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Nitroxyl radicals are known to electrochemically oxidize thiols as well as alcohols and amines. In this study, a preliminary investigation of the electrochemical reaction of thiols with 9-azabicyclo[3.3.1]nonane N-oxyl (ABNO), 2-azaadamantane N-oxyl (AZADO), and nortropine N-oxyl (NNO), which are highly active due to their bicyclo structures, for use in electrochemical analysis was performed and the results were compared with those for a typical nitroxyl radical compound, 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO). Mercaptopropane sulfonic acid (MPS) was used as a model compound to investigate the electrochemical response in aqueous solution. In addition, electrochemical detection of glutathione, a biological thiol molecule, was performed.

The mitochondria of adult and plerocercoid Spirometra mansoni were characterized in isolated mitochondria and in situ by electron microscopic histochemistry with special attention to the respiratory chain. Although the specific activities of the constituent enzyme complexes of succinate oxidase are fairly similar in adult and plerocercoid mitochondria, those of succinate oxidase and NADH-FRD are approximately 4- and 25-fold higher in adult mitochondria than in plerocercoid mitochondria, respectively. Quinone analysis by high performance liquid chromatography and mass spectrometry showed that adult and plerocercoid mitochondria contained both rhodoquinone-10 and ubiquinone-10 at concentrations of 4.98 and 0.106 nmol mg

Herein, it is reported that p-acetamidophenylboronic acid can be electrolytic cleavage of the carbon-boron bond to p-acetamidophenol at an electric potential of 1.2 V vs. Ag/AgCl in 100 mM phosphate buffer of pH 7.4 (containing 10% acetonirile). The electrochemical reaction was investigated by HPLC, LC with tandem mass spectrometry, and cyclic voltammetry. This electrochemical reaction could be useful in the development of electrical controlled drug delivery systems under neutral pH conditions.

A modified electrode was developed by immobilizing poly(azure A) (pAA) onto the surface of a glassy carbon electrode via the electropolymerization of azure A (AA). The pAA immobilized on the electrode exhibited redox response during cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The redox reaction obeyed the Nernst equation because of the involvement of H+ ions. In addition, the peak potential was shifted according to the solution pH. The shifts of the oxidation peak potential could be more easily observed using DPV than when using CV, indicating that the developed electrode could be useful as a pH sensor. This pH measurement method can be successfully applied in the pH range of 1 to 10 and can be successfully repeated more than 50 times.

Binding assays are widely used to study the estrogenic activity of compounds targeting the estrogen receptor (ER). The fluorescence properties of benzofurazan (BD), an environmentally sensitive fluorophore, are affected by solvent polarity. In this study, we synthesized BD-labeled estradiol (E2) derivatives hoping to develop a fluorescent ligand to be used in ER binding assays, without the separation of free- from bound-ligand. Three fluorescent ligands with a BD skeleton were obtained and their fluorescence properties were investigated. Analysis of the fluorescent ligands and human recombinant ERα (hr-ERα) interactions revealed that the fluorescence intensity increased in hydrophobic environments, such as the receptor-binding site. In saturation binding assays, ABD-E2 derivative 2c showed positive cooperative binding, and its dissociation constant (Kd) and Hill coefficient were 23.4 nM and 1.34, respectively. The estrogenic compounds affinity, assessed by competitive binding assays was well correlated with the results obtained by conventional studies, using the fluorescence polarization method. Overall, the developed assay using BD-labeled ligands was a simple, rapid, and reliable method for the evaluation of ER binding affinity.

Purpose Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. Methods We investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. Results Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. Conclusion These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Dynactin is a principal regulator of the minus-end directed microtubule motor dynein. The sidearm of dynactin is essential for binding to microtubules and regulation of dynein activity. Although our understanding of the structure of the dynactin backbone (Arp1 rod) has greatly improved recently, structural details of the sidearm subcomplex remain elusive. Here, we report the flexible nature and diverse conformations of dynactin sidearm observed by electron microscopy. Using nanogold labeling and deletion mutant analysis, we determined the domain organization of the largest subunit p150 and discovered that its coiled-coil (CC1), dynein-binding domain, adopted either a folded or an extended form. Furthermore, the entire sidearm exhibited several characteristic forms, and the equilibrium among them depended on salt concentrations. These conformational diversities of the dynactin complex provide clues to understanding how it binds to microtubules and regulates dynein.

