The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection.

Salmonella species translocate effector proteins into the host cell cytoplasm using a type III secretion system (TTSS). The translocation machinery probably contacts the eukaryotic cell plasma membrane to effect protein transfer. Data presented here demonstrate that both SspB and SspC, components of the translocation apparatus, are inserted into the epithelial cell plasma membrane 15 min after Salmonella typhimurium infection. In addition, a yeast two-hybrid interaction between SspC and an eukaryotic intermediate filament protein was identified. Three individual carboxyl-terminal point mutations within SspC that disrupt the yeast two-hybrid interaction were isolated. Strains expressing the mutant SspC alleles were defective for invasion, translocation of effector molecules and membrane localization of SspC. These data indicate that insertion of SspC into the plasma membrane of target cells is required for invasion and effector molecule translocation and that the carboxyl terminus of SspC is essential for these functions.

Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable.

Protein arginine N-methyltransferases have been implicated in a variety of processes, including cell proliferation, signal transduction, and protein trafficking. In this study, we have characterized essentially a null mutation induced by insertion of the U3betaGeo gene trap retrovirus into the second intron of the mouse protein arginine N-methyltransferase 1 gene (Prmt1). cDNAs encoding two forms of Prmt1 were characterized, and the predicted protein sequences were found to be highly conserved among vertebrates. Expression of the Prmt1-betageo fusion gene was greatest along the midline of the neural plate and in the forming head fold from embryonic day 7.5 (E7.5) to E8.5 and in the developing central nervous system from E8.5 to E13.5. Homozygous mutant embryos failed to develop beyond E6.5, a phenotype consistent with a fundamental role in cellular metabolism. However, Prmt1 was not required for cell viability, as the protein was not detected in embryonic stem (ES) cell lines established from mutant blastocysts. Low levels of Prmt1 transcripts (approximately 1% of the wild-type level) were detected as assessed by a quantitative reverse transcription-PCR assay. Total levels of arginine N-methyltransferase activity and asymmetric N(G), N(G)-dimethylarginine were reduced by 85 and 54%, respectively, while levels of hypomethylated substrates were increased 15-fold. Prmt1 appears to be a major type I enzyme in ES cells, and in wild-type cells, most substrates of the enzyme appear to be maintained in a fully methylated state.

SspA is required for lethal Salmonella enterica serovar Typhimurium infections in calves but is not essential for diarrhea.

Salmonella pathogenicity island 1 (SPI-1) encodes virulence determinants, which are important for enteropathogenicity in calves. To determine whether the Salmonella enterica serovar Typhimurium SPI-1 effector proteins SspA and SptP are important for enteropathogenicity, strains lacking these proteins were tested during oral infection of calves. Calves infected with a sptP mutant or its isogenic parent developed diarrhea and lethal morbidity. In contrast, calves infected with an sspA mutant developed diarrhea, which resolved within 10 days but did not result in mortality. The sspA mutant was recovered from bovine intestinal tissues at numbers similar to those obtained for its isogenic parent and caused marked intestinal lesions. Thus, the severity of pathological changes caused by serovar Typhimurium strains or their ability to cause diarrhea were not predictive of their ability to cause lethal morbidity in calves. We conclude that factors other than or in addition to bacterial colonization, intestinal lesions, or electrolyte loss contribute to lethal morbidity in calves infected with serovar Typhimurium.

Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems.

Salmonellae encode two virulence-associated type III secretion systems (TTSS) within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Two Salmonella typhimurium genes, sspH1 and sspH2, that encode proteins similar to the Shigella flexneri and Yersinia species TTSS substrates, IpaH and YopM, were identified. SspH1 and SspH2 are proteins containing leucine-rich repeats that are differentially targeted to the SPI1 and SPI2 TTSS. sspH2 transcription was induced within RAW264.7 macrophages, and was dependent upon the SPI2-encoded regulator ssrA/ssrB. In contrast, sspH1 transcription is independent of SPI2, and is not induced after bacterial phagocytosis by eukaryotic cells. Infection of eukaryotic cells with strains expressing a SspH2-CyaA fusion protein resulted in SPI2 TTSS-dependent cAMP increases. In contrast, SspH1-CyaA-mediated cAMP increases were both SPI1 and SPI2 TTSS dependent. sspH2-like sequences were found in most Salmonella serotypes examined, whereas sspH1 was detected in only one S. typhimurium isolate, indicating that the copy number of sspH genes can be variable within Salmonella serotypes. S. typhimurium deleted for both sspH1 and sspH2 was not able to cause a lethal infection in calves, indicating that these genes participate in S. typhimurium virulence for animals.

Transcriptional specificity of the pluripotent embryonic stem cell.

