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Objective Synovial fibroblasts (SFs) in rheumatoid arthritis (RA) and osteoarthritis (OA) play biphasic roles in joint destruction and regeneration of bone/cartilage as mesenchymal stem cells (MSCs). Although MSCs contribute to joint homeostasis, such function is impaired in arthritic joints. We have identified functionally distinct three SF subsets characterized by the expression of CD34 and THY1 as follows: CD34+THY1+, CD34−THY1−, and CD34−THY1+. The objective of this study was to clarify the differentiation potentials as MSCs in each SF subset since both molecules would be associated with the MSC function. Methods SF subsets were isolated from synovial tissues of 70 patients (RA: 18, OA: 52). Expressions of surface markers associated with MSCs (THY1, CD34, CD73, CD271, CD54, CD44, and CD29) were evaluated in fleshly isolated SF subsets by flow cytometry. The differentiation potentials of osteogenesis, chondrogenesis, and adipogenesis were evaluated with histological staining and a quantitative polymerase chain reaction of differentiation marker genes. Small interfering RNA was examined to deplete THY1 in SFs. Results The expression levels of THY1+, CD73+, and CD271+ were highest and those of CD54+ and CD29+ were lowest in CD34+THY1+ among three subsets. Comparing three subsets, the calcified area, alkaline phosphatase (ALP)-stained area, and cartilage matrix subset were the largest in the CD34+THY1+ subset. Consistently, the expressions of differentiation markers of the osteoblasts (RUNX2, ALPL, and OCN) or chondrocytes (ACAN) were the highest in the CD34+THY1+ subset, indicating that the CD34+THY1+ subset possessed the highest osteogenic and chondrogenic potential among three subsets, while the differentiation potentials to adipocytes were comparable among the subsets regarding lipid droplet formations and the expression of LPL and PPARγ. The knockdown of THY1 in bulk SFs resulted in impaired osteoblast differentiation indicating some functional aspects in this stem-cell marker. Conclusion The CD34+THY1+ SF subset has high osteogenic and chondrogenic potentials. The preferential enhancement of MSC functions in the CD34+THY1+ subset may provide a new treatment strategy for regenerating damaged bone/cartilage in arthritic joints.

Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors-such as aging, pregnancy, and genetic polymorphisms-influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure-activity relationship studies, 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.

In the face of emerging infectious diseases, there remains an unmet need for vaccine development where adjuvants that enhance immune responses to pathogenic antigens are highly desired. Using high-throughput screens with a cell-based nuclear factor κB (NF-κB) reporter assay, we identified a sulfamoyl benzamidothiazole bearing compound 1 that demonstrated a sustained activation of NF-κB after a primary stimulus with a Toll-like receptor (TLR)-4 agonist, lipopolysaccharide (LPS). Here, we explore systematic structure-activity relationship (SAR) studies on compound 1 that indicated the sites on the scaffold that tolerated modification and yielded more potent compounds compared to 1. The selected analogs enhanced release of immunostimulatory cytokines in the human monocytic cell line THP-1 cells and murine primary dendritic cells. In murine vaccination studies, select compounds were used as co-adjuvants in combination with the Food and Drug Administration approved TLR-4 agonistic adjuvant, monophosphoryl lipid A (MPLA) that showed significant enhancement in antigen-specific antibody titers compared to MPLA alone. Additionally, our SAR studies led to identification of a photoaffinity probe which will aid the target identification and mechanism of action studies in the future.