Autophagic dysfunction is associated with the progression of various liver diseases, including nonalcoholic fatty liver disease (NAFLD). However, serum markers for evaluating autophagic function have not been reported. Highly insoluble nuclear proteins participate in many cellular functions and are potential diagnostic markers for cancer. We performed a proteomic analysis of the hepatic nuclear insoluble fraction to identify novel autophagy-related diagnostic biomarkers.The insoluble nuclear protein fraction was extracted from the livers of Atg7The levels of insoluble nuclear proteins 14-3-3ζ and importin α4 were upregulated following hepatic autophagy dysfunction and were detectable in serum. Under normal conditions, these proteins are mainly distributed in the cytoplasm, whereas autophagic dysfunction induces their translocation to the nucleus. Incubation with an autophagy inhibitor up-regulated these proteins expression in the insoluble nuclear fraction of primary hepatocytes. Treatment with EGF or insulin enhanced 14-3-3ζ expression in the nuclear insoluble fraction; in contrast, the addition of rapamycin downregulated 14-3-3ζ expression. Importin α4 expression was increased in the nuclear insoluble fraction after incubation with tunicamycin or hydrogen peroxide.Accumulation of 14-3-3ζ and importin α4 as nuclear-insoluble proteins may be associated with autophagic dysfunction. Our findings indicate that these proteins might be useful diagnostic biomarkers for liver diseases with autophagic disorders.

Herein, it is reported that p-acetamidophenylboronic acid can be electrolytic cleavage of the carbon-boron bond to p-acetamidophenol at an electric potential of 1.2 V vs. Ag/AgCl in 100 mM phosphate buffer of pH 7.4 (containing 10% acetonirile). The electrochemical reaction was investigated by HPLC, LC with tandem mass spectrometry, and cyclic voltammetry. This electrochemical reaction could be useful in the development of electrical controlled drug delivery systems under neutral pH conditions.

Nitroxyl radicals, such as 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO), can catalyze the electrochemical oxidation of alcohols and amines. Because the oxidation current obtained in this process depends on the concentration of alcohols and amines, this process can be applied to their sensing. However, the relatively high oxidation potentials required by nitroxyl radicals can induce interfering oxidation currents from various reductive substances in biological samples, which affects the accuracy of analyte measurements. In this study, we examined the electrooxidation of alcohols and amines at a low potential by applying cooperative oxidation catalysis using a nitroxyl radical and a copper salt. Nortropine N-oxyl (NNO), which showed higher catalytic activity than TEMPO was used as the nitroxyl radical. An increase in the oxidation current was observed at the low potential, and this increase depended on the alcohol concentration. In the case of the electrooxidation of amines, a positive correlation between oxidation current and amine concentration was observed at low amine concentrations. Therefore, low-potential cooperative catalysis can be applied to alcohol and amine electrooxidation for the development of accurate sensors suitable for clinical settings.

