RESUMO
Canonical Wnt signaling critically regulates cell fate and proliferation in developmental stages and adult tissues. Redox regulation through nucleoredoxin (NRX) has recently been identified in canonical Wnt signaling. However, the source of reactive oxygen species (ROS) affecting the redox state of NRX remains elusive. Our principal aim in this study was to investigate whether superoxide-generating NADPH oxidase1 (Nox1) is involved in NRX-regulated Wnt signaling in intestinal and colon epithelial cells. Here, we demonstrate that Wnt treatment of mouse intestinal cells induces production of ROS through Nox1. This Nox1 action is regulated by Rac1 GTPase through Wnt-induced activation of the Rac1 guanine nucleotide exchange factor Vav2 by Src-mediated tyrosine phosphorylation. Nox1-generated ROS oxidize and inactivate NRX, thereby releasing the NRX-dependent suppression of Wnt-ß-catenin signaling through dissociation of NRX from Dvl. Nox1 small-interference RNA inhibits cell response to Wnt, including stabilization of ß-catenin, expression of cyclin D1 and c-Myc via the TCF transcription factor, and accelerated cell proliferation. Nox1 mediates Wnt-induced cell growth in colon cancer cells with the normal Wnt pathway, but not in APC-deficient colon cancer cells, which are constitutively active in Wnt signaling. Together, these results suggest the mediating role of Nox1 in redox-dependent regulation of canonical Wnt-ß-catenin signaling and provide further insight into the regulatory mechanism of the Wnt pathway.
Assuntos
NADH NADPH Oxirredutases/fisiologia , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Camundongos , NADPH Oxidase 1 , Oxirredução , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismoRESUMO
The major objective of this study was to investigate the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in different types of food samples and to compare their genetic relatedness with STEC strains previously isolated from animal sources in Bangladesh. We investigated a total of 213 food samples, including 90 raw meat samples collected from retail butcher shops, 20 raw milk samples from domestic cattle, and 103 fresh juice samples from street vendors in Dhaka city. We found that more than 68% (n = 62) of the raw meat samples were positive for the stx gene(s); 34% (n = 21) of buffalo meats and 66% (n = 41) of beef. Approximately 10% (n = 2) of the raw milk and 8% (n = 8) of the fresh juice samples were positive for stx. We isolated STEC O157 from seven meat samples (7.8%), of which two were from buffalo meats and five from beef; and no other STEC serotypes could be isolated. We could not isolate STEC from any of the stx-positive raw milk and juice samples. The STEC O157 isolates from raw meats were positive for the stx(2), eae, katP, etpD, and enterohemorrhagic E. coli hly virulence genes, and they belonged to three different phage types: 8 (14.3%), 31 (42.8%), and 32 (42.8%). Pulsed-field gel electrophoresis (PFGE) typing revealed six distinct patterns among seven isolates of STEC O157, suggesting a heterogeneous clonal diversity. Of the six PFGE patterns, one was identical and the other two were ≥90% related to PFGE patterns of STEC O157 strains previously isolated from animal feces, indicating that raw meats are readily contaminated with fecal materials. This study represents the first survey of STEC in the food chain in Bangladesh.
Assuntos
Bebidas/microbiologia , Microbiologia de Alimentos/métodos , Carne/microbiologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Bangladesh , Búfalos , Bovinos , DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado , Escherichia coli O157/isolamento & purificação , Fezes/microbiologia , Manipulação de Alimentos/métodos , Leite/microbiologia , Reação em Cadeia da Polimerase , Sorotipagem , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/classificaçãoRESUMO
To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx(1) and/or stx(2), respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx(2), eae, katP, etpD, and enterohemorrhagic E. coli hly (hly(EHEC)) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx(1). Only 7.0% (n = 5) of the isolates were positive for hly(EHEC), and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpf(O113), saa, lpfA(O157/01-141), and lpfA(O157/OI-154) genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.
