Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice.

BACKGROUND: Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca2+ channels and Ca2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca(2+)-induced Ca(2+) release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. METHODS AND RESULTS: To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma-sarcoplasmic reticulum distance. CONCLUSIONS: Loss of JPH2 had profound effects on Ca2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA-mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca2+ release in the heart.

Genome sequence of the emerging pathogen Aeromonas caviae.

Aeromonas caviae is a Gram-negative, motile and rod-shaped facultative anaerobe that is increasingly being recognized as a cause of diarrhea in children. Here we present the first genome sequence of an A. caviae strain that was isolated as the sole pathogen from a child with profuse diarrhea.

Ryanodine receptor phosphorylation by calcium/calmodulin-dependent protein kinase II promotes life-threatening ventricular arrhythmias in mice with heart failure.

BACKGROUND: approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca(2+) remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. METHODS AND RESULTS: mice in which the S2814 Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca(2+) release events, resulting in reduced sarcoplasmic reticulum Ca(2+) load on confocal microscopy. These Ca(2+) release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. CONCLUSIONS: our results suggest that Ca(2+)/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca(2+) release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

Accelerated development of pressure overload-induced cardiac hypertrophy and dysfunction in an RyR2-R176Q knockin mouse model.

In response to chronic hypertension, the heart compensates by hypertrophic growth, which frequently progresses to heart failure. Although intracellular calcium (Ca(2+)) has a central role in hypertrophic signaling pathways, the Ca(2+) source for activating these pathways remains elusive. We hypothesized that pathological sarcoplasmic reticulum Ca(2+) leak through defective cardiac intracellular Ca(2+) release channels/ryanodine receptors (RyR2) accelerates heart failure development by stimulating Ca(2+)-dependent hypertrophic signaling. Mice heterozygous for the gain-of-function mutation R176Q/+ in RyR2 and wild-type mice were subjected to transverse aortic constriction. Cardiac function was significantly lower, and cardiac dimensions were larger at 8 weeks after transverse aortic constriction in R176Q/+ compared with wild-type mice. R176Q/+ mice displayed an enhanced hypertrophic response compared with wild-type mice as assessed by heart weight:body weight ratios and cardiomyocyte cross-sectional areas after transverse aortic constriction. Quantitative PCR revealed increased transcriptional activation of cardiac stress genes in R176Q/+ mice after transverse aortic constriction. Moreover, pressure overload resulted in an increased sarcoplasmic reticulum Ca(2+) leak, associated with higher expression levels of the exon 4 splice form of regulator of calcineurin 1, and a decrease in nuclear factor of activated T-cells phosphorylation in R176Q/+ mice compared with wild-type mice. Taken together, our results suggest that RyR2-dependent sarcoplasmic reticulum Ca(2+) leak activates the prohypertrophic calcineurin/nuclear factor of activated T-cells pathway under conditions of pressure overload.

High frequency of hemolytic and cytotoxic activity in Aeromonas spp. isolated from clinical, food and environmental in Rio de Janeiro, Brazil.

Molecular study of aerolysin and cytotonic enterotoxin genes by PCR and colony blot hybridization was performed in 117 strains of Aeromonas spp. isolated from different sources. Homogeneous distribution of these genes in A. hydrophila complex strains was observed. For A. caviae and A. sobria complex strains, aerolysin genes were more frequent than cytotonic enterotoxins genes. Of 64 A. caviae complex strains, only one (1.5%) amplified the 451 bp product for the aer gene, however, the same primers detected a 400 bp product in 50 (78%) strains. This product was sequenced and had two short regions with homology to several hemolysin genes. The genotype aer (+)/aerA(+)/hly (+)/ast (+)/alt (+) was detected in six A. hydrophila strains from food and environmental source. The most common genotype found in A. hydrophila strains was hly (+) (85%) and aerA(+) (78.7%), while in A. caviae complex strains was aerA(+) (32.8%). All A. veronii complex sobria strains were aer (+)/aerA(+). All A. caviae and A. hydrophila were positive when tested with aer probe using the colony blot test. Thirty-seven percent of A. hydrophila and 53% of A. caviae tested were positive for ast probe. Eighty-nine percent of samples were cytotoxic in Vero cells. Our data demonstrated that Aeromonas spp. can harbor and express virulence genes and reinforce the potential of Aeromonas as a human pathogen.