The specificity of gene expression in embryonic stem (ES) cells was analyzed both under in vitro culture conditions and during early embryogenesis. ES cells were infected with U3 beta geo, a U3 gene trap retrovirus that contains coding sequences for a beta-galactosidase-neomycin phosphotransferase hybrid protein. Integrated proviruses, which disrupted expressed cellular genes, were selected in the presence of G418. ES clones expressing regulated beta geo fusion genes were identified by changes in 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining after in vitro differentiation. Thirty-one of 191 clones tested (16%) exhibited regulated expression of beta geo protein. Seven genes disrupted by U3 beta geo were passed into the germline, and expression of the beta geo fusion genes was analyzed in vivo, including inserts disrupting the Eck and REX-1 genes. In each case, genes trapped in cultured ES cells were expressed in the inner cell mass of preimplantation embryos, and changes in lacZ expression during in vitro differentiation were also observed during early development. Thus, cultured ES cells maintain, to a considerable extent, the transcriptional specificity of the pluripotent cells of the preimplantation embryo. As a consequence, in vitro screens utilizing gene traps provide a rapid and accurate means to identify and disrupt developmentally regulated genes.

Refluxo gastroesofágico na criança: tratamento cirúrgico pela técnica de Nissen / Gastroesophageal reflux in children: surgical treatment by Nissen's technique

Objetivo. Analisar os resultados da fundoclipatura de Nissen no tratamento do refluxo gastroesofágico (RGE) na criança. Material e Métodos. Foi realizado um estudo retrospectivo de 185 crianças portadoras de RGE, diagnosticado através de seriografia esofagogastroduodenal (SEGD), e internadas no Hospital Infantil Joana de Gusmao, Florianópolis, SC, no período de fevereiro de 1981 a junho de 1989. Foram revisados os seguintes dados dos prontuários: idade, sexo, quadro clínico, exames complementares, indicaçoes cirúrgicas, tratamento pré-operatório, tratamento cirúrgico pela tecnica de Nissen, complicaçoes trans e pós-operatórias e resultados divididos em bom (ausência de RGE), mau (recidiva), desconhecido (sem acompanhamento) e óbitos. Resultados. A idade variou de trinta dias a 7 anos e houve prevalência do sexo masculino (2:1). As manifestaçoes clínicas mais frequentes foram vômitos (85,40 por cento), distúrbios respiratórios (64,32 por cento) e desnutriçao (55,13 por cento). A SEGD demonstrou refluxo isolado (40 por cento), associado hérnia hiatal (18,91 por cento) e complicado com esofagite (29,18 por cento) e estenose (11,89 por cento). No transoperatório houve perfuraçao inadvertida do esôfago terminal em um caso (0,54 por cento). As complicaçoes pós-operatórias mais frequentes foram a síndrome da bolha gasosa (7,02 por cento) e o edema do esôfago terminal (5,94 por cento). O resultado foi bom para 159 crianças (85,94 por cento), mau em sete (3,78 por cento) e desconhecido em 11 (5,94 por cento). Ocorreram oito óbitos (4,32 por cento): um (0,54 por cento) relacionado a afecçao, um (0,54 por cento) com a cirurgia e seis (3,24) atribuidos a causas clínicas.

[Gastroesophageal reflux in children. Surgical treatment by the Nissen's technique]. / Refluxo gastroesofágico na criança. Tratamento cirúrgico pela técnica de Nissen.

BACKGROUND: To analyse the results of Nissen's fundoplication in the treatment of gastro-esophageal reflux (GER) in children. MATERIAL AND METHODS: A retrospective study was undertaken in 185 children with GER, diagnosed through upper gastrointestinal (GI) tract barium examination, and admitted at Hospital Infantil Joana de Gusmão-Florianópolis, SC, Brazil, from February 1981 to June 1989. From the charts the following data were reviewed: age, sex, clinical picture, subsidiary tests, surgical indications, surgical treatment by Nissen's fundoplication, pre and transoperative treatment, post operative complications and results classified as good (no reflux), bad (relapse), unknow (no follow-up) and deaths. RESULTS: The age ranged from 30 days to seven years and sex incidence was predominant in males (2:1). The main clinical manifestation were vomits (85.40%), respiratory distress (64.32%) and malnutrition (55.13%). Upper GI tract examination revealed isolated GER (40.00%), associated to hiatal hernia (18.91%) and complicated with esophagitis (29.18%) and peptic stenosis (11.89%). In the transoperative period there was terminal esophagus perforation in one case. The main postoperative complications were gas bloat syndrome (7.02%) and edema of the terminal esophagus (5.94%). The result was good in 159 children (85.94%), bad in 7 (3.78%) and unknown in 11 (5.94%). There were 8 deaths (4.32%): 1 (0.54%) related to the disease, 1 (0.54%) to the surgery and 6 (3.24%) related to medical causes. CONCLUSIONS: Based on these results, Nissen's fundoplication is an adequated operation for treatment of children with GER.