estudio prospectivo multicéntrico.

OBJECTIVE: The purpose of this study was to know the etiology, clinical background, treatment an evolution of severe infectious diseases in children admitted to Pediatric Intensive Care Units (PICUs). PATIENTS AND METHODS: A multicenter prospective study was carried out. Children with respiratory infections admitted to 10 PICUs throughout Spain between May 1994 and April 1995 were included in a long term survey. The nosocomial infections were not included. Student's t and Wilcoxon tests were used for quantitative variables and Chi square with Yates correction and Fisher's test for the qualitative variables. RESULTS: One hundred twenty-two patients with acute respiratory infections were studied. The mean value on Downes score at admittance was 5.2 +/- 2.3. Diagnosis were allocated as follows: 47 bronchopneumonia (38.5%), 40 bronchiolitis (33%), 15 epiglotitis (12%), 14 laryngitis (11.5%) and "others" 6 (5%). Etiologic agents were identified in 69 cases (56.5%), with respiratory syncytial virus being the most frequently isolated agent (35 cases, 51%), followed by Hemophilus influenzae in 13 cases (19%). The mean PICU stay was 5.8 +/- 7.9 days (1-67 days). Of these cases, 112 (92%) recovered completely and 9 (7%) died (8 with bronchopneumonia and 1 with epiglotitis). A significant association could be seen between the increase in mortality and the variables Downes' score and diagnosis of bronchopneumonia. CONCLUSIONS: The most frequent respiratory infections in the PICU were pneumonia and bronchopneumonia. Viral etiology, with a frequency of 54%, was the main cause of respiratory infection. Bacterial etiology represented 46% of the total cases, with Hemophilus influenzae as the most frequent etiologic agent.