Detection of superoxide anion radical in phospholipid liposomal membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran

AbstractUtilization of a fluorescence dye, 1,3-diphenylisobenzofuran (DPBF) as a detector of superoxide anion radical (O⋅−2) was examined. The fluorescence intensity of DPBF incorporated in phospholipid liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) is effectively quenched by incubation with xanthine/xanthine oxidase system. On the other hand, xanthine or xanthine oxidase alone did not induce quenching of the DPBF fluorescence in the liposomes. Xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes was almost completely protected by the addition of superoxide dismutase (SOD, 1 U/ml), but not by heat-denatured SOD (10 min boiling) at the same concentration. On the other hand, catalase (1 U/ml), and hydroxyl radical and singlet oxygen scavengers (10 mM sodium benzoate, 300 mM mannitol, 1 mM tryptophan and 1 mM sodium azide) did not protect xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes. The concentration dependence profiles of xanthine oxidase on the DPBF fluorescence quenching and O⋅−2 generation showed that there is a good correlation between these parameters. Under the present experimental conditions, approximately 7 μM H2O2/30 min were produced, but the addition of H2O2 (1 mM) to DPBF-labeled liposomes did not quench the dye fluorescence in the liposomes. Temperature dependence profiles of the DPBF fluorescence quenching induced by xanthine/xanthine oxidase treatment and the excimer fluorescence formation of pyrene molecules embedded in the liposomal membrane suggested that the quenching efficiency of the DPBF fluorescence is largely dependent on their lipid dynamics. Based on these results, we proposed the possibility that DPBF fluorescence quenching method is able to be used as a simple method for detecting O⋅−2 inside the membrane lipid layer and that DPBF fluorescence quenching by O⋅−2 is controlled by the physical state of membrane lipids

Vascular Access Intervention by Nephrologist in Hiroshima

Background: Vascular access intervention (VAI) is a procedure essential to the maintenance of patency in vascular access and has become indispensable to nephrologists. This procedure has changed nephrology to a more exciting field. Concept: This report describes the VAI technique which is based on 15 years of treatment experience of the author of this report and which the author has taught to young nephrologists in Hiroshima

Klotho as a Therapeutic Target during the Development of Renal Fibrosis

Systemic symptoms such as the ectopic calcification, atrophy of skin and muscle, and impaired sexual function observed in chronic kidney diseases (CKD) have been reported to coincide with those observed in geriatric symptoms. Regarding the kidney, clinical/pathological characteristics in CKD patients also coincide with those in the aging kidney. These findings suggest common mechanisms in the development of both CKD and aging. Our investigation of aging factors associated with renal fibrosis in IgA nephropathy patients revealed a significant correlation between accumulation of cells with an arrested cell cycle and decreased expression of Klotho protein. Because cell cycle arrest has a protective effect on organs in the acute phase, the proposed therapeutic target against the aging process is to maintain expression of Klotho protein. In addition, it is recognized that TGF-β1 plays a central role in the development of renal fibrosis. However, TGF-β1 has also been reported to decrease expression of Klotho protein. In this report, we provide an interpretation of our new treatment strategy which involves controlling histone methylation.Scientific Aspects of Dialysis Therapy: JSDT/ISBP Anniversary Edition.This work was supported by the Grant-in-Aid for Scientific Research (KAKENHI) No.16K09610 (to S.D.) in Hiroshima University

Inductive asymmetric cell division: The WRM leads the way

C. elegans, with its invariant cell lineage, provides a powerful model system in which to study signaling-dependent asymmetric cell division. The C. elegans β-catenin-related protein, WRM-1, specifies endoderm at the 4-cell stage during the first cell signaling-induced asymmetric cell division of embryogenesis. During this interaction, Wnt signaling and the cell cycle regulator CDK-1 act together to induce the asymmetric cortical release of WRM-1 at prophase of the EMS cell cycle. Genetic studies suggest that release of WRM-1 unmasks a cortical site that drives EMS spindle rotation onto the polarized axis of the cell, simultaneously making WRM-1 available for nuclear translocation, and downstream signaling to specify endoderm. These studies suggest a general paradigm for how cortical factors like WRM-1 can function at the cell cortex to mask potentially confounding polarity cues, and when released with appropriate cell cycle timing, can also function downstream to define cell fate

Decomposition and ergosterol content of the moss Hylocomium splendens litter under various climatic conditions

We examined the differences in the decomposition rate and fungal biomass in the litter of Hylocomium splendens among forests under different climatic conditions. The samples were collected from one boreal forest in Canada, three subalpine forests on Mt. Fuji and one cool temperate forest on Mt. Tsurugi, Shikoku in Japan. The decomposition rate in the cool temperate forest was much faster than those in the boreal and subalpine forests. Ergosterol, which is a component of fungal cell membranes, was used as an indicator of fungal biomass. Ergosterol was detected not only from brown moss litter but also from green shoots of the moss. In spite of the faster decomposition rate, ergosterol content of the moss litter of the cool temperate forest was about one half of those of the boreal and subalpine forests. The results suggest that the relationship between fungal biomass and decomposition rate differs significantly among forest types

Cytoskeletal inhibitors, anti-adhesion molecule antibodies, and lectins inhibit hepatocyte spheroid formation.

We investigated the role of cytoskeletons, adhesion molecules, membrane-glycosylations, and proteoglycans in forming the shape of adult rat hepatocyte spheroids. Isolated hepatocytes were cultured on dishes coated with chondroitin sulfate phosphatidyl ethanolamine (CS-PE). Spheroid-forming ability was observed after adding cytoskeletal inhibitors (cytochalasin D, colchicine, okadaic acid, mycalolide B), anti-adhesion molecule antibodies (anti-E-cadherin, anti-connexin 32, anti-zo-1), a glycosphingolipid synthetic inhibitor (N-butyldeoxynojirimycin), a proteoglycan synthetic inhibitor (p-nitrophenyl-beta-D-xylopyranoside), and several lectins. Localization of actin was studied using confocal microscopy after rhodamine-phalloidin staining. Adding cytoskeletal inhibitors on the initial day resulted in weakly clustered cell aggregates rather than smoothly formed spheroids. These effects disappeared at lower reagent concentrations. When reagents were added on day 3, after the formation of spheroids, only mycalolide B was associated with an irregular spheroid surface; the others had no effect. Adding the anti-E-cadherin, anti-connexin 32 on the initial day showed inhibition of spheroid formation, but anti-zo-1 and proteoglycan synthetic inhibitor had no effects. Among the several lectins, only Wheat Germ Agglutinin (WGA), Ricinus communis Agglutinin I (RCA-I), and Concanavalin A (ConA) showed inhibition. These results suggest that cytoskeletal conformation and some adhesion molecules are necessary to form spheroids. Based on the interactions between lectins and hepatocytes in the present study, hepatocytes appear to contain an N-linked complex or N-linked hybrid glycosylated chains