SPIRIT Women, evaluation of the safety and efficacy of the XIENCE V everolimus-eluting stent system in female patients: referral time for coronary intervention and 2-year clinical outcomes.

AIMS: SPIRIT Women is the first interventional trial dedicated exclusively to women, focusing on symptoms at presentation, referral time to coronary intervention and the safety and performance of the XIENCE V stent. METHODS AND RESULTS: SPIRIT Women is a prospective, open-label, multicentre study in which 1,573 women were enrolled at 73 sites outside the United States. The primary endpoint is the composite of all death, Academic Research Consortium (ARC) defined myocardial infarction (MI) and target vessel revascularisation (TVR) at one year. Data collected included symptoms at presentation and referral to coronary intervention. To allow comparison by gender, the latter were compared to data from male patients from the SPIRIT V study. The one- and two-year composite of all death, MI and TVR was 12% and 15%, respectively. Target lesion revascularisation (TLR) and stent thrombosis (definite and probable) rates were 2.4% and 0.59%, respectively, at one year and 3.6% and 0.73%, at two years. The total referral time for coronary intervention in women was four days longer than for men in the SPIRIT V study. CONCLUSIONS: The XIENCE V stent is safe and effective with low TLR and stent thrombosis rates. More efforts remain to be made to increase the awareness of women and physicians of the risk for coronary artery disease (CAD).

Molecular cloning and characterization of European seabass (Dicentrarchus labrax) and Gilthead seabream (Sparus aurata) complement component C3.

We present the complete C3 cDNA sequence of Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) and its molecular characterization with a descriptive analysis of their structural elements. We obtained one sequence for Gilthead seabream (gsbC3) which encodes a predicted protein of 1656 amino acids, and two sequences for European seabass (esbC3_1 and esbC3_2) which encode two predicted proteins of 1654 and 1587 amino acids respectively. All sequences present the characteristic structural features of C3 but interestingly esbC3_2 lacks the anaphylotoxin domain and the cysteine residue responsible for thiolester bond formation. Moreover, we have detected and quantified (by real-time PCR-based absolute quantification) specific isoform expression in European seabass depending on pathogen and density conditions in vivo. In addition, we have analyzed the tissue distribution pattern of European seabass and Gilthead seabream C3 genes under crowding stress and under pathological challenges in vivo, and we have observed that crowding and infection status provoke changes in expression levels, tissue expression pattern and C3 isoform expression balance.

Changes in complement responses in Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) under crowding stress, plus viral and bacterial challenges.

Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) were subjected to either experimental infection with Photobacterium damselae subsp. piscicida or Nodavirus after a period of 2 weeks of crowding in which fish were subjected to a 5-fold increase in density (10-50 kg/m(3)). Samples were obtained before the crowding period (0 h or control) and at 24h and 72 h after crowding from both groups of infected fish. The Complement haemolytic activity and the expression of the C3 gene were evaluated in blood and liver samples respectively. The bacteriolytic and lysozyme activities were also assessed. The results showed that Complement haemolytic activity was reduced at 72 h with both bacteria and virus in high density Gilthead seabream, and a similar increase was observed at low density. Bacteriolytic activity under both bacterial and viral challenges for both species was increased at 24h, under low density. At high density, the bacterial challenge did not induce significant changes. C3 mRNA abundance was substantially increased after pathogen treatments in low density groups at 24h but no significant changes were detected at high densities. These results support the idea of the suppressor effect of stressors on the immune system since a reduction of Complement activity under virus and high density, or lack of response in C3 expression under high density were observed.

Tratamiento quirúrgico de la obesidad: ¿a quién?, ¿qué técnica?, ¿es necesario el seguimiento postoperatorio? / Surgical treatment: of obesity what patient?, what technique?, is postoperative follow-up mandatory?

La obesidad mórbida (índice de masa corporal igual o superior a 40 kg/m2) implica un riesgo elevado de morbimortalidad y suele ser resistente al tratamiento médico. El abordaje quirúrgico puede conseguir una pérdida de peso efectiva a largo plazo, con mejoría de las comorbilidades y de la calidad de vida, pero presenta el riesgo de que se produzcan complicaciones potencialmente graves, si bien constituye una opción válida en pacientes seleccionados con criterios de inclusión en un programa multidisciplinario protocolizado. Las técnicas quirúrgicas actuales pueden ser simples (restrictivas) o complejas, asociando derivación gástrica y/o malabsorción intestinal. En general, las técnicas complejas producen mejores resultados en cuanto a pérdida de peso, pero tienen mayor riesgo de que se produzcan deficiencias nutricionales, por lo que la elección del tipo de intervención debe individualizarse en función de la experiencia del equipo quirúrgico y de las características del paciente. El seguimiento postoperatorio es imprescindible, no sólo para detectar cuanto antes la aparición de complicaciones, sino para validar los resultados de la intervención. Deben valorarse la evolución de los índices ponderales, la mejoría de comorbilidades, la calidad de vida, los parámetros nutricionales y la aparición de complicaciones. Es necesaria una educación nutricional específica para facilitar la tolerancia digestiva y conseguir una buena adherencia al tratamiento (AU)

¿Qué población infantil entre 2 meses y 2 años debe recibir la vacuna antineumocócica? / Which child population between 2 months and 2 years of age needs to be vaccinated against pneumococci?