Assuntos
Doenças dos Bovinos/epidemiologia , Infecções por Escherichia coli/epidemiologia , Doenças das Cabras/epidemiologia , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Matadouros , Animais , Tipagem de Bacteriófagos , Bangladesh/epidemiologia , Búfalos/microbiologia , Bovinos/microbiologia , Doenças dos Bovinos/microbiologia , DNA Bacteriano/genética , Reservatórios de Doenças/microbiologia , Eletroforese em Gel de Campo Pulsado , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Fezes/microbiologia , Microbiologia de Alimentos , Genes Bacterianos , Doenças das Cabras/microbiologia , Cabras/microbiologia , Carne/microbiologia , Prevalência , Sorotipagem , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/classificação , Fatores de Virulência/genéticaRESUMO
Every year, around 3 % of isolates from patients with diarrhoea at Dhaka Hospital, ICDDR,B, are identified as Shigella-like organisms (SLOs) based on their activity in biochemical tests. These isolates do not react with any of the current Shigella antisera including all existing and provisional serotypes. Among these SLOs, a unique cluster of seven isolates with an identical plasmid profile was found and these isolates were further characterized by phenotypic and genotypic techniques. All were nonlactose fermenters, with an identical biochemical pattern typical of Shigella dysenteriae. They were classified as invasive since they harboured the 140 MDa invasive plasmid, were able to bind Congo red, produced keratoconjunctivitis in the guinea pig eye, and were positive by PCR for the ipaH gene and Shigella enterotoxin 2 [ShET-2] gene. All isolates were resistant to ampicillin, tetracycline and sulfamethoxazole-trimethoprim but were susceptible to mecillinam, nalidixic acid, ceftriaxone and ciprofloxacin. Six of the isolates were identical in DNA pattern by PFGE with the seventh exhibiting a closely related pattern; both patterns were distinguishable from all other Shigella and Escherichia coli patterns. An antiserum prepared against one of the isolates reacted with all isolates and did not cross-react with other Shigella and E. coli serotype reference strains. It is therefore proposed that these isolates represent a new provisional serovar of S. dysenteriae, type strain KIVI 162.
Assuntos
Diarreia/microbiologia , Disenteria Bacilar/microbiologia , Shigella dysenteriae/classificação , Shigella dysenteriae/isolamento & purificação , Animais , Antibacterianos/farmacologia , Anticorpos Antibacterianos/metabolismo , Antígenos de Bactérias/genética , Técnicas de Tipagem Bacteriana , Bangladesh , Vermelho Congo/metabolismo , Reações Cruzadas , Impressões Digitais de DNA , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Eletroforese em Gel de Campo Pulsado , Enterotoxinas/genética , Cobaias , Humanos , Ceratoconjuntivite/microbiologia , Plasmídeos , Sorotipagem , Shigella dysenteriae/patogenicidade , Shigella dysenteriae/fisiologiaRESUMO
Glioblastoma multiforme is a common primary brain tumor in adults and one of the most devastating human cancers. Reactive oxygen species (ROS) generated by NADPH oxidase (Nox) 4 have recently been a focus of attention in the study of glioblastomas, but the molecular mechanisms underlying the actions of Nox4 remain elusive. In this study, we demonstrated that silencing of Nox4 expression by Nox4-targeted siRNA suppressed cell growth and motility of glioblastoma U87 cells, indicating the involvement of Nox4. Furthermore, Nox4-derived ROS oxidized and inactivated protein tyrosine phosphatase (PTP):1B: PTP1B in its active form downregulates cell proliferation and migration. By affinity purification with the substrate-trapping mutant of PTP1B, tyrosine-phosphorylated coronin-1C was identified as a substrate of PTP1B. Its tyrosine phosphorylation level was suppressed by Nox4 inhibition, suggesting that tyrosine phosphorylation of coronin-1C is regulated by the Nox4-PTP1B pathway. Finally, ablation of coronin-1C attenuated the proliferative and migratory activity of the cells. Collectively, these findings reveal that Nox4-mediated redox regulation of PTP1B serves as a modulator, in part through coronin-1C, of the growth and migration of glioblastoma cells, and provide new insight into the mechanistic aspect of glioblastoma malignancy.