Intestinal barrier function is closely related to intestinal health and diseases. Recent studies demonstrate that some probiotic and commensal bacteria secrete metabolites that are capable of affecting the intestinal functions. The present study examined an enhancing effect of bioactive factors secreted by Bifidobacterium breve strain B-3 on the intestinal tight junction (TJ) barrier integrity in human intestinal Caco-2 cells. Administration of conditioned medium obtained from B. breve strain B-3 (B3CM) to Caco-2 cells for 24 h increased trans-epithelial electrical resistance (TER), a TJ barrier indicator, across their monolayers. Immunoblot, immunofluorescence, and qPCR analyses demonstrated that B3CM increased an integral TJ protein, claudin-4 expression. In luciferase reporter assay, the administration of B3CM enhanced the claudin-4 promoter activity, indicating the transcriptional upregulation of claudin-4. Site-directed mutation of specificity protein 1 (Sp1) binding sites in the claudin-4 promoter sequence and suppression of Sp1 expression by siRNA technology clearly reduced the enhancing effect of B3CM on claudin-4 promoter activity. Liquid chromatography/mass spectrometry detected a significant amount of acetic acid in B3CM (28.3 mM). The administration of acetic acid to Caco-2 cells partially mimicked a B3CM-mediated increase in TER, but failed to increase claudin-4 expression. Taken together, bioactive factors secreted by B. breve B-3 enhanced the TJ barrier integrity in intestinal Caco-2 cells. Transcriptional regulation of claudin-4 through Sp1 is at least in part one of the underlying molecular mechanisms. In addition, acetic acid contributes to the B3CM-mediated barrier effect independently of claudin-4 expression.
Gastrulation is a universal process in the morphogenesis of many animal embryos. Although morphological and molecular events in gastrulation have been well studied, the mechanical driving forces and underlying regulatory mechanisms are not fully understood. Here, we investigated the gastrulation of embryos of a sea urchin, Hemicentrotus pulcherrimus, which involves the invagination of a single-layered vegetal plate into the blastocoel. We observed that omeprazole, a proton pump inhibitor capable of perturbing the left-right asymmetry of sea urchin embryo, induced "partial exogastrulation" where the secondary invagination proceeds outward. During early gastrulation, intracellular apical-basal polarity of F-actin distribution in vegetal half was higher than those in animal half, while omeprazole treatment disturbed the apicalbasal polarity of F-actin distribution in vegetal half. Furthermore, gastrulation stopped and even partial exogastrulation did not occur when F-actin polymerization or degradation in whole embryo was partially inhibited via RhoA or YAP1 knockout. A mathematical model of the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin. Additionally, such cell positiondependent intracellular F-actin distributions might be regulated by intracellular pH distributions. Therefore, apical-basal polarity of F-actin distribution disrupted by omeprazole may induce the partial exogastrulation via anomalous secondary invagination.
Gastrulation is a universal process in the morphogenesis of many animal embryos. In sea urchin embryos, it involves the invagination of a single-layered vegetal plate into the blastocoel. Although morphological and molecular events in gastrulation have been well studied, the mechanical driving forces and the regulatory mechanism underlying gastrulation is not fully understood. In this study, structural features and cytoskeletal distributions were studied in sea urchin embryos using an “exogastrulation” model induced by inhibiting the H+/K+ ion pump with omeprazole. The vegetal poles of the exogastrulating embryos showed reduced roundness indices, intracellular pH polarization, and intracellular F-actin polarization at the pre-early gastrulation stage compared with normal embryos. Gastrulation stopped when F-actin polymerization or degradation was inhibited via RhoA or YAP1 knockout, although pH distributions were independent of such a knockout. A mathematical model of sea urchin embryos at the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin and pH distributions. Thus, gastrulation required appropriate cell position-dependent intracellular F-actin distributions regulated by the H+/K+ ion pump through pH control.
Gastrulation is a universal process in the morphogenesis of many animal embryos. In sea urchin embryos, it involves the invagination of single-layered vegetal plate into blastocoel. Although morphological and molecular events have been well studied for gastrulation, the mechanical driving forces and their regulatory mechanism underlying the gastrulation is not fully understood. In this study, structural features and cytoskeletal distributions were studied in sea urchin embryo using an "exogastrulation" model induced by inhibiting the H+/K+ ion pump with omeprazole. The vegetal pole sides of the exogastrulating embryos had reduced roundness indices, intracellular pH polarization, and intracellular F-actin polarization at the pre-early gastrulation compared with the normal embryo. Gastrulation stopped when F-actin polymerization or degradation was inhibited by RhoA or YAP1 knockout, although pH distributions were independent of such a knockout. A mathematical model of sea urchin embryos at the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin and pH distributions. Thus, gastrulation required appropriate cell position-dependent intracellular F-actin distributions regulated by the H+/K+ ion pump through pH control.
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