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.

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.

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.

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.

Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met----Ala168 mutant penicillin acylases from Kluyvera citrophila.

The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters Km and kcat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met----Ala168 mutation unstabilizes the first transition-state (E..S not equal to) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.

Genetic and molecular analysis of new female-specific lethal mutations at the gene Sxl of Drosophila melanogaster.

We have isolated three female-specific lethal mutations at the gene Sex-lethal (Sxl): Sxlfb, Sxlfc and Sxlfd. We have carried out the complementation analysis between these mutations and other previously reported Sxlf mutations. It is possible to classify the alleles tested in this report into two complementation groups: the bc group defined by Sxlfb, and Sxlfc, and the LS group defined by SxlfLS. The other alleles tested affect both complementation groups albeit with different degrees. Contrary to what happens with mutations at the LS group, mutations at the bc group do not affect sex determination, nor late dosage compensation nor oogenesis. Both Sxlfb and Sxlfc present a DNA insertion of at least 5 kb between position -10 and -11 on the molecular map, within the fourth intron. On the contrary, Sxlfd, a strong mutation affecting all Sxl functions, is not associated to any detectable DNA alteration in Southern blots, so that it seems to be a "point" mutation. In agreement with their phenotypes, both Sxlfc/SxlfLS and Sxlfc homozygous female larvae express only the late Sxl transcripts characteristic of females, while females homozygous for SxlfLS express only the late Sxl transcripts characteristic of males. Moreover, Sxlfc presents a lethal synergistic interaction with mutations at either da or the X:A ratio, two signals that define the initial activity state of Sxl, while SxlfLS do not. These data suggest that the two complementation groups are related to the two sets of early and late Sxl transcripts, which are responsible for the early and late Sxl functions, respectively: Sxlfb and Sxlfc would affect the early functions and SxlfLS would affect the late Sxl functions.

Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila.

The penicillin acylase (PAC) from Kluyvera citrophila ATCC21285 has been purified to homogeneity and found to be composed of two non-identical subunits of 23 and 62 kDa, in contrast with the previous findings [Shimizu et al., Agr. Biol. Chem. 39 (1975) 1655-1661]. The nucleotide (nt) sequence of the K. citrophila pac gene contained in the 3-kb PvuI-HindIII fragment of pKAP1 [García and Buesa, J. Biotechnol. 3 (1986) 187-195] has been determined, showing that it encodes a protein of 844 amino acid (aa) residues. The aa analysis of the N-terminal and C-terminal sequences of the purified subunits showed that they were derived from a common precursor protein of 93.5 kDa, from which a signal peptide of 26 aa, responsible for the periplasmic translocation of the protein, and an internal connecting polypeptide of 54 aa, have been removed in the maturation of the PAC. The comparison of the nt sequences of the pac genes from K. citrophila and Escherichia coli ATCC11105 [Schumacher et al., Nucl. Acids Res. 14 (1986) 5713-5727] revealed 80% homology, suggesting a common ancestral pac gene origin. The results reported here should allow investigation of the unusual mechanism of maturation of this prokaryotic protein, as well as manipulation, using DNA recombinant techniques, of the catalytic properties of this industrially important enzyme.

Overproduction and purification of biologically active native fungal alpha-sarcin in Escherichia coli.

An efficient system was developed to produce, in Escherichia coli, large amounts of native alpha-sarcin (alpha Sar), a cytotoxin from the mold Aspergillus giganteus. The protein has been purified to homogeneity with a yield of 1.5 micrograms/ml of original culture. The constructed expression vector (pINPG alpha S) is based on the synthesis of a fusion protein between alpha Sar and a modified version of the OmpA signal peptide. This peptide seems to favour the postranslational processing of the fusion protein. The purified recombinant alpha-sarcin (re-alpha Sar) is structurally identical to the mature fungal protein according to the following criteria: N-terminal amino acid (aa) sequence, aa composition, electrophoretic mobility, chromatographic behaviour, immunoreactivity and spectroscopic features. Indeed, the recombinant protein recovered is completely functional, since it cleaves, in vitro, eukaryotic rRNA and it is able to interact with phospholipid vesicles with the same specificity as the native fungal alpha Sar.

SA-1, a nuclear protein encoded by one member of a novel gene family: molecular cloning and detection in hemopoietic organs.

We report the molecular cloning of a novel gene family. The first member of this family was cloned from a mouse lambda gt11 cDNA library using the B92 monoclonal antibody (mAb) raised against stromal cell extracts. This was followed by RACE-PCR using mRNA from the stromal cell line. A 4 kb cDNA was obtained encoding a unique protein sequence of 1258 aa, that we designate stromal antigen (SA)-1. The human SA-1 gene was cloned by homology from a thymus cDNA library and the sequence of the predicted protein was found to be highly homologous to the murine SA-1 (>98.9%). Another cDNA was cloned and the deduced protein (SA-2) was 71% homologous to SA-1. Northern blot and PCR analysis indicated that on the mRNA level the SA-1 gene is expressed in all tissues analyzed and probably encodes a single transcript. The identification of SA-1 protein in tissues and cells required combined immunoprecipitation and Western blotting using a polyclonal antiserum raised against a predicted peptide of SA-1 and the B92 mAb. Using this assay we identified a protein of about 120 kDa in hemopoietic organs. Subcellular fractionation indicated that SA-1 is a nuclear protein. Thus, despite the ubiquitous expression on the mRNA level, the protein was predominantly detected in hemopoietic organs and may therefore be controlled on a post-transcriptional level. The SA-1 gene described in this study is highly conserved between mouse and man. This implies a crucial function for this protein.

