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

Interconversion of Two GDP-Bound Conformations and Their Selection in an Arf-Family Small G Protein

SummaryADP-ribosylation factor (Arf) and other Arf-family small G proteins participate in many cellular functions via their characteristic GTP/GDP conformational cycles, during which a nucleotide∗Mg2+-binding site communicates with a remote N-terminal helix. However, the conformational interplay between the nucleotides, the helix, the protein core, and Mg2+ has not been fully delineated. Herein, we report a study of the dynamics of an Arf-family protein, Arl8, under various conditions by means of NMR relaxation spectroscopy. The data indicated that, when GDP is bound, the protein core, which does not include the N-terminal helix, reversibly transition between an Arf-family GDP form and another conformation that resembles the Arf-family GTP form. Additionally, we found that the N-terminal helix and Mg2+, respectively, stabilize the aforementioned former and latter conformations in a population-shift manner. Given the dynamics of the conformational changes, we can describe the Arl8 GTP/GDP cycle in terms of an energy diagram

A new perspective on phylogeny and evolution of tetraodontiform fishes (Pisces: Acanthopterygii) based on whole mitochondrial genome sequences: Basal ecological diversification?

<p>Abstract</p> <p>Background</p> <p>The order Tetraodontiformes consists of approximately 429 species of fishes in nine families. Members of the order exhibit striking morphological diversity and radiated into various habitats such as freshwater, brackish and coastal waters, open seas, and deep waters along continental shelves and slopes. Despite extensive studies based on both morphology and molecules, there has been no clear resolution except for monophyly of each family and sister-group relationships of Diodontidae + Tetraodontidae and Balistidae + Monacanthidae. To address phylogenetic questions of tetraodontiform fishes, we used whole mitochondrial genome (mitogenome) sequences from 27 selected species (data for 11 species were newly determined during this study) that fully represent all families and subfamilies of Tetraodontiformes (except for Hollardinae of the Triacanthodidae). Partitioned maximum likelihood (ML) and Bayesian analyses were performed on two data sets comprising concatenated nucleotide sequences from 13 protein-coding genes (all positions included; third codon positions converted into purine [R] and pyrimidine [Y]), 22 transfer RNA and two ribosomal RNA genes (total positions = 15,084).</p> <p>Results</p> <p>The resultant tree topologies from the two data sets were congruent, with many internal branches showing high support values. The mitogenomic data strongly supported monophyly of all families and subfamilies (except the Tetraodontinae) and sister-group relationships of Balistidae + Monacanthidae and Tetraodontidae + Diodontidae, confirming the results of previous studies. However, we also found two unexpected basal splits into Tetraodontoidei (Triacanthidae + Balistidae + Monacanthidae + Tetraodontidae + Diodontidae + Molidae) and Triacanthodoidei (Ostraciidae + Triodontidae + Triacanthodidae).</p> <p>Conclusion</p> <p>This basal split into the two clades has never been reported and challenges previously proposed hypotheses based on both morphology and nuclear gene sequences. It is likely that the basal split had involved ecological diversification, because most members of Tetraodontoidei exclusively occur in shallow waters (freshwater, brackish and coastal waters, and open seas), while those of Triacanthodoidei occur mainly in relatively deep waters along continental shelves and slopes except for more derived ostraciids. This suggests that the basal split between the two clades led to subsequent radiation into the two different habitats.</p

Development of two types of mite-allergen induced murine models of chronic asthma with different severity

Asthma is an allergic disease characterized by chronic airway inflammation, hyper-responsiveness (AHR), and reversible obstruction. The main inflammatory changes are induced by infiltration of eosinophils into the airway. Few animal models resemble the spontaneous history of asthma due to variations in the selection of the mouse strain, appropriate antigen, and exposure methods. Here, we prepared two different mouse models in which the mechanism was close to that of human asthma. We transnasally administered mite Dermatophagoides farinae (Df) allergen to BALB/c mice 10 times (Df-2) or 25 times (Df-5). After comparison with mice administered phosphate-buffered saline, the AHR and immediate asthmatic response were evaluated, in addition to the number of eosinophils in the bronchoalveolar lavage fluid (BALF). Df-specific IgE and IgG1 levels in the serum, and Th2 cytokines (interleukin [IL]-5, IL-13) in the BALF were measured by enzymelinked immunosorbent assay. Immediate asthmatic response and AHR were enhanced in mite allergen-treated mice (Df-2 and Df-5) compared to PBS-treated mice. The number of eosinophils and IL-13 levels in the BALF, and specific IgE in the serum were greater in Df-5 than in Df-2 mice. We established two different murine chronic asthma models, in which the severity depended on the number of exposures to Df. Greater intranasal exposure to a Df allergen resulted in more severe asthma in a BALB/c mouse model

Sequence divergence and retrotransposon insertion underlie interspecific epigenetic differences in primates

内在性レトロウイルス配列によってヒトのエピゲノムが変化してきたことを発見！ --ヒトとチンパンジーのiPS細胞の比較解析から--. 京都大学プレスリリース. 2022-10-12.Changes in the epigenome can affect the phenotype without the presence of changes in the genomic sequence. Given the high identity of the human and chimpanzee genome sequences, a substantial portion of their phenotypic divergence likely arises from epigenomic differences between the two species. In this study, the transcriptome and epigenome were determined for induced pluripotent stem cells (iPSCs) generated from human and chimpanzee individuals. The transcriptome and epigenomes for trimethylated histone H3 at lysine-4 (H3K4me3) and lysine-27 (H3K27me3) showed high levels of similarity between the two species. However, there were some differences in histone modifications. Although such regions, in general, did not show significant enrichment of interspecies nucleotide variations, gains in binding motifs for pluripotency-related transcription factors, especially POU5F1 and SOX2, were frequently found in species-specific H3K4me3 regions. We also revealed that species-specific insertions of retrotransposons, including the LTR5_Hs subfamily in human and a newly identified LTR5_Pt subfamily in chimpanzee, created species-specific H3K4me3 regions associated with increased expression of nearby genes. Human iPSCs have more species-specific H3K27me3 regions, resulting in more abundant bivalent domains. Only a limited number of these species-specific H3K4me3 and H3K27me3 regions overlap with species-biased enhancers in cranial neural crest cells, suggesting that differences in the epigenetic state of developmental enhancers appear late in development. Therefore, iPSCs serve as a suitable starting material for studying evolutionary changes in epigenome dynamics during development

Establishment of an asthma model by sensitization with mite antigen alone in C57BL/6J mice

Bronchial asthma is characterized by the bronchial hyperresponsiveness and airway obstruction related to airway smooth muscle contraction. Eosinophilic airway inflammation is involved in its pathogenesis. To reproduce the condition, various animal models have been prepared. However, there are many models that do not reflect the spontaneous history of bronchial asthma onset in humans due to the mouse strain, sensitizing antigen, or administration method. In this study, we prepared a mouse model of which the mechanism is similar to that of human bronchial asthma. Mite Extract-Dermatophagoides farinae (Derf) antigen was transnasally administered to wild-type C57BL/6J mice (WT) 13 times. Subsequently, an airway hypersensitivity test (Mch PC_200), specific antigen exposure test (ΔSRaw), bronchoalveolar lavage (BAL), and blood collection were performed to examine the presence or absence of asthma acquisition and differences in the local pulmonary levels of cytokines/chemokines in comparison with the physiological saline-treated group. In the mite antigen-treated mice (WT/-Derf), bronchial hyperresponsiveness was enhanced, antigen-specific was increased airway resistance in comparison with physiological salinetreated mice (WT/-Saline). In addition, the number of eosinophils in BAL fluid (BALF) was greater. Furthermore, there was a correlation among leukotrienes, eotaxin, and tissue inhibitors of metalloproteinase 1 in BALF, suggesting that the mechanism concerning eosinophilic airway inflammation involving in human bronchial asthma was reproduced. In this study, we successfully established a mouse bronchial asthma model in which the pathogenesis resembles that in humans in comparison with conventional models, using Derf antigen alone and C57BL/6J mice

Destruxin E Decreases Beta-Amyloid Generation by Reducing Colocalization of Beta-Amyloid-Cleaving Enzyme 1 and Beta-Amyloid Protein Precursor

Alzheimer-disease-associated beta-amyloid (A beta) is produced by sequential endoproteolysis of beta-amyloid protein precursor (beta APP): the extracellular portion is shed by cleavage in the juxtamembrane region by beta-amyloid-cleaving enzyme (BACE)/beta-secretase, after which it is cleaved by presenilin (PS)/gamma-secretase near the middle of the transmembrane domain. Thus, inhibition of either of the secretases reduces A beta generation and is a fundamental strategy for the development of drugs to prevent Alzheimer disease. However, it is not clear how small compounds reduce A beta production without inhibition of the secretases. Such compounds are expected to avoid some of the side effects of secretase inhibitors. Here, we report that destruxin E (Dx-E), a natural cyclic hexadepsipeptide, reduces A beta generation without affecting BACE or PS/gamma-secretase activity. In agreement with this, Dx-E did not inhibit Notch signaling. We found that Dx-E decreases colocalization of BACE1 and beta APP, which reduces beta-cleavage of beta APP. Therefore, the data demonstrate that Dx-E represents a novel A beta-reducing process which could have fewer side effects than secretase inhibitors. Copyright (C) 2009 S. Karger AG, Base