Single GUV method reveals interaction of tea catechin (-)-epigallocatechin gallate with lipid membranes

Tea catechins, which are flavonoids and the main components of green tea extracts, are thought to have antibacterial and antioxidant activity. Several studies indicate that lipid membranes are one of the targets of the antibacterial activity of catechins. Studies using a suspension of large unilamellar vesicles (LUVs) indicate that catechin causes gradual leakage of internal contents from LUVs. However, the detailed characteristics of the interaction of catechins with lipid membranes remain unclear. In this study, we investigated the interaction of (-)-epigallocatechin gallate (EGCg), a major catechin in tea extract, with single giant unilamellar vesicles (GUVs) of egg phosphatidylcholine (egg PC) using phase-contrast fluorescence microscopy and the single GUV method. We prepared GUVs of lipid membranes of egg PC in a physiological ion concentration ( approximately 150 mM NaCl) using the polyethylene glycol-lipid method. Low concentrations of EGCg at and above 30 muM induced rapid leakage of a fluorescent probe, calcein, from the inside of single egg PC-GUVs; after the leakage, the GUVs changed into small lumps of lipid membranes. On the other hand, phase-contrast microscopic images revealed the detailed process of the EGCg-induced burst of GUVs, the decrease in their diameter, and their transformation into small lumps. The dependence of the fraction of burst GUVs on EGCg concentration was almost the same as that of the fraction of leaked GUV. This correlation strongly indicates that the leakage of calcein from the inside to the outside of the GUV occurred as a result of the burst of the GUV. The fraction of completely leaked GUV and the fraction of the burst GUV increased with time and also increased with increasing EGCg concentration. We compared the EGCg-induced leakage from single GUVs with EGCg-induced leakage from a LUV suspension. The analysis of the EGCg-induced shape changes shows that the binding of EGCg to the external monolayer of the GUV increases its membrane area, inducing an increase in its surface pressure. Small angle x-ray scattering experiments indicate that the intermembrane distance of multilamellar vesicles of PC membrane greatly decreased at EGCg concentrations above the threshold, suggesting that neighboring membranes came in close contact with each other. On the basis of these results, we discuss the mechanism of the EGCg-induced bursting of vesicles.     

Magnesium attenuates isoproterenol-induced acute cardiac dysfunction and beta-adrenergic desensitization

Sympathetic nervous activation is a crucial compensatory mechanism in heart failure. However, excess catecholamine may induce cardiac dysfunction and beta-adrenergic desensitization. Although magnesium is known to be a cardioprotective agent, its beneficial effects on acute cardiac dysfunction remain to be elucidated. We examined the effects of magnesium on left ventricular (LV) dysfunction induced by a large dose of isoproterenol in dogs. Sixteen anesthetized dogs underwent a continuous infusion of isoproterenol (1 micro g.kg(-1).min(-1)) with or without a magnesium infusion (1 mg.kg(-1).min(-1)). The dose response to small doses of isoproterenol (0.025-0.2 micro g.kg(-1).min(-1)) was tested hourly. A large dose of isoproterenol decreased LV systolic function, increased the time constant of LV isovolumic relaxation, and suppressed the dose response to small doses of isoproterenol in a time-dependent manner. Magnesium significantly attenuated isoproterenol-induced LV systolic and diastolic dysfunction and preserved the dose response to isoproterenol. Serum-ionized calcium significantly decreased with a large dose of isoproterenol but was fully maintained at baseline level with magnesium. A large dose of isoproterenol increased serum lipid peroxide levels and serological markers of myocardial damage, which were significantly suppressed by magnesium. In conclusion, magnesium significantly attenuated excess isoproterenol-induced acute cardiac dysfunction and beta-adrenergic desensitization.     

PTEN reduces cuff-induced neointima formation and proinflammatory cytokines

An inflammatory response followed by vascular injury plays an important role in neointima formation and development of atherosclerotic lesions, which are in part mediated by proinflammatory cytokines. Using a cuff injury model, we examined the effects of adenovirus-mediated overexpression of phosphatase and tensin homology deleted on chromosome 10 (PTEN) on neointima formation and the proinflammatory response. A cuff was placed around the femoral artery, and adenovirus expressing human PTEN type 1 (AdPTEN) or Escherichia coli beta-galactosidase (AdLacZ) was injected between the cuff and the adventitia. After 14 days, the arteries were examined histopathologically and by Western blotting. The significant reduction of neointima formation by AdPTEN compared with AdLacZ was accompanied by reduced cell proliferation and increased adventitial cell apoptosis. AdPTEN also reduced expression of phosphorylated I kappa B-alpha, but not nonphosphorylated I kappa B-alpha. Western blotting revealed that AdPTEN reduced the cuff injury-induced expression levels of monocyte chemoattractant protein-1, TNF-alpha, and IL-1 beta and their expression in all layers of the arterial wall. In contrast, cuff-induced macrophage invasion, which was also inhibited by AdPTEN, was detected only at the intimal surface and in the adventitia. In cultured vascular smooth muscle cells, PTEN directly inhibited ANG II-induced monocyte chemoattractant protein-1 expression as quantified by real-time PCR and Western blotting. Our results suggest that overexpression of PTEN reduces neointima formation, possibly in part through inhibition of the inflammatory response by macrophage invasion and proinflammatory cytokine expression.     