As viruses continue to mutate the need for rapid high titer neutralizing antibody responses has been highlighted. To meet these emerging threats, agents that enhance vaccine adjuvant activity are needed that are safe with minimal local or systemic side effects. To respond to this demand, we sought small molecules that would sustain and improve the protective effect of a currently approved adjuvant, monophosphoryl lipid A (MPLA), a Toll-like receptor 4 (TLR4) agonist. A lead molecule from a high-throughput screen, (N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide, was identified as a hit compound that sustained NF-κB activation by a TLR4 ligand, lipopolysaccharide (LPS), after an extended incubation (16 h). In vitro, the resynthesized compound (2D216) enhanced TLR4 ligand-induced innate immune activation and antigen presenting function in primary murine bone marrow-derived dendritic cells without direct activation of T cells. In vivo murine vaccination studies demonstrated that compound 2D216 acted as a potent co-adjuvant when used in combination with MPLA that enhanced antigen-specific IgG equivalent to that of AS01B. The combination adjuvant MPLA/2D216 produced Th1 dominant immune responses and importantly protected mice from lethal influenza virus challenge. 2D216 alone or 2D216/MPLA demonstrated minimal local reactogenicity and no systemic inflammatory response. In summary, 2D216 augmented the beneficial protective immune responses of MPLA as a co-adjuvant and showed an excellent safety profile.

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z′ factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.

Autophagy regulates fatty acid and triglyceride synthesis at the transcriptional level by fine-tuning the levels of NCoR1, a negative regulator of nuclear receptors. Defective autophagy impairs physiological steatosis both under fasting conditions and after hepatectomy., Lipid droplets (LDs) are dynamic organelles that store neutral lipids during times of energy excess, such as after a meal. LDs serve as an energy reservoir during fasting and have a buffering capacity that prevents lipotoxicity. Autophagy and the autophagic machinery have been proposed to play a role in LD biogenesis, but the underlying molecular mechanism remains unclear. Here, we show that when nuclear receptor co-repressor 1 (NCoR1), which inhibits the transactivation of nuclear receptors, accumulates because of autophagy suppression, LDs decrease in size and number. Ablation of ATG7, a gene essential for autophagy, suppressed the expression of gene targets of liver X receptor α, a nuclear receptor responsible for fatty acid and triglyceride synthesis in an NCoR1-dependent manner. LD accumulation in response to fasting and after hepatectomy was hampered by the suppression of autophagy. These results suggest that autophagy controls physiological hepatosteatosis by fine-tuning NCoR1 protein levels.

Lipid droplets (LDs) are dynamic organelles that store neutral lipids during times of energy excess, such as following a meal. LDs serve as an energy reservoir during fasting and have a buffering capacity that prevents lipotoxicity. Autophagy and the autophagic machinery have been proposed to play a role in LD biogenesis but the underlying molecular mechanism remains unclear. Here, we show that when nuclear receptor co-repressor 1 (NCoR1), which inhibits the transactivation of nuclear receptors, accumulates due to autophagy suppression, LD biogenesis is blocked. Ablation of ATG7, a gene essential for autophagy, suppressed the expression of gene targets of liver X receptor α (LXRα), a nuclear receptor responsible for fatty acid and triglyceride synthesis in an NCoR1-dependent manner. LD biogenesis in response to fasting and after hepatectomy was hampered by the suppression of autophagy. These results indicate that autophagy controls physiological hepatosteatosis by fine-tuning NCoR1 protein levels.

Selective autophagy ensures the removal of specific soluble proteins, protein aggregates, damaged mitochondria, and invasive bacteria from cells. Defective autophagy has been directly linked to metabolic disorders. However how selective autophagy regulates metabolism remains largely uncharacterized. Here we show that a deficiency in selective autophagy is associated with suppression of lipid oxidation. Hepatic loss of Atg7 or Atg5 significantly impairs the production of ketone bodies upon fasting, due to decreased expression of enzymes involved in β-oxidation following suppression of transactivation by PPARα. Mechanistically, nuclear receptor co-repressor 1 (NCoR1), which interacts with PPARα to suppress its transactivation, binds to the autophagosomal GABARAP family proteins and is degraded by autophagy. Consequently, loss of autophagy causes accumulation of NCoR1, suppressing PPARα activity and resulting in impaired lipid oxidation. These results suggest that autophagy contributes to PPARα activation upon fasting by promoting degradation of NCoR1 and thus regulates β-oxidation and ketone bodies production., Defective autophagy has been associated with metabolic disorders. Here Saito et al. show that autophagy promotes the selective degradation of NCoR1, a repressor of lipid metabolism regulator PPARα, in response to starvation, and thus induces the expression of enzymes involved in lipid oxidation and the production of ketone bodies.