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Brain temperature, body core temperature, and intracranial pressure in acute cerebral damage.

OBJECTIVES: To assess the frequency of hyperthermia in a population of acute neurosurgical patients; to assess the relation between brain temperature (ICT) and core temperature (Tc); to investigate the effect of changes in brain temperature on intracranial pressure (ICP). METHODS: The study involved 20 patients (10 severe head injury, eight subarachnoid haemorrhage, two neoplasms) with median Glasgow coma score (GCS) 6. ICP and ICT were monitored by an intraventricular catheter coupled with a thermistor. Internal Tc was measured in the pulmonary artery by a Swan-Ganz catheter. RESULTS: Mean ICT was 38.4 (SD 0.8) and mean Tc 38.1 (SD 0.8) degrees C; 73% of ICT and 57.5% of Tc measurements were > or =38 degrees C. The mean difference between ICT and Tc was 0.3 (SD 0.3) degrees C (range -0.7 to 2.3 degrees C) (p=0. 0001). Only in 12% of patients was Tc higher than ICT. The main reason for the differences between ICT and Tc was body core temperature: the difference between ICT and Tc increased significantly with body core temperature and fell significantly when this was lowered. The mean gradient between ICT and Tc was 0.16 (SD 0.31) degrees C before febrile episodes (ICT being higher than Tc), and 0.41 (SD 0.38) degrees C at the febrile peak (p<0.05). When changes in temperature were considered, ICT had a profound influence on ICP. Increases in ICT were associated with a significant rise in ICP, from 14.9 (SD 7.9) to 22 (SD 10.4) mm Hg (p<0.05). As the fever ebbed there was a significant decrease in ICP, from 17.5 (SD 8.62) to 16 (SD 7.76) mm Hg (p=0.02). CONCLUSIONS: Fever is extremely frequent during acute cerebral damage and ICT is significantly higher than Tc. Moreover, Tc may underestimate ICT during the phases when temperature has the most impact on the intracranial system because of the close association between increases in ICT and ICP.

Cloning and characterization of mouse UBPy, a deubiquitinating enzyme that interacts with the ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1.

We used yeast "two-hybrid" screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25(Mm). Three independent overlapping clones were isolated from a mouse embryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction. It encodes a large protein (1080 amino acids) highly homologous to the human deubiquitinating enzyme hUBPy and contains a well conserved domain typical of ubiquitin isopeptidases. Therefore we called this new protein mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA present in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25(Mm) is exclusively expressed. Using a glutathione S-transferase fusion protein, we demonstrated an "in vitro" interaction between mUBPy and the N-terminal half (amino acids 1-625) of CDC25(Mm). In addition "in vivo" interaction was demonstrated after cotransfection in mammalian cells. We also showed that CDC25(Mm), expressed in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decreases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of the human homolog hUBPy is not defined, although its expression was correlated with cell proliferation. Our results suggest that mUBPy may play a role in controlling degradation of CDC25(Mm), thus regulating the level of this Ras-guanine nucleotide exchange factor.

The overexpression of the CDC25 gene of Saccharomyces cerevisiae causes a derepression of GAL system and an increase of GAL4 transcription.

The CDC25 gene product is an exchange factor for Ras proteins and it activates the Ras/cAMP pathway in the yeast Saccharomyces cerevisiae. The overexpression of the CDC25 gene in S. cerevisiae cells causes a partial glucose-derepressed phenotype which is particularly evident for expression of invertase. To define domains of Cdc25 protein relevant for this derepression and to test another glucose repressed system, different to invertase, we have overexpressed different regions of the CDC25 gene under the control of a GAL-promoter. We found that a derepression of both GAL regulated promoters and invertase was related to the overexpression of CDC25 regions that contain a functional guanine nucleotide exchange (GEF) domain. The effect on GAL-promoters was particular evident when the CDC25 gene was under the control of a UASgal element and operates at transcriptional level, although a moderate derepression was found also for UASgal/lacZ reporter gene. Finally, the overexpression of the GEF domain of CDC25 also caused an increase in the expression of the GAL4 regulatory gene, while a constitutive activation of the Ras/cAMP pathway did not produce any increase in GAL4 expression. These findings indicate that the overexpression of the catalytic domain of CDC25 gene is necessary and sufficient to give a glucose-derepression of GAL promoters and of invertase. They also suggest that the derepression of GAL promoters occurs through an increase of GAL4 expression in a Ras cAMP independent way.