Lectin-binding properties of Aeromonas caviae strains / Propriedades lectínicas de amostras de Aeromonas caviae

The cell surface carbohydrates of four strains of Aeromonas caviae were analyzed by agglutination and lectin-binding assays employing twenty highly purified lectins encompassing all sugar specificities. With the exception of L-fucose and sialic acid, the sugar residues were detected in A. caviae strains. A marked difference, however, in the pattern of cell surface carbohydrates in different A. caviae isolates was observed. Specific receptors for Tritricum vulgaris (WGA), Lycopersicon esculentum (LEL) and Solanum tuberosum (STA) (D-GlcNAc-binding lectins) were found only in ATCC 15468 strain, whereas Euonymus europaeus (EEL, D-Gal-binding lectin) sites were present exclusively in AeQ32 strain, those for Helix pomatia (HPA, D-GalNAc-binding lectin) in AeC398 and AeV11 strains, and for Canavalia ensiformes (Con A, D-Man-binding lectin) in ATCC 15468, AeC398, AeQ32 and AeV11 strains, after bacterial growing at 37ºC. On the other hand, specific receptors for WGA and EEL were completely abrogated growing the bacteria at 22ºC. Binding studies with 125I- labeled lectins from WGA, EEL and Con A were performed. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the A. caviae strains.

Os carboidratos de superfície celular de quatro amostras de Aeromonas caviae foram analisados por aglutinação e ensaios de ligação de lectinas empregando vinte lectinas altamente purificadas com especificidade para açúcares. Com exceção da L-fucose e do ácido siálico, os resíduos de açúcar foram detectados em amostras de A. caviae. Entretanto, foi observada uma diferença marcante no padrão de carboidratos de superfície celular em diferentes amostras de A. caviae. Receptores específicos para Tritricum vulgaris (WGA), Lycopersicon esculentum (LEL) e Solanum tuberosum (STA), lectinas de ligação a D-GlcNAc, foram encontrados apenas na amostra ATCC 15468, enquanto sítios de Euonymus europaes (EEL), lectina de ligação a D-Gal, estavam presentes exclusivamente na amostra AeQ32, sítios de Helix pomatia (HPA), lectina de ligação a D-GalNac, nas amostras AeC398 e AeV11 e de Canavalia ensiformis (Com A), lectina de ligação a D-Man, nas amostras ATCC 15468, AeC398, AeQ32 e AeV11, após crescimento bacteriano a 37ºC. Por outro lado, receptores específicos para WGA e EEL foram completamente abolidos após o crescimento das bactérias a 22ºC. Estudos de ligação com lectinas WGA, EEL e Con A marcadas com 125I também foram realizados. Esses ensaios confirmaram a seletividade, demonstrada em ensaios de aglutinação dessas lectinas para as amostras de A. caviae.

Lectin-binding properties of Aeromonas caviae strains.

The cell surface carbohydrates of four strains of Aeromonas caviae were analyzed by agglutination and lectin-binding assays employing twenty highly purified lectins encompassing all sugar specificities. With the exception of L-fucose and sialic acid, the sugar residues were detected in A. caviae strains. A marked difference, however, in the pattern of cell surface carbohydrates in different A. caviae isolates was observed. Specific receptors for Tritricum vulgaris (WGA), Lycopersicon esculentum (LEL) and Solanum tuberosum (STA) (D-GlcNAc-binding lectins) were found only in ATCC 15468 strain, whereas Euonymus europaeus (EEL, D-Gal-binding lectin) sites were present exclusively in AeQ32 strain, those for Helix pomatia (HPA, D-GalNAc-binding lectin) in AeC398 and AeV11 strains, and for Canavalia ensiformes (Con A, D-Man-binding lectin) in ATCC 15468, AeC398, AeQ32 and AeV11 strains, after bacterial growing at 37°C. On the other hand, specific receptors for WGA and EEL were completely abrogated growing the bacteria at 22°C. Binding studies with (125)I- labeled lectins from WGA, EEL and Con A were performed. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the A. caviae strains.

Influence of polarisation and differentiation on interaction of 43-kDa outer-membrane protein of Aeromonas caviae with human enterocyte-like Caco-2 cell line.