The dynamics of a hydrogen bonding network (HBN) relating to macroscopic properties of hydrogen bonding liquids were observed as a significant relaxation process by dielectric spectroscopy measurements. In the cases of water and water rich mixtures including biological systems, a GHz frequency relaxation process appearing at around 20 GHz with the relaxation time of 8.2 ps is generally observed at 25 &deg, C. The GHz frequency process can be explained as a rate process of exchanges in hydrogen bond (HB) and the rate becomes higher with increasing HB density. In the present work, this study analyzed the GHz frequency process observed by suitable open-ended coaxial electrodes, and physical meanings of the fractal nature of water structures were clarified in various aqueous systems. Dynamic behaviors of HBN were characterized by a combination of the average relaxation time and the distribution of the relaxation time. This fractal analysis offered an available approach to both solution and dispersion systems with characterization of the aggregation or dispersion state of water molecules. In the case of polymer-water mixtures, the HBN and polymer networks penetrate each other, however, the HBN were segmented and isolated more by dispersed and aggregated particles in the case of dispersion systems. These HBN fragments were characterized by smaller values of the fractal dimension obtained from the fractal analysis. Some examples of actual usages suggest that the fractal analysis is now one of the most effective tools to understand the molecular mechanism of HBN in aqueous complex materials including biological systems.

Two major bile acids were isolated from the gallbladder bile of two hornbill species from the Bucerotidae family of the avian order Bucerotiformes Buceros bicornis (great hornbill) and Penelopides panini (Visayan tarictic hornbill). Their structures were determined to be 3α,7α,24-dihydroxy-5β-cholestan-27-oic acid and its 12α-hydroxy derivative, 3α,7α,12α,24-tetrahydroxy-5β-cholestan-27-oic acid (varanic acid, VA), both present in bile as their corresponding taurine amidates. The four diastereomers of varanic acid were synthesized and their assigned structures were confirmed by X-ray crystallographic analysis. VA and its 12-deoxy derivative were found to have a (24R,25S)-configuration. 13 additional hornbill species were also analyzed by HPLC and showed similar bile acid patterns to B. bicornis and P. panini. The previous stereochemical assignment for (24R,25S)-VA isolated from the bile of varanid lizards and the Gila monster should now be revised to the (24S,25S)-configuration.

Synovial fibroblasts play crucial roles in inflammation and joint destruction in rheumatoid arthritis (RA). How they accumulate in the RA joints remains unclear. This study was conducted to discern whether cellular influx from the outside of the joints and local proliferation are responsible for synovial fibroblast accumulation in an animal model of RA. We found that synovial fibroblasts were identified as GFP+ cells using collagen type I alpha 2 (Col1a2)-GFP transgenic reporter mice. Then, bone marrow transplantation and parabiosis techniques were utilized to study the cellular influx. Irradiated wild-type mice were transplanted with bone marrow from Col1a2-GFP mice. Col1a2-GFP and wild-type mice were conjoined for parabiosis. The transplanted mice and the parabionts were subjected to collagen antibody-induced arthritis (CAIA). We found no GFP+ cells in the hyperplastic synovial tissues from the transplanted mice with CAIA and from the wild-type parabionts with CAIA. Furthermore, normal and CAIA synovial tissues from Col1a2-GFP mice and from fluorescent ubiquitination-based cell cycle indicator (Fucci) transgenic mice, in which cells in S/G2/M phases of the cell cycle express Azami-Green, were studied for Ki67, a cellular proliferation marker, and vimentin, a fibroblast marker, expression. The percentages of Ki67+/GFP+ and Azami-Green+/vimentin+ cells in the CAIA synovial tissues were higher than those in the untreated synovial tissues (34% vs. 0.40% and 19% vs. 0.26%, respectively). These findings indicate that local fibroblast proliferation but not cellular influx is responsible for the synovial hyperplasia in CAIA. Suppression of proliferation of the local synovial fibroblasts should be a promising treatment for RA.

