RESUMO
OBJECTIVES: Early weaning (EW) increases proliferation of the gastric epithelium in parallel with higher expression of transforming growth factor alpha and its receptor epidermal growth factor receptor (EGFR). The primary objective of the present study was to examine involvement of EGFR signalling in regulating mucosal cell proliferation during the early weaning period. MATERIALS AND METHODS: Fifteen-day-old rats were split into two groups: suckling (control) and EW, in which pups were separated from the dam. Animals were killed daily until the 18th day, 3 days after onset of treatment. To investigate the role of EGFR in proliferation control, EW pups were injected with AG1478, an EGFR inhibitor; signalling molecules, proliferative indices and cell cycle-related proteins were evaluated. RESULTS: EW increased ERK1/2 and Src phosphorylation at 17 days, but p-Akt levels were unchanged. Moreover, at 17 days, AG1478 administration impaired ERK phosphorylation, whereas p-Src and p-Akt were not altered. AG1478 treatment reduced mitotic and DNA synthesis indices, which were determined on HE-stained and BrdU-labelled sections. Finally, AG1478 injection decreased p21 levels in the gastric mucosa at 17 days, while no changes were detected in p27, cyclin E, CDK2, cyclin D1 and CDK4 concentrations. CONCLUSIONS: EGFR is part of the mechanism that regulates cell proliferation in rat gastric mucosa during early weaning. We suggest that such responses might depend on activation of MAPK and/or Src signalling pathways and regulation of p21 levels.
Assuntos
Receptores ErbB/metabolismo , Mucosa Gástrica/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Quinases da Família src/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Mitose , Fosforilação , Quinazolinas , Ratos , Ratos Wistar , Transdução de Sinais , Tirfostinas/farmacologiaRESUMO
AIMS: Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H-) on Caco 2 and HT-29-human epithelial intestinal cells. METHODS AND RESULTS: The Caco 2 and HT-29 cells, which were treated with Enterohemolysin (EHly) within 10-15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. CONCLUSIONS: Enterohemolysin induced apoptosis on human epithelial intestinal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC.
Assuntos
Apoptose , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Mucosa Intestinal/microbiologia , Fosfatase Alcalina/metabolismo , Células CACO-2 , Sobrevivência Celular , Diarreia/metabolismo , Diarreia/microbiologia , Escherichia coli/química , Células HT29 , Humanos , Mucosa Intestinal/citologia , L-Lactato Desidrogenase/metabolismoRESUMO
AIMS: To determine the potential virulence factors produced by culture supernatants of clinical isolates of Stenotrophomonas maltophilia. METHODS AND RESULTS: Culture supernatants of clinical isolates of S. maltophilia were assayed for haemolytic, enzymatic (lipase, protease and phospholipase) and cytotoxic activity. Cytotoxic activity was assayed in Vero (African green monkey), HeLa (human cervix) and HEp-2 (human larynx epidermoid carcinoma) cells. Microscopic analyses revealed intensive rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp-2 cells. In Vero and HeLa cells, the cytotoxic effects were characterized by vigorous endocytosis and cell aggregation. The viability of cultured mammalian cells was determined with neutral red and demonstrated that the sensitivity among the cells was different. This activity was inactivated by heating at 56 degrees C for 15 min and protease inhibitors did not inhibit cytotoxic activity. The clinical S. maltophilia presented a cell-free haemolytic activity similar to the 'hot-cold' haemolysins. CONCLUSIONS: S. maltophilia culture supernatants caused vigorous endocytosis and cell aggregation in HeLa and Vero cells, produced haemolytic and enzymatic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Stenotrophomonas maltophilia strains.
Assuntos
Citotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Stenotrophomonas maltophilia/metabolismo , Animais , Cálcio/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Citotoxinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Proteínas Hemolisinas/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Ferro/farmacologia , Ovinos , Células Vero , Zinco/farmacologiaRESUMO
Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 æg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 æg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.
Assuntos
Animais , Masculino , Camundongos , Toxinas Bacterianas , Eritrócitos , Escherichia coli , Células Eucarióticas , Toxinas Bacterianas , Membrana Celular , Colesterol , Eletroforese em Gel de Poliacrilamida , Hemólise , Ligação ProteicaRESUMO
Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 microg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 microg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.