It has been recognised that adherence and invasion to host cells are important steps in the pathogenesis of entero-pathogenic bacteria, including Aeromonas caviae. However, the virulence factors of A. caviae remain, for the most part, poorly known. This study examined the interaction of A. caviae isolates to Caco-2 cells in different polarisation and differentiation conditions. The adherence of A. caviae may be related to accessibility of host cell basolateral receptors. Aggregative A. caviae isolates, grown at 22 degrees C, were more adherent in both non-polarised and undifferentiated Caco-2 cells and EGTA-treated polarised and differentiated Caco-2 cells. Furthermore, monolayers pre-incubated with 43-kDa outer-membrane protein (OMP) or A. caviae strains pre-incubated with rabbit IgG anti-43-kDa OMP decreased adherence of some A. caviae strains to EGTA-treated polarised and differentiated Caco-2 cells, suggesting an interaction of 43-kDa OMP with basolateral cell receptors. Bacterial cells were observed adhering to microvilli and to plasma membrane on both the apical and basal surfaces of the monolayer. Pedestal-like formation with cytoskeletal rearrangement was also observed. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. Furthermore, A. caviae were observed free in the cytosol of Caco-2 cells, suggesting escape form cytoplasmatic vacuoles.

Aeromonas hydrophila vacuolating activity in the Caco-2 human enterocyte cell line as a putative virulence factor.

A non-enterotoxigenic strain of Aeromonas hydrophila isolated from diarrheic stools of an 8-month-old child was found to cause vacuolation in Caco-2 cells. The vacuoles became prominent 60 min after addition of the bacterial culture to the cell monolayers and, after 120 min, a complete disruption of the monolayers was observed. Cell vacuolation was not detected when Caco-2 monolayers were tested with sterile filtrates of overnight cultures of the A. hydrophila vacuolating strain AH14846. This strain produced a diffuse adherence pattern in Caco-2 cell monolayers, but did not produce detectable cytotonic enterotoxin in the suckling mouse test and only produced small quantities of aerolysin. By demonstrating the ability to induce vacuolation in mammalian cells of enterocytic lineage, the current study raises the possibility that such activity might contribute to gastrointestinal symptoms in infections involving Aeromonas strains which do not express well-established enterotoxins.

Isolation and serological identification of enteropathogenic Escherichia coli in pasteurized milk in Brazil

OBJECTIVE: To evaluate the microbiological quality of pasteurized milk commercialized in Rio de Janeiro, Brazil, and determine serologically enteropathogenic Escherichia coli (EPEC) strains in E. coli isolates obtained from milk samples. METHODS: Ninety samples of pasteurized milk ó types B and C ó of three different commercial brands, purchased in supermarkets and bakeries in Rio de Janeiro, were examined. The amount of total and fecal coliform bacteria was estimated using the Most Probable Number technique. Mesophilic, psychrotrophic, and thermoduric microorganism counts were determined by the Standard Plate Count technique. Isolation and identification of E. coli were carried out using conventional physiological tests. Commercial antisera were used for serological characterization of EPEC. RESULTS: The three milk brands analyzed revealed bacterial counts above the regulated values of the Brazilian government. It was found that among 208 strains of E. coli isolated, 46 (22.1 por cento) were serologically classified as EPEC. The most common EPEC serogroup was O55 (15.2 por cento). CONCLUSIONS: Though recent studies on virulence factors indicate that not all strains serologically classified as EPEC are able to attaching/effacing lesion, it is believed that the isolation of EPEC serogroups from pasteurized milk represent a potential risk for children, as well as an indicative of the presence of other enteropathogens

Aspectos psicológicos e psiquiátricos do transplante de medula óssea / Psychological and psychiatric aspects of bone marrow transplantation

A intensidade e a complexidade envolvidas no transplante de medula óssea (TMO) em seus vários níveis, produz profundos efeitos psicológicos no paciente, na família e na equipe profissional. Ignorar esta realidade e reduzir os problemas do TMO a seus aspectos puramente técnicos pode trazer consequências catastróficas para o paciente e seus familiares e ameaçar a sobrevivência da equipe. Neste trabalho, säo discutidos três aspectos muito importantes de saúde mental no TMO, enfatizando a prática desenvolvida na Unidade de TMO do HCFMRP: 1) os múltiplos papéis desempenhados pela assistência psiquiátrica na Unidade de TMO, desde a prescriçäo de psicofármacos até o apoio à equipe multiprofissional em grupos de reflexäo; 2) uma análise dos sentimentos vivenciados pelo paciente submetido ao TMO nas diferentes fases do transplante (pré, intra e pós-TMO) sob a perspectiva da esperança de cura; 3) uma avaliaçäo da qualidade de vida dos pacientes submetidos ao TMO, a curto e médio prazos, integrando vários instrumentos de análise, incluindo técnicas projetivas.