Urothelial carcinoma of the bladder (UCB) is potentially life-threatening; therefore, we aimed to discover a novel urine biomarker for diagnosis and prognostication of UCB. This is a retrospective case-control study. Exploration of a new biomarker using urine from 20 UCB patients in the present study revealed that urinary level of lactoferrin (LF), a multifunctional glycoprotein released from neutrophils, was higher in 11 of 15 with invasive/high-grade UCB than 5 with non-invasive one, and 2 healthy adults. We therefore focused on LF and assessed the value of urine LF normalized by urine creatinine concentration (LF/Cr) using an enzyme-linked immunosorbent assay. Diagnostic performance of urine LF/Cr was examined using urine from 92 patients with primary (newly diagnosed) untreated UCB and 166 controls without UCB, including 62 patients with pyuria, and 104 subjects without pyuria consisting of 84 patients and 20 healthy adults. However, the diagnostic accuracies were accompanied by the risk of bias. In 92 primary UCB patients, both pyuria and tumor-infiltrating neutrophils (TINs) were independent predictors for urine LF/Cr. In contrast, TINs or urine LF/Cr were independent predictors for invasive histology, whereas pyuria was not. In terms of prognostication, urine LF/Cr and nodal metastasis were independent predictors of disease-specific survival in 22 patients with muscle-invasive bladder cancer, characterized by a high mortality rate, in the Cox proportional hazards model. In conclusion, urine LF/Cr linked to TINs was a predictor of both invasive histology and prognosis in UCB. Urine LF/Cr is a potential biomarker reflecting the degree of malignancy in UCB., 論文

Quantifying drug concentrations in vivo quickly and easily is possible using electrochemical methods. The present study describes the electrochemical detection of vancomycin (VCM) and other antibiotics from the current obtained using nitroxyl radicals as electrocatalysts. Nortropine N-oxyl (NNO), which is more active than 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), a typical nitroxyl radical compound, produced greater current values for drugs with intramolecular hydroxy groups and secondary and tertiary amines. However, because the catalytic action of NNO is inactivated by primary amines in the substrate, VCM and teicoplanin with primary amines could not be detected. TEMPO was less active than NNO but not inactivated against primary amines. Therefore, electrochemical sensing of vancomycin was done using 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl (A-TEMPO), which has a greater oxidation capacity than TEMPO due to its electron-withdrawing groups. As a result, the current of A-TEMPO increased in the low concentration range of VCM as compared to TEMPO. This method also was able to quantify VCM in the concentration range of 10–100 μM, which is an important concentration range for drug monitoring in blood.

Oxidative stress, an inducer of apoptosis, plays a critical role in ischemia/reperfusion injury and atherosclerosis. We previously identified an apoptosis-inducing ligand, the post-translationally modified secreted form of eukaryotic translation initiation factor 5A (eIF5A), ‘oxidative stress-responsive apoptosis-inducing protein’ (ORAIP). In this study, we investigated the role of ORAIP in patients with heterozygous familial hypercholesterolemia (HeFH), a leading cause of premature cardiovascular disease. We analyzed plasma ORAIP and oxidized low-density lipoprotein (oxLDL) levels in 60 patients with HeFH (60% male, 57.0 ± 13.6 years of age) and 20 patients with LDL-C hypercholesterolemia (DL, 85% male, 64.1 ± 13.3 years of age). The coronary artery atherosclerosis from the patients with HeFH who had a coronary artery bypass graft was investigated by double immunostaining. The plasma ORAIP levels in the patients with HeFH were significantly elevated compared to those in the patients with DL (73.5 ± 46.0 vs. 48.3 ± 21.4 ng/mL, p = 0.0277). The plasma oxLDL levels in HeFH patients were also elevated (156.8 ± 65.2 vs. 123.7 ± 46.6 mg/dL, p = 0.0461) compared to those in DL patients and correlated with maxLDL-C levels (R = 0.4454, p = 0.00648). Double-immunostaining of ORAIP and oxLDL in the coronary artery from patients with HeFH who had a coronary artery bypass graft showed that ORAIP and oxLDL were colocalized with apoptotic vascular smooth muscle cells in the atherosclerotic plaque. ORAIP plays a role in the development of oxidative stress-induced atherosclerosis and may be an important therapeutic target for plaque rupture in patients with HeFH.

In this paper we propose a novel measurement for NO and ·OH by electrochemical detection using sesamol. Standard samples of the sesamol monomer and dimer were subjected to differential pulse voltammetry, resulting in their peaks being clearly separated and detected. Based on the oxidative dimerization of sesamol, the current simple, sensitive and selective method was successfully applied to preliminary measurements for NO and ·OH, respectively.

