RESUMEN
The effects of marine substances with various cytotoxic mechanisms on the integrity of the human intestinal Caco-2 cell monolayer were examined by measuring the transepithelial electrical resistance (TEER). TEER was rapidly decreased by apical exposure of the monolayers to discodermin A, a membrane pore-forming substance. The decrease in TEER occurred in an earlier stage of incubation than the release of intracellular lactate dehydrogenase (LDH) which is commonly used as a parameter of cell damage or death. Mycalolide B (an actin-depolymerizing substance), calyculin A and okadaic acid (protein phosphatase inhibitors) also rapidly decreased the TEER value, although no cell membrane damage or resultant LDH release by these toxicants were detected. The TEER decrease caused by the toxicants was associated with the increased transepithelial permeability of the cell monolayer. Treatment with these toxicants, except calyculin A, caused morphological changes in the intracellular actin filament, suggesting that these toxicants altered the cytoskeletal structure, by which the tight junction was opened. Calyculin A was likely to loosen the cellular junctions rapidly and induce cell detachment from the monolayer. Although onnamide A, a protein synthesis inhibitor, did not cause any decrease in TEER, at least during a 90-min incubation, TEER sensitively reflects the cytotoxic effects of various types of toxicants with acute toxicity.
Asunto(s)
Mucosa Intestinal/efectos de los fármacos , Toxinas Marinas/toxicidad , Actinas/efectos de los fármacos , Células CACO-2 , Citoesqueleto/efectos de los fármacos , Impedancia Eléctrica , Humanos , Oxazoles/toxicidad , Inhibidores de la Síntesis de la Proteína/toxicidad , Uniones Estrechas/efectos de los fármacosRESUMEN
A new protein that decreases transepithelial electrical resistance (TEER) in the human intestinal Caco-2 cell monolayer was found in a water-soluble fraction of the mushroom Flammulina velutipes. This protein, termed TEER-decreasing protein (TDP), is not cytotoxic and does not induce cell detachment, but rapidly increases the tight junctional permeability for water-soluble marker substances such as Lucifer Yellow CH (Mr 457) through the paracellular pathway. TDP was isolated and purified from the aqueous extract of F. velutipes by chromatographic means. Purified TDP was found to be a simple, nonglycosylated protein without intermolecular disulfide bonds, and the apparent molecular mass as estimated by SDS/PAGE and gel filtration is 30 kDa. It was revealed that the N-terminal amino-acid sequence of purified TDP is identical to the recently reported N-terminal sequence of flammutoxin, a membrane-perturbing hemolytic protein, for which the complete primary structure has not yet been reported [Tomita, T., Ishikawa, D., Noguchi, T., Katayama, E., and Hashimoto, Y. (1998) Biochem. J. 333, 24794-24799]. The cDNA coding for TDP was cloned by 5' and 3' rapid amplification of cDNA ends. The ORF encodes a protein with 272 amino-acid residues showing no homology to known proteins. Relevant studies using TDP cDNA will provide insight into the structure-function relationships of membrane pore-forming toxins.
Asunto(s)
Basidiomycota/química , Células CACO-2/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , ADN Complementario/aislamiento & purificación , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/farmacología , Micotoxinas , Secuencia de Aminoácidos , Secuencia de Bases , Basidiomycota/genética , Células CACO-2/efectos de los fármacos , Clonación Molecular , ADN de Hongos/aislamiento & purificación , Impedancia Eléctrica , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Datos de Secuencia MolecularRESUMEN
BACKGROUND: Although retinal breaks occur frequently during vitrectomy, the postoperative occurrence of new retinal holes close to the vascular arcade after vitrectomy for rhegmatogenous retinal detachment rarely has been reported. METHODS: Three patients with rhegmatogenous, retinal detachment were treated by vitrectomy. More than 49 days after vitrectomy, posterior retinal holes with no retinal detachment occurred halfway between the vascular arcade and the chorioretinal scar around the extrusion hole or the primary retinal tear. RESULTS: These new holes were effectively managed with photocoagulation. CONCLUSION: New hole formation could be caused by the technique of the internal drainage, the contraction of the photocoagulation scar, or epiretinal membrane contraction. Another possibility is that new holes occur through two opposite tangential traction contractile forces: one induced by the contraction of the photocoagulation scar, the other caused by the contraction of the premacular cortical vitreous attached to the vascular arcade.
Asunto(s)
Complicaciones Posoperatorias/etiología , Desprendimiento de Retina/cirugía , Perforaciones de la Retina/etiología , Vitrectomía/efectos adversos , Anciano , Femenino , Estudios de Seguimiento , Fondo de Ojo , Humanos , Coagulación con Láser/métodos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/cirugía , Reoperación , Desprendimiento de Retina/patología , Perforaciones de la Retina/patología , Perforaciones de la Retina/cirugía , Agudeza VisualRESUMEN
The transepithelial electrical resistance (TEER) of human intestinal Caco-2 cell monolayers cultured on a permeable filter rapidly decreased on apical exposure of the monolayer cells to such cytotoxic membrane perturbents as benzalkonium chloride and saponin. This decrease in TEER occurred at lower concentrations of these toxicants and at earlier stages of incubation than those for the release of intracellular lactate dehydrogenase, which is commonly used as a parameter for assessing cell membrane damage. The TEER decrease caused by the toxicants was associated with increased transepithelial permeability of the cell monolayer, as well as with subtle changes in the cytoskeletal structure, suggesting that membrane perturbation by the toxicants induced cytoskeletal changes that resulted in disruption of the tight junction and a subsequent increase in paracellular permeability. The rapid TEER change caused by the cytotoxic membrane perturbents suggests that TEER measurement would provide a simple and sensitive method for evaluating membrane-perturbing toxicants.