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.

Different PrlA proteins increase the efficiency of periplasmic production of human interleukin-6 in Escherichia coli.

The export efficiency of a fusion of the Escherichia coli preOmpA signal peptide to human interleukin-6 can be significantly raised by coexpressing three different prlA alleles of sec Y along with wild type secE. The effect seems prlA-specific, as prlG1 (a prl allele of secE) does not affect the export of preOmpA-hIL-6. Coexpression of secD and secF also stimulates the export of the fusion protein.

Production and secretion of human interleukin 6 into the periplasm of Escherichia coli: efficient processing of N-terminal variants of hIL6 by the E. coli signal peptidase.

We have developed a system for expressing human interleukin 6 into the periplasmic space of Escherichia coli. The method is based on the expression of the hIL6 gene under the control of the regulatory signals of plasmid pINIII-OMPA3, i.e., lpp-lac promoter and the E. coli OMPA ribosome binding site and leader sequence. Since microheterogeneity is known to occur in the amino end of the cytokine, we tested different 'natural' versions of the protein, and we found that the secretion process was only efficient when the N-terminal amino acid was not proline. In flask experiments this procedure yields about 8-10 mg of biologically active hIL6 per liter.

[Alterations of fibrinolysis in stroke]. / Alteraciones de la fibrinolisis en los ACVA.

The fibrinolytic system has an important role as a controller of the coagulation, since plasmin digests and dissolves the fibrin clot. In this way, they have described many alterations in this system that are responsible of the thromboembolic disease, moreover stroke. This study tries to show the incidence of these alterations in a group of patients suffering from stroke, and the difference of this incidence between men and women. We obtained that the fibrinogen, tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) levels are significantly increased (p < 0.01) in the group of patients, and in the same way, there are also differences (p < 0.01) between men and women, so in the control group as in the patients group; these differences should mean that women have a major risk for suffering an stroke, but it doesn't fits the fact that stroke is more frequent in men and that they are younger when they suffer from this disease.

[Recurrent ARDS in varicella pneumonia complicated by disseminated candidiasis in a pregnant woman]. / SDRA recurrente tras neumonía varicelosa complicado con candidiasis diseminada en mujer gestante.

Adult respiratory distress syndrome (ARDS) seems to be the common way from different etiologies. We describe the clinical evolution of an ARDS in a pregnant woman, initially due to Varicella Pneumonia which was complicated with Disseminated Candidiasis and recurrent ARDS. We review the nosocomial infection with Candida in ICU patients: the growing incidence, the diagnostic problems and the new standards for treatment.

[Antiphospholipid antibodies in megakaryocytic thrombopenias]. / Anticuerpos antifosfolípidos en las trombopenias megacariocíticas.

We investigated the prevalence of antiphospholipid antibodies (APA) in megakaryocytic thrombopenias in order to establish their clinical significance and prognostic value. We studied 82 thrombopenic patients: 38 women and 44 men (age: 17-91 years). We assessed circulating lupus antibody (LA) through thromboplastin inhibition test (TIT), anticardiolipin antibodies (ACL) through ELISA and antiplatelet antibodies through flow cytometer. We conducted a statistical study RSIGMA, comparing patients with ACL + against ACL-. We found ACL positive in 21%, LA in 33% and antiplatelet antibodies in 42%. We observed a direct correlation (p < 0.05) between ACL and LA and between ACL and anticore antibodies, but not between ACL and antiplatelet antibodies. There was also a significant correlation between ACL and abortion, thrombosis and bleeding in thrombopenic patients. We conclude that APAs are present in 39% of the megakaryocytic thrombopenias and that they are not correlated with antiplatelet antibodies. Positivity of APA in thrombopenic patients is associated to poor prognosis and long term follow-up.

Increasing the efficiency of protein export in Escherichia coli.

Export of recombinant proteins to the periplasm of Escherichia coli is in many cases preferable to cytoplasmic production. However, when the protein is overexpressed, export efficiency decreases significantly and some advantages of the system are lost. This is what happens when attempting to produce recombinant human interleukin-6 (hIL-6) as a pre(OmpA) fusion in E. coli. Assuming that the host protein export machinery becomes overloaded, we have tested the effect of providing the host with additional copies of two key components of that machinery. Supplementation with a plasmid bearing prlA4 (secY allele) and secE genes increased the ratio of mature to precursor hIL-6 from 1.2 to 10.8. The increase in processing ratio was associated with the accumulation of a larger amount of total (mature plus precursor forms) hIL-6. Providing a plasmid-borne wild-type prlA was ineffective compared to prlA4 allele. This suggests that the PrlA protein, a component of the translocator, recognizes features at the mature portion of secretory substrates independently of those at the signal peptide portion.

[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.