RESUMO
Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.
Assuntos
Colo/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Subunidades Proteicas/toxicidade , Toxina Shiga II/toxicidade , Água/metabolismo , Adulto , Animais , Chlorocebus aethiops , Colo/metabolismo , Diarreia/microbiologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Humanos , Mucosa Intestinal/metabolismo , Transporte de Íons/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Células VeroRESUMO
Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.
Assuntos
Humanos , Animais , Masculino , Adulto , Ratos , Toxinas Bacterianas , Colo , Escherichia coli , Mucosa Intestinal , Transporte de Íons , Água , Diarreia , Eletroforese em Gel de Poliacrilamida , Ratos Sprague-DawleyRESUMO
Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (approximately 98.5%) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (approximately 76%) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V.
Assuntos
Inibidores de Adenilil Ciclases , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Animais , Células COS/metabolismo , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/biossíntese , Isoenzimas/metabolismo , Rim/citologia , Plasmídeos , TransfecçãoRESUMO
Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (98.5 percent) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (76 percent) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V
Assuntos
Animais , Adenilil Ciclases , Óxido Nítrico , Doadores de Óxido Nítrico , Nitroprussiato , Células COS , AMP Cíclico , Isoenzimas , Rim , Mamíferos , Plasmídeos , TransfecçãoRESUMO
Shiga toxin-producing Escherichia coli (STEC) colonize the lower segments of the human gastrointestinal tract, causing gastrointestinal and systemic diseases. In this study, the effects of Shiga toxin 2 (Stx2) on fluid absorption and ion transport in the human colon were examined. Net water movement (Jw) and short-circuit current (Isc) were simultaneously measured across the colonic mucosa incubated with crude or purified Stx2. Stx2 significantly inhibited the absorptive J(w) with no effect on the basal I(sc) after 60 min of exposure. These effects may be due to the inhibition of a nonelectrogenic transport system present in the surface colonic villus cells. Morphological studies of the colonic mucosa treated with crude or purified Stx2 demonstrated a selective damage in the absorptive villus epithelial cells. These findings suggest that Stx2 inhibits water absorption across the human colon by acting on a specific cell population: the mature, differentiated absorptive villus epithelium.
Assuntos
Toxinas Bacterianas/farmacologia , Colo/efeitos dos fármacos , Colo/metabolismo , Enterotoxinas/farmacologia , Escherichia coli , Transporte de Íons/efeitos dos fármacos , Água/metabolismo , Colo/patologia , Humanos , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Toxinas ShigaRESUMO
Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.
Assuntos
Aquaporinas/fisiologia , Colo/química , Absorção Intestinal/fisiologia , Adulto , Sequência de Aminoácidos , Animais , Aquaporina 3 , Aquaporinas/química , Aquaporinas/genética , Northern Blotting , Permeabilidade da Membrana Celular , Criança , Pré-Escolar , Células Epiteliais/química , Fluorimunoensaio , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Oócitos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xenopus laevisRESUMO
Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98 percent identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells
Assuntos
Adulto , Pré-Escolar , Criança , Humanos , Aquaporinas/fisiologia , Colo/química , Células Epiteliais/química , Absorção Intestinal/fisiologia , Sequência de Aminoácidos , Aquaporinas/química , Aquaporinas/genética , Northern Blotting , Permeabilidade da Membrana Celular , Imunofluorescência , Fluorimunoensaio , Imuno-Histoquímica , Oócitos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/análise , Xenopus laevisRESUMO
The recent spread of El Tor cholera in Latin America highlights the need for a safe and economical vaccine. The main approach for developing live recombinant vaccines has been to disarm known pathogenic strains of cholera toxin leaving intact antigens involved in protection. These recombinant vaccine candidates do not cause severe diarrhea, but they are too reactogenic for wide scale usage. We describe here a test capable of determining the diarrheagenic potential of attenuated V. cholerae strains. The functional test consists in the simultaneous recording of net water movement, electrical potential difference and short-circuit current across the human intestine ex vivo. We found that human tissues incubated with supernatants from the attenuated 638, 413 and 251a V. cholerae strains caused no changes in the ion conductances and water absorption in ileal and colon tissues allowing them to be assayed in volunteers.
Assuntos
Vacinas contra Cólera/toxicidade , Diarreia/imunologia , Mucosa Intestinal/microbiologia , Vibrio cholerae/imunologia , Animais , Diarreia/metabolismo , Diarreia/microbiologia , Modelos Animais de Doenças , Humanos , Íleo/imunologia , Íleo/metabolismo , Íleo/microbiologia , Técnicas In Vitro , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Dose Letal Mediana , Camundongos , Coelhos , Vacinas Atenuadas/toxicidade , Vibrio cholerae/patogenicidadeRESUMO
PIP: "The article examines the possibilities of systematically exploiting nominative document corpuses related to immigration in Buenos Aires [Argentina] in the period of mass migration [1885-1910], and proposes to focus on the occupations declared by the Italian immigrants. After reviewing the formal aspects of the documental series used (missing information, polysemy of professional aggregates) the immigration of masons is analyzed, taking into account demographic characteristics, annual rhythms of arrival, [and] migration typologies." (EXCERPT)^ieng
Assuntos
Coleta de Dados , Emigração e Imigração , Ocupações , Projetos de Pesquisa , América , Argentina , Demografia , Países Desenvolvidos , Países em Desenvolvimento , Economia , Europa (Continente) , Mão de Obra em Saúde , Itália , América Latina , População , Dinâmica Populacional , Pesquisa , América do Sul , Estatística como Assunto , MigrantesRESUMO
The infusion of certain amino acids, such as serine, alanine, and proline (SAP), has been shown to increase the glomerular filtration rate, whereas branched chain amino acids (BCAA) leucine, isoleucine, and valine fail to modify the glomerular filtration rate. It has been suggested that this effect of amino acids on the glomerular filtration rate is mediated by the action of the hormone glomerulopressin. The purpose of this work was to study the action of SAP and BCAA on glomerulopressin production. Livers isolated from rats were perfused with (i) Krebs-Ringer-Bicarbonate, (ii) SAP, or (iii) BCAA. Results indicate that glomerulopressin production is stimulated by SAP, but inhibited by BCAA.
Assuntos
Aminoácidos/farmacologia , Glucuronatos/metabolismo , Fígado/metabolismo , Alanina/farmacologia , Aminoácidos de Cadeia Ramificada/farmacologia , Análise de Variância , Animais , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Feminino , Infusões Intravenosas , Masculino , Tono Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Prolina/farmacologia , Ratos , Ratos Endogâmicos , Serina/farmacologiaRESUMO
Amino acid infusion induces a rise in glomerular filtration rate (GFR) in normal subjects, but the mechanism is as yet unknown. Glomerulopressin infused into the renal arteries of rats and dogs increases GFR. The aim of this study was to ascertain whether amino acid infusion raised glomerulopressin production and GFR. Accordingly, before renal arteriovenography, in 11 potential kidney donors, the caval catheter was introduced into the right hepatic vein and 60-ml blood samples were collected at the beginning and end of each experiment; six patients received amino acid infusion and five a saline infusion. Glomerulopressin in ultrafiltrates from hepatic vein plasma was measured by toad bioassay and GFR determined with diethylenetriamine pentaacetic acid-Tc99. The amino acid-infused group showed significant glomerulopressin activity in ultrafiltrates, as well as a significant GFR increase, whereas in the control group no glomerulopressin activity was observed, and there was no change in GFR. These findings suggest that intravenous amino acid infusion stimulates glomerulopressin production, which may in turn induce an increase in GFR.