Three new phthalide derivatives, emefuranones A1, A2 and B (1-3); six new phthalane derivatives, emefuran A, B1, B2, C1, C2 and D (4-9); three new farnesylated phthalide derivatives, farnesylemefuranones A-C (10-12); xylarinol C (13); and emericelloxide (14), along with four known compounds (dustanin, sorbicillin, aspergillodiol and xylarinol A), were isolated from the culture extracts of Emericella sp. IFM57991. Structures of 1-14 were elucidated on the basis of spectroscopic analysis and chemical evidence. Compounds 4-7 and 13 showed moderate antibacterial activities against Bacillus subtilis.

Two novel N-acyl amidated bile acids, N-methyltaurine conjugated cholic acid and N-methyltaurine conjugated deoxycholic acid, were found to be major biliary bile acids in two species of angelfish the regal (Pygoplites diacanthus) and the blue-girdled (Pomacanthus navarchus) angelfish. The identification was based on their having MS and NMR spectra identical to those of synthetic standards. A survey of biliary bile acids of 10 additional species of angelfish found 7 with N-methyltaurine conjugation. In all 12 species, conjugated deoxycholic acid (known to be formed by bacterial 7-dehydroxylation of cholic acid) was a major bile acid. In all previous studies of biliary bile acids in fish, deoxycholic acid has been present in only trace proportions. In addition, bile acid conjugation with N-methyltaurine has not been detected previously in any known vertebrate. N-methyltaurine conjugated bile acids are resistant to bacterial deconjugation and dehydroxylation, and such resistance to bacterial enzymes should aid in the maintenance of high concentrations of bile acids during lipid digestion. Our findings suggest that these species of angelfish have a novel microbiome in their intestine containing anaerobic bacteria, and describe the presence of N-methyltaurine conjugated bile acids that are resistant to bacterial attack.

Although postoperative liver dysfunction (LD) following left ventricular assist device (LVAD) implantation is associated with high mortality, outcome is difficult to predict in patients with liver dysfunction. We aimed to clarify factors affecting recovery from LD after VAD implantation. A total of 167 patients underwent LVAD implantation, of whom 101 developed early postoperative LD, defined as maximum total bilirubin (max T-bil) greater than 5.0 mg/dl within 2 weeks. We set two different end-points, unremitting LD, and 90-day mortality. The rates of early mortality (90 days) and recovery from LD were 36 % (36/101) and 72 % (73/101), respectively. Univariate analysis showed that preoperative body weight, preoperative mechanical support, preoperative T-bil and creatinine, left ventricular diastolic dimension, right VAD (RVAD) insertion, cardiopulmonary bypass time, postoperative cardiac index, and postoperative T-bil and central venous pressure (CVP) on postoperative day (POD) 3 (non-recovered vs recovered, 12.4 ± 4.5 vs 9.5 ± 3.6 mmHg) were higher in patients with unremitting LD. Preoperative T-bil, RVAD insertion, and T-bil and CVP on POD 3 (non-survivor vs survivor, 12.4 ± 4.4 vs 9.4 ± 3.6 mmHg) were also higher in non-survivors. Multivariate analysis demonstrated that CVP on POD 3 was predictive of recovery from postoperative LD (OR 0.730, P < 0.05) and 90-day mortality (OR 0.730, P < 0.05). A key outcome factor in patients who developed early postoperative LD after LVAD implantation was postoperative liver congestion with high CVP. To overcome postoperative LD, appropriate management of postoperative CVP level is important.

Vascular endothelial function is impaired in hypercholesterolemia partly because of injury by modified LDL. In addition to modified LDL, free cholesterol (FC) is thought to play an important role in the development of endothelial dysfunction, although the precise mechanisms remain to be elucidated. The aim of this study was to clarify the mechanisms of endothelial dysfunction induced by an FC-rich environment. Loading cultured human aortic endothelial cells with FC induced the formation of vesicular structures composed of FC-rich membranes. Raft proteins such as phospho-caveolin-1 (Tyr-14) and small GTPase Rac were accumulated toward FC-rich membranes around vesicular structures. In the presence of these vesicles, angiotensin II-induced production of reactive oxygen species (ROS) was considerably enhanced. This ROS shifted endothelial NOS (eNOS) toward vesicle membranes and vesicles with a FC-rich domain trafficked toward perinuclear late endosomes/lysosomes, which resulted in the deterioration of eNOS Ser-1177 phosphorylation and NO production. Angiotensin II-induced ROS decreased the bioavailability of eNOS under the FC-enriched condition.