Identification of gene encoding a putative RNA-helicase, homologous to SKI2, in chromosome VII of Saccharomyces cerevisiae.

We have determined the nucleotide sequence of a segment of VII of the yeast Saccharomyces cerevisiae contained in the cosmid clone pEGH101 for a total of 7 kbp. This sequence contains a large open reading frame (ORF) called G9365, coding for a protein of 1967 amino acids that shows a significant homology with the product of the SKI2 gene of S. cerevisiae and contains domains characteristics of RNA-helicases. The ORF is transcribed in vegetative cells but it is not essential for viability as demonstrated by gene disruption.

The minimal active domain of the mouse ras exchange factor CDC25Mm.

The minimal active domain of the mouse CDC25Mm, a GDP/GTP exchange factor (GEF) active on H-ras protein, was determined by constructing several deletion mutants of the C-terminal domain of the protein. The functional activity of these fragments was analyzed for the ability to complement the yeast temperature sensitive mutation cdc25-1 and to catalyze the GDP/GTP exchange on Ras proteins in vitro. A C-terminal domain of 256 residues (CDC25Mm 1005-1260) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25Mm 1005-1233) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25Mm 1030-1260 corresponding to the most conserved domain) led to a complete loss of expression. The results in vivo were supported by experiments in vitro. Highly purified CDC25Mm 1005-1260, expressed in E. coli using the pMAL system, enhanced the GDP release from both H-ras p21 and S. cerevisiae Ras2p and its activity was nearly as high as that of CDC25Mm 974-1260. Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25Mm is shorter than the yeast one.

Functional expression of the transcriptional activator Opaque-2 of Zea mays in transformed yeast.

The aim of this research was to determine whether the structural homology between the O2 gene, a maize transcriptional activator, and the GCN4 gene, a yeast transcriptional factor, is reflected at the level of function. The O2 cDNA was cloned in the yeast expression vector pEMBLyex4 under the control of a hybrid inducible promoter, and used to transform the yeast Saccharomyces cerevisiae. Transformed yeast cells produced O2 mRNA and a polypeptide immunoreactive with anti-O2 antibodies during growth in galactose. The heterologous protein was correctly translocated into the yeast nuclei, as demonstrated by immunofluorescence, indicating that the nuclear targeting sequences of maize are recognized by yeast cells. Further experiments demonstrated the ability of O2 to rescue a gcn4 mutant grown in the presence of aminotriazole, an inhibitor of the HIS3 gene product, suggesting that O2 activates the HIS3 gene, gene normally under control of GCN4. It was shown that the O2 protein is able to trans-activate the HIS4 promoter in yeast cells and binds to it in vitro. The sequence protected by O2, TGACTC, is also the binding site for GCN4. Finally, the expression of O2 protein in yeast did not produce alterations during batch growth at 30 degrees C, while transformants expressing O2 protein showed a conditionally lethal phenotype when grown in galactose at 36 degrees C; this phenotype mimics the behaviour of gcd mutants. The results support the idea that basic mechanisms of transcription control have been highly conserved in eukaryotes.

Expression of high levels of human tissue plasminogen activator in yeast under the control of an inducible GAL promoter.

The human tissue plasminogen activator (h-tPA) cDNA was fused either with the leader sequence of the killer toxin of Kluyveromyces lactis or with the Saccharomyces diastaticus glucoamylase leader peptide and cloned in the yeast expression vector under the control of the inducible USAgal/CYC1 promoter. The recombinant tPA is produced in yeast as a single-chain glycosylated polypeptide of 66-72 kDa, which accumulates intracellularly associated with a membrane fraction. Using two-step fed-batch fermentation, a productivity up to 100 mg/l of active intracellular tPA was obtained.

The sequence of 8.8 kb of yeast chromosome III cloned in lambda PM3270 contains an unusual long ORF (YCR601).

We have determined the nucleotide sequence of a segment of chromosome III contained in the right part of the lambda PM3270 clone, for a total of 8824 bp. This sequence contains an unusual long open reading frame, YCR601, of 6501 bp that encodes for a protein of 2167 amino acids that show no homology with other known proteins. YCR601 was disrupted by internal deletion and insertion of LEU2 gene and is a non-essential gene, however, it is transcribed during vegetative growth yielding a polyadenylated mRNA of approximately 7 kb.