Nitroxyl radical catalysts oxidize alcohols under an applied electric potential. It is possible to quantify the alcohol concentration from the resulting oxidation current. In this work, we evaluated the catalytic activity of nitroxyl radicals (or their corresponding hydroxylamines), including 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) as representative nitroxyl radicals, acetoamido-TEMPO, which shows higher oxidation potential than TEMPO owing to the acetoamido group, AZADO, Nor-AZADO, and NNO as less-hindered bicyclic nitroxyl radicals, and NHPI as an N,N-diacyl-type hydroxylamine, in acetonitrile solution. TEMPO, AZADO and NNO were also evaluated for their ability to oxidize alcohols in organic solvents, and their reactivity was compared with the electrochemical response. The most active NNO was used for electrochemical detection of cyclosporin A, a drug with a hydroxyl group.

Phenylboronic acid-bearing polyamidoamine dendrimer (PBA-PAMAM)/poly(vinyl alcohol) (PVA) multilayer films were prepared through the layer-by-layer (LbL) deposition of PBA-PAMAM solution and PVA solution. PBA-PAMAM/PVA films were constructed successfully through the formation of boronate ester bonds between the boronic acid moiety in PBA and 1,3-diol units in PVA. When the (PBA-PAMAM/PVA)5 films were immersed in rose bengal (RB) solution, RB was adsorbed onto the LbL films. The amount of RB adsorbed was higher in the LbL films immersed in acidic solution than in basic solution. The release of RB from the LbL films was also promoted in the basic solution, while it was suppressed in the acidic solution. The boronic acid ester is oxidized to phenol by hydrogen peroxide (H2O2) and the carbon-boron bond is cleaved, so that the (PBA-PAMAM/PVA)5 films can be decomposed by immersion in H2O2 solution. Therefore, when RB-adsorbed (PBA-PAMAM/PVA)5 films were immersed in H2O2 solution, the release of RB was moderately promoted when the solution was weakly acidic.

Background Specific species of ceramides (Cer), major constituents of lipids in the stratum corneum (SC), are decreased and are correlated with SC barrier and water-holding functions in the skin of patients with atopic dermatitis (AD) or psoriasis (Pso). However, possible correlations between Cer subclass ratios and skin properties in barrier-disrupted skin and in healthy skin remain unclear. The objective of this study was to identify a new marker to evaluate skin properties and epidermal differentiation in SC not only in barrier-disrupted skin but also in healthy skin. Methods The Cer subclass ratios in the SC of healthy control subjects and in patients with AD or Pso were evaluated. Correlations with candidate markers and facial skin features of healthy Japanese females (20–74 years old, n = 210) were investigated. Variations of markers during epidermal differentiation were studied in human epidermis and in cultured keratinocytes. Results The ratios of Cer [NP]/[NS], Cer [NH]/[NS], Cer [NP]/[AS], Cer [NH]/[NS], Cer [NDS]/[AS], Cer [AH]/[AS] and Cer [EOP]/[AS] showed significant differences between non-lesional skin of AD patients and normal skin of healthy control subjects, as well as Pso patients and their healthy control subjects. The Cer [NP]/[NS] ratio was correlated with SC functional parameters (transepidermal water loss and capacitance) and with skin appearance (texture, scaling and color) even in the cheek skin of healthy female subjects. The Cer [NP]/[NS] ratio in the SC was approximately 18-times higher than in living keratinocytes, and it increased as they differentiated. Conclusions The Cer [NP]/[NS] ratio in the SC is a potential marker for skin properties and epidermal differentiation in barrier-disrupted skin as well as in healthy skin.