A new threespine stickleback,Gasterosteus nipponicus sp. nov. (Teleostei: Gasterosteidae), from the Japan Sea region

Gasterosteus nipponicus sp. nov. is described from the holotype, 35 paratypes, and 60 additional specimens. The species differs from congeners in the following combination of characters: lateral plates complete, abruptly reducing in size above the anus, depth of lateral plate above the anus < 60 % that of the deepest plate; caudal keels thin, membranous. The new species is distributed in coastal Japan facing the Sea of Japan from Kyushu to Hokkaido Islands, along the Pacific coast of northern Japan from the Chiba Prefecture to Hokkaido, along the Sea of Okhotsk of Hokkaido, west to the southern and eastern coasts of Korean Peninsula, the Maritime Territory and north to Sakhalin Island, Russia.     

Detailed Localization of Augmented Angiotensinogen mRNA and Protein in Proximal Tubule Segments of Diabetic Kidneys in Rats and Humans

In the intrarenal renin-angiotensin system, angiotensinogen levels are well known to be increased in diabetes, and these enhanced intrarenal angiotensinogen levels may initiate the development and accelerate the progression of diabetic nephropathy. However, the specific localization of the augmented angiotensinogen in proximal tubule segments in diabetes is still unknown. We investigated the detailed localization of angiotensinogen in 3 proximal tubule segments in the diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and the control Long-Evans Tokushima Otsuka (LETO) rats. We also prepared OLETF rats treated with angiotensin II type 1 receptor blocker, olmesartan or with a combination of vasodilator agents. Moreover, biopsied samples of human kidney cortex were used to confirm the results of animal studies. We examined the co-localization of angiotensinogen with segment-specific markers by double staining using fluorescence in situ hybridization and/or immunofluorescence. Angiotensinogen mRNA expression was barely detectable in segment 1. In segment 3, the area of angiotensinogen mRNA expression was augmented in the OLETF rats compared with the LETO rats. Angiotensinogen protein expression areas in segments 1 and 3 were also increased in the OLETF rats compared with the LETO rats. Chronic treatment with olmesartan ameliorated these areas of augmented angiotensinogen expression. Biopsied human kidney samples showed similar results. These data suggest that the augmented angiotensinogen mRNA levels in segment 3 and angiotensinogen protein levels in segments 1 and 3 may contribute to the progression of diabetic nephropathy.     

Recovery of electric energy from formate by using a recombinant strain of Escherichia coli

Recombinant Escherichia coli cells were applied for the recovery of electric energy from formate. Initially, the fdh gene, which encodes formate dehydrogenase (FDH) of Mycobacterium vaccae, was introduced into E. coli cells to allow efficient degradation of formate. The constructed microbial fuel cell (MFC) with E. coli BW25113 cells carrying fdh gene showed appreciable generation of current density in the presence of formate as a substrate. Current density and polarization curves revealed that the performance of MFC under examined conditions was limited by the electron transfer from bulk liquid to the electrode surface; accordingly, agitation resulted in an increase in the current density and achieved a coulombic efficiency of 21.7 % on the basis of formate consumed. Thus, gene recombination enables E. coli cells to utilize formate as a fuel for MFC.     

Influence of surface topography on the human epithelial cell response to micropatterned substrates with convex and concave architectures

Background

Understanding the fundamental mechanisms underlying the cellular response to topographical surface features will extend our knowledge regarding the regulation of cell functions. Analyzing the cellular response to different topographical features, over multiple temporal and spatial scales, is central to understanding and guiding several biological functions. We used micropatterned substrates with convex and concave architectures to evaluate the behaviors of human epithelial cells on these substrates.

Results

Pillar and pit substrates caused heterogeneous spatial growth and distribution, with differences in cell density, over 48 h. Regional densities and distribution were significantly increased at pillar sidewalls, and at pit sidewalls and bottoms compared with those on flat unpatterned areas. Time-lapse observations revealed that different mechanisms of cell migration were dependent upon pillar and pit features. Cells on pillar substrate migrated towards the sidewall, whereas cells on pit substrate tended to move towards the sidewalls and bottom. Cytoskeletal staining of F-actin and vinculin showed that this migration can be attributed to difference in spatial reorganization of actin cytoskeleton, and the formation of focal adhesions at various points on the at the convex and concave corners of pillar and pit substrates. Cells cultured on the pillar substrate had stress fibers with extended filopodia and immature focal contacts at the sidewalls and convex corners, similar to those on the flat unpatterned substrate. Cells at the sidewalls and concave corners of pit substrate had more contractile stress fibers and stable focal contacts compared with cells on the pillar substrate. We also found that the substrate structures affect cell-cell contact formation via E-cadherin, and that this was associated with reorganization of the actin cytoskeleton at the sidewall, and at the convex and concave corners of the substrate.

Conclusion

Migration is an important factor affecting spatial growth and distribution. Heterogeneity at various locations was caused by different migratory behaviors at the convex and concave corners of pillar and pit substrates. We propose that this investigation is a valuable method for understanding cell phenotypes and the heterogeneity during spatial growth and distribution of epithelial cells during culture.

     

Rapid generation of knockdown transgenic mice by silencing lentiviral vectors

Lentiviral vectors are potent gene delivery vehicles that enable stable expression of transgenes in both dividing and postmitotic cells, including preimplantation embryos. We have developed lentiviral vectors carrying silencing cassettes consisting of an RNA polymerase III promoter expressing short hairpin RNAs. Transgenic mice can be generated rapidly by transduction of early embryos with lentiviral silencing vectors, resulting in mice with downregulated target genes. We describe two alternative early embryo transduction protocols (removal of zona pellucida and subzonal microinjection). These methodologies offer the possibility of large-scale generation of knockdown transgenic mice for functional genomic studies and enable the production of transgenic mice in 7 weeks.