Cooling Treatment Transiently Increases the Permeability of Brain Capillary Endothelial Cells Through Translocation of Claudin-5

The blood–brain-barrier (BBB) is formed by different cell types, of which brain microvascular endothelial cells are major structural constituents. The goal of this study was to examine the effects of cooling on the permeability of the BBB with reference to tight junction formation of brain microendothelial cells. The sensorimotor cortex above the dura mater in adult male Wistar rats was focally cooled to a temperature of 5 °C for 1 h, then immunostaining for immunoglobulin G (IgG) was performed to evaluate the permeability of the BBB. Permeability produced by cooling was also evaluated in cultured murine brain endothelial cells (bEnd3) based on measurement of trans-epithelial electric resistance (TEER). Immunocytochemistry and Western blotting of proteins associated with tight junctions in bEnd3 were performed to determine protein distribution before and after cooling. After focal cooling of the rat brain cortex, diffuse immunostaining for IgG was observed primarily around the small vasculature and in the extracellular spaces of parenchyma of the cortex. In cultured bEnd3, TEER significantly decreased during cooling (15 °C) and recovered to normal levels after rewarming to 37 °C. Immunocytochemistry and Western blotting showed that claudin-5, a critical regulatory protein for tight junctions, was translocated from the membrane to the cytoplasm after cooling in cultured bEnd3 cells. These results suggest that focal brain cooling may open the BBB transiently through an effect on tight junctions of brain microendothelial cells, and that therapeutically this approach may allow control of BBB function and drug delivery through the BBB.     

Horizontal gene transfer of a genetic island encoding a type III secretion system distributed in Vibrio cholerae

Twelve Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected among 110 environmental and 14 clinical isolates. T3SS‐related genes were distributed among the various serogroups and pulsed‐field gel electrophoresis of NotI‐digested genomes showed genetic diversity in these strains. However, the restriction fragment length polymorphism profiles of the T3SS‐related genes had similar patterns. Additionally, naturally competent T3SS‐negative V. cholerae incorporated the ca. 47 kb gene cluster of T3SS, which had been integrated into a site on the chromosome by recombination. Therefore, it is suggested that horizontal gene transfer of T3SS‐related genes occurs among V. cholerae in natural ecosystems.     

Implication of Catecholamines During Spawning in Marine Bivalve Molluscs

Summary

During the tail-bud stage of Ascidiella aspersa embryogenesis, the test cells or innermost cells of the egg envelope manifest locomotive activities. Light microscopy further reveals that the adhesive behaviour of test cells changes in the course of embryogenesis. Mechanical dechorionation experiments performed on 846 embryos demonstrate that up to the tail-bud stage all test cells are attached to the inner surface of the chorion. The embryo is completely devoid of test cells. At the onset of larval tunic secretion, increasing numbers of test cells settle on the embryo until all test cells adhere to it. This switch in adhesive properties is completed within 65 min. The hatched larva carries the entire complement of test cells until the onset of metamorphosis. SEM and observations show that test cells do not establish direct cell-to-cell contacts to ectodermal cells but attach to the larval tunic.     

Phosphopeptides designed for 5-methylcytosine recognition

An artificial phosphopeptide has been developed through rational design of the interaction with 5-methylcytosine in duplex DNA. The peptide consists of two tandem zinc finger motifs, in one of which the glutamate was replaced with a phosphotyrosine, the phosphotyrosine in the peptide being effective for methylcytosine selectivity of DNA binding. The flexible modulation of the target methylated sequence by rearrangement of zinc finger peptides is possible, and the phosphopeptide provided us an important hint for expansion of the codes for the interactions of zinc fingers with DNA to methylated DNA sequences. The fluorescence-labeled phosphopeptide provided information on the methylation status of genomic DNA through fluorescence anisotropy after a 10 min incubation.     

Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces intimal thickening after vascular injury

Glucagon-like peptide-1 is a hormone secreted by L cells of the small intestine and stimulates glucose-dependent insulin response. Glucagon-like peptide-1 receptor agonists such as exendin-4 are currently used in type 2 diabetes, and considered to have beneficial effects on the cardiovascular system. To further elucidate the effect of glucagon-like peptide-1 receptor agonists on cardiovascular diseases, we investigated the effects of exendin-4 on intimal thickening after endothelial injury. Under continuous infusion of exendin-4 at 24 nmol/kg/day, C57BL/6 mice were subjected to endothelial denudation injury of the femoral artery. Treatment of mice with exendin-4 reduced neointimal formation at 4 weeks after arterial injury without altering body weight or various metabolic parameters. In addition, in vitro studies of isolated murine, rat and human aortic vascular smooth muscle cells showed the expression of GLP-1 receptor. The addition of 10 nM exendin-4 to cultured smooth muscle cells significantly reduced their proliferation induced by platelet-derived growth factor. Our results suggested that exendin-4 reduced intimal thickening after vascular injury at least in part by the suppression of platelet-derived growth factor-induced smooth muscle cells proliferation.     

Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca²⁺ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser⁶²¹ and Thr⁶²⁶ were O-GlcNAcylated and that Thr⁶²⁶ was O-GlcNAcylated in the steady state but Ser⁶²¹ was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser⁶²¹. Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser⁶²¹. These data suggest that both decrease in O-GlcNAcylation at Thr⁶²⁶ and increase in O-GlcNAcylation at Ser⁶²¹ in STIM1 lead to impairment of SOCE activity through decrease in Ser⁶²¹ phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.