A number of N-acyl-L-proline derivatives were synthesized and their biological activities were investigated by using lettuce (Lactuca sativa L. cv. Sacramento) seedling test. A wide variety of these compounds promoted root growth at 25°C both under light and in darkness. Of the compounds tested, N-(2-ftuorobenzoyl)-L-proline methyl ester (4) showed the highest activity and caused a 270% increase in the root elongation compared to the control. N-(2-Naphthoyl)-L-proline methyl ester (14) promoted the root growth, while N-(1-naphthoyl)-L-proline methyl ester inhibited it. L-Proline, benzoic acid, and 2-naphthoic acid had no significant effect on lettuce seedlings. Compounds 4 and 14, and N-(2-chlorobenzoyl)-L-proline methyl ester (7) reduced the inhibitory effect of 1 ppm ABA on the root growth, while the D-isomer of 4 was less activite than compound 4. Compounds 4, 7, and 14 did not show any rescue-activity for the complete inhibition of germination that was caused by treating 10 ppm of ABA.

Bile alcohols and bile acids from gallbladder bile of the Arapaima gigas, a large South American freshwater fish, were isolated by reversed-phase high-performance liquid chromatography. The structures of the major isolated compounds were determined by electrospray-tandem mass spectrometry and nuclear magnetic resonance using (1)H- and (13)C-NMR spectra. The novel bile salts identified were six variants of 2-hydroxy bile acids and bile alcohols in the 5α- and 5β-series, with 29% of all compounds having hydroxylation at C-2. Three C27 bile alcohols were present (as ester sulfates): (24ξ,25ξ)-5α-cholestan-2α,3α,7α,12α,24,26-hexol; (25ξ)-5β-cholestan-2β,3α,7α,12α,26,27-hexol, and (25ξ)-5α-cholestan-2α,3α,7α,12α,26,27-hexol. A single C27 bile acid was identified: (25ξ)-2α,3α,7α,12α-tetrahydroxy-5α-cholestan-26-oic acid, present as its taurine conjugate. Two novel C24 bile acids were identified: the 2α-hydroxy derivative of allochenodeoxycholic acid and the 2β-hydroxy derivative of cholic acid, both occurring as taurine conjugates. These studies extend previous work in establishing the natural occurrence of novel 2α- and 2β-hydroxy-C24 and C27 bile acids as well as C27 bile alcohols in both the normal (5β) as well as the (5α) "allo" A/B-ring juncture. The bile salt profile of A. gigas appears to be unique among vertebrates.

Biliverdin, the precursor of bilirubin, was adsorbed onto the synthetic clay mineral, smectite, in a mixed solution of benzene and methyl alcohol (7:3 v/v) to form a biliverdin–smectite conjugate. The adsorption of biliverdin to smectite followed the Freundlich equation with an adsorption coefficient of 1.0. The water-soluble conjugate had a green color with the absorbance maxima at 679 and 378 nm. The biliverdin–smectite conjugate became photostable in water to irradiation with UV, although the biliverdin in benzyl alcohol was rapidly decolorized.

Phycocyanin with blue color, phycocyanobilin bound to protein, with the absorption maximum at 614 nm is quite unstable against urea-treatment because of the denaturation of the protein conformation. To prevent the bleaching of phycocyanin by urea, amino groups of lysine residues in the phycocyanin molecule were modified with DSP [dithiobis(succinimidyl propionate)] to cross-link amino groups in the protein molecule in order to maintain the high order structure of phycocyanin. The color development of DSP-modified phycocyanin became stable, but unmodified phycocyanin was markedly decreased to 20% of the original absorbance at 614 nm against urea-treatment. Splitting of the S–S linkage of DSP-modified phycocyanin molecule by 2-mercaptoethanol caused the reduction of color development of phycocyanin under urea-treatment. The color development of phycocyanin is closely associated with its high order structure of protein.