BACKGROUND Prostate-specific antigen (PSA) has been the most popular diagnostic marker for prostate cancer. The frequent occurrence of low PSA values (

Purpose Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. Methods We investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. Results Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. Conclusion These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Background Skin occlusion caused by the use of diapers or sanitary napkins often results in irritant contact dermatitis. Furthermore, prolonged occlusion and exposure to body fluids are known to increase skin hydration and permeability, thus leading to irritant contact dermatitis. Objective To investigate the effects of water exposure on the skin and its barrier functions, in order to obtain more insights into the mechanisms of irritant contact dermatitis. Methods Water patches were applied to the volar forearm skin of 10 human subjects for 3 hours. Permeability of the stratum corneum (SC) was examined with methyl nicotinate (MN). Alterations in the hydration and ultrastructure of the SC were measured with Raman spectroscopy and multiphoton microscopy, respectively. Results Water profiles found with Raman spectroscopy showed notable increases in water content throughout the SC and skin surface. Multiphoton microscopy showed morphological changes in the intercellular space of the SC. Emerged pools seemed to contribute to increased MN absorption. Conclusion Excessive skin hydration leading to changes in the SC ultrastructure might result in increased skin permeability to skin irritants and allergens.

The yeast L-A double-stranded RNA virus synthesizes capped transcripts by a unique cap-snatching mechanism in which the m7 Gp moiety of host mRNA (donor) is transferred to the diphosphorylated 5' end of the viral transcript (acceptor). This reaction is activated by viral transcription. Here, we show that cap snatching can be reversible. Because only m7 Gp is transferred during the reaction, the resulting decapped donor, as expected, retained diphosphates at the 5' end. We also found that the 5' terminal nucleotide of the acceptor needs to be G but not A. Interestingly, the A-initiated molecule when equipped with a cap structure (m7 GpppA…) could work as cap donor. Because the majority of host mRNAs in yeast have A after the cap structures at the 5' ends, this finding implies that cap-snatching in vivo is virtually a one-way reaction, in favor of furnishing the viral transcript with a cap. The cap-snatching sites are located on the coat protein Gag and also the Gag domain of Gag-Pol. Here, we demonstrate that both sites are functional, indicating that activation of cap snatching by transcription is not transmitted through the peptide bonding between the Gag and Pol domains of Gag-Pol.

Nonalcoholic fatty liver disease (NAFLD) plays a crucial role in type 2 diabetes and hepatocellular carcinoma. The major underlying pathogenesis is hepatic insulin resistance. The aim of the present study was to characterize patients with NAFLD with paradoxically normal hepatic insulin sensitivity relative to patients with NAFLD with hepatic insulin resistance. We recruited 26 patients with NAFLD and divided them into three groups ranked by the level of hepatic insulin sensitivity (HIS; high-HIS, mid-HIS, low-HIS), as assessed by the hyperinsulinemic-euglycemic clamp studies using stable isotope. Hepatic insulin sensitivity of the high-HIS group was identical to that of the non-NAFLD lean control (clamped percent suppression of endogenous glucose production, 91.1% ± 5.2% versus 91.0% ± 8.5%, respectively) and was significantly higher than that of the low-HIS group (66.6% ± 7.5%; P < 0.01). Adiposity (subcutaneous, visceral, intrahepatic, and muscular lipid content), hepatic histopathology, and expression levels of various genes by using liver biopsies, muscle, and adipose tissue insulin sensitivity, plasma metabolites by metabolomics analysis, putative biomarkers, and lifestyles were assessed and compared between the high-HIS and low-HIS groups. Among these, adipose tissue insulin sensitivity assessed by clamped percent suppression of free fatty acid, serum high molecular weight adiponectin, and plasma tricarboxylic acid cycle metabolites, such as citric acid and cis-aconitic acid, were significantly higher in the high-HIS group compared to the low-HIS group. In contrast, there were no differences in adiposity, including intrahepatic lipid content assessed by proton magnetic resonance spectroscopy (28.3% ± 16.1% versus 20.4% ± 9.9%, respectively), hepatic histopathology, other putative biomarkers, and lifestyles. Conclusion: High levels of adipose tissue insulin sensitivity, serum high molecular weight adiponectin, and plasma tricarboxylic acid cycle metabolites are unique characteristics that define patients with hepatic insulin-sensitive NAFLD regardless of intrahepatic lipid content. (Hepatology Communications 2017;1:634-647).

Histamine is a biogenic amine that is chiefly produced in mast cells and basophils and elicits an allergic response upon stimulation. Histidine decarboxylase (HDC) is a unique enzyme that catalyzes the synthesis of histamine. Therefore, the spatiotemporally specific Hdc gene expression profile could represent the localization of histamine-producing cells under various pathophysiological conditions. Although the bioactivity of histamine is well defined, the regulatory mechanism of Hdc gene expression and the distribution of histamine-producing cell populations in various disease contexts remains unexplored. To address these issues, we generated a histidine decarboxylase BAC (bacterial artificial chromosome) DNA-directed GFP reporter transgenic mouse employing a 293-kb BAC clone containing the entire Hdc gene locus and extended flanking sequences (Hdc-GFP). We found that the GFP expression pattern in the Hdc-GFP mice faithfully recapitulated that of conventional histamine-producing cells and that the GFP expression level mirrored the increased Hdc expression in lipopolysaccharide (LPS)-induced septic lungs. Notably, a CD11b+Ly6G+Ly6Clow myeloid cell population accumulated in the lung during sepsis, and most of these cells expressed high levels of GFP and indeed contain histamine. This study reveals the accumulation of a histamine-producing myeloid cell population during sepsis, which likely participates in the immune process of sepsis.

Oxidative stress is known to play a critical role in the pathogenesis of various disorders, especially in ischemia/reperfusion (I/R) injury. We identified an apoptosis-inducing humoral factor and named this novel post translationally modified secreted form of eukaryotic translation initiation factor 5A (eIF5A) “oxidative stress-responsive apoptosis inducing protein” (ORAIP). The purpose of this study was to investigate the role of ORAIP in the mechanisms of cerebral I/R injury. Hypoxia/reoxygenation induced expression of ORAIP in cultured rat cerebral neurons, resulting in extensive apoptosis of these cells, which was largely suppressed by neutralizing anti-ORAIP monoclonal antibody (mAb) in vitro. Recombinant-ORAIP induced extensive apoptosis of cerebral neurons. Cerebral I/R induced expression of ORAIP in many neurons in a rat tandem occlusion model in vivo. In addition, we analyzed the effects of intracerebroventricular administration of neutralizing anti-ORAIP mAb on the development of cerebral infarction. Cerebral I/R significantly increased ORAIP levels in cerebrospinal fluid. Treatment with intracerebroventricular administration of neutralizing anti-ORAIP mAb reduced infarct volume by 72%, and by 55% even when started after reperfusion. These data strongly suggest that ORAIP plays a pivotal role and will offer a critical therapeutic target for cerebral I/R injury induced by thrombolysis and thrombectomy for acute ischemic stroke.

Vzb22, an amino-group carrier protein from Streptomyces, is required for biosynthesis of the noncanonical amino acid DADH, a biosynthetic precursor of vazabitide A and related azabicyclohexane natural products. Amino-group carrier proteins (AmCPs) mediate the biosynthesis of lysine and arginine in some bacteria and archaea. Here we demonstrate that an uncharacterized AmCP-mediated biosynthetic system functions to biosynthesize the previously uncharacterized and nonproteinogenic amino acid (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH) in Streptomyces sp. SANK 60404. DADH is incorporated into a novel peptide metabolite, vazabitide A, featuring an azabicyclo-ring structure, by nonribosomal peptide synthetases and successive modification enzymes in this bacterium. As the AmCP-mediated machinery for DADH biosynthesis is widely distributed in bacteria, further analysis of uncharacterized AmCP-containing gene clusters will lead to the discovery of novel bioactive compounds and novel biosynthetic enzymes.

Background/Purpose Irritancy levels of surfactants on human skin have not been clarified completely. The relationships between skin damage and changes of skin properties caused by various surfactants were investigated using non-invasive measurements. Methods Aqueous solutions of seven kinds of anionic, non-ionic, and amphoteric surfactants were exposed to the inside of forearm skin of 20 human subjects in two separate studies using the cup method. Hydration of the stratum corneum (SC), transepidermal water loss (TEWL), pH, skin surface roughness, and contents of the SC were measured before and after one exposure and after five and nine consecutive exposures to various surfactants. The discontinuation ratio of subjects for testing in each surfactant was determined by skin irritation symptoms and was defined as the degree of skin damage. Results Significant changes were observed only in hydration, TEWL, and natural moisturizing factors (NMF) content in the SC following surfactant exposure. A significant correlation was observed between the discontinuation ratio of each surfactant and the changes of hydration, TEWL, and NMF. Especially, the change of SC hydration showed an excellent correlation with the discontinuation ratio both for single (r = 0.942, P < 0.001) and for chronic exposures (r = 0.934, P < 0.001). Conclusion Our results indicate that the change of hydration of the SC is equivalent to the skin damage caused by surfactants, and therefore is the most suitable indicator to evaluate the irritation of surfactants on the skin.

The 5' end of RNA conveys important information on self-identity. In mammalian cells, double-stranded RNA (dsRNA) with 5' di- or triphosphates generated during virus infection is recognized as foreign and elicits the host innate immune response. Here we analyze the 5' ends of the dsRNA genome of the yeast L-A virus. The positive strand has largely diphosphates with a minor amount of triphosphates, while the negative strand has only diphosphates. Although the virus can produce capped transcripts by cap snatching, neither strand carried a cap structure, suggesting that only non-capped transcripts serve as genomic RNA for encapsidation. We also found that the 5' diphosphates of the positive but not the negative strand within the dsRNA genome are crucial for transcription in vitro. Furthermore, the presence of a cap structure in the dsRNA abrogated its template activity. Given that the 5' diphosphates of the transcripts are also essential for cap acquisition and that host cytosolic RNAs (mRNA, rRNA and tRNA) are uniformly devoid of 5' pp-structures, the L-A virus takes advantage of its 5' terminal diphosphates, using them as a self-identity tag to propagate in the host cytoplasm. This article is protected by copyright. All rights reserved.

We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respectively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b5, an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b5 and the canonical cytochrome b5 proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cytochrome b5-like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b5 in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b5, which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b5 precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b5 and highlight the crucial function of this A. suum adult-specific secretory cytochrome b5 in parasitic adaptation.

Ethambutol is a common medicine used for the treatment of tuberculosis, which can have serious side effects, such as retinal and liver dysfunction. Although ethambutol has been reported to impair autophagic flux in rat retinal cells, the precise molecular mechanism remains unclear. Using various mammalian cell lines, we showed that ethambutol accumulated in autophagosomes and vacuolated lysosomes, with marked Zn2+ accumulation. The enlarged lysosomes were neutralized and were infiltrated with Zn2+ accumulations in the lysosomes, with simultaneous loss of acidification. These results suggest that EB neutralizes lysosomes leading to insufficient autophagy, implying that some of the adverse effects associated with EB in various organs may be of this mechanism.

pH is one of the most important properties associated with an aqueous solution and various pH measurement techniques are available. In this study, Azure A-modified poly(methacrylic acid) (AA-PMA) was synthesized used to prepare a layer-by-layer deposited film with poly(allylamine hydrochloride) (PAH) on a glassy carbon electrode via electrostatic interactions and the multilayer film-immobilized electrode was used to measure pH. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurement were performed. Consequently, the oxidation potential of AA on the electrode changed with pH. As per Nernst&rsquo, s equation, because H+ ions are involved in the redox reaction, the peak potential shifted depending on the pH of the solution. The peak potential shifts are easier to detect by DPV than CV measurement. Accordingly, using electrochemical responses, the pH was successfully measured in the pH range of 3 to 9, and the electrodes were usable for 50 repeated measurements. Moreover, these electrochemical responses were not affected by interfering substances.