Of the cytochrome P450 (CYP) family of carcinogen-activating enzymes, CYP3A is the major form found in human livers. The purpose of this study was to investigate the cytotoxic effects of dental resin monomers after being metabolized by CYP3A4 and CYP3A7, using a colony formation assay and a neutral red assay. Specimen wells were plated with transfected cells derived from the Chinese hamster lung at 100 cells well(-1). The experimental group consisted of CYP-producing 3A4-10 and 3A7-40 cells, while the control group consisted of non-CYP-producing CR-119 cells. Bisphenol A (BPA) and bisphenol A glycidyl methacrylate (Bis-GMA) and a positive control (Aflatoxine Bl) were added separately to each well and cultured for 7 days. After cultivation, the number of the colonies was counted and IC50 values were determined. The data were statistically analysed by a Student's t-test. The resultant of IC50 values indicated that the monomers were not metabolically activated by CYP3A4 or CYP3A7 as compared with the control (P < 0.05). We also confirmed that these monomers act neither as activators nor as inhibitors of CYP3A4 and CYP3A7.

Biopterin-α-glucoside purified from Spirulina platensis is stable to irradiation by visible and ultraviolet light. Its absorption spectrum shows maxima at 268 nm and 340 nm and a fluorescence band appears at 447 nm. Although cyanidin-3-galactoside with an absorption maximum at 514 nm (red colour) is unstable and is decolourized by UV light, the pigment becomes stable in the presence of biopterin-α-glucoside. Similar photostabilization phenomenon was observed for phycocyanin (λ max 614 nm) consisting of phycocyanobilin as chromophore and protein subunits α 3 β 3 in the presence of biopterin-α-glucoside. Although the chromophore of phycocyanin is related to phycocyanobilin and protein conformation, the chromophore can be stabilized to UV light in the presence of biopterin-α-glucoside.

The ether extract of the liverwort Chiloscyphus polyanthos afforded sesquiterpenoids which are enantiomeric to those found in another liverwort Lepidozia vitrea. The absolute configurations of the sesquiterpenoids found in the title species were determined by spectroscopic evidence, chemical derivatization and /or X-ray crystallographic analysis.

Four non-bridged dimethylzirconocene complexes. i.e., bis(η 5 -cyclopentadienyl )dimethylzirconium (1), (η 5 -cyclopentadienyl) (η-fluorenyl)dimethylzirconium (2), bis(η 3 -indenyl)dimethylzirconium (3) and (η 5 -cyclopentadienyl)(η 5 -pentamethylcyclopentadienyl)dimethylzirconium (4) were synthesized and used as a catalyst for the methyl methacrylate polymerization combined with triphenylmethyl tetrakis(pentafluorophenyl)borate in the presence of ZnEt 2 or Al(i-Bu) 3 . The catalyst activity strongly depends on the zirconocene compound and decreases in the order 2 > 1 >> 3 4. The polymerization proceeds in a living manner when 1 and 2 are used combined with ZnEt 2 . On the other hand, stereoregularities of the resulting polymers are independent of the zirconocene compound, and syndiotactic-rich polymers are formed by a chain-end controlled mechanism.

A focused ion beam (FIB) interface attached to a column of 200 keV transmission electron microscope (TEM) was developed for in situ micropatterning to semiconductors. TEM specimens of Si and GaAs, and those of a thin Ni2Si layer on a Si substrate were micromilled in the TEM during observation. A set of 6 x 6-um squares and alphabet letters were patterned with a 25 keV Ga+-FIB of 0.2-μm beam diameter at room temperature. The effect of FIB irradiation on the structural evolution was observed simultaneously by a TV-rate video camera and sequentially by regular film. FIB micropatterning to semiconductor specimens caused amorphization and Ga injection. The excess Ga in the specimens precipitated as metastable solid γ-phase for Si and as liquid phase for GaAs. Ni2Si/Si specimens lost silicide crystallinity after FIB patterning. Annealing of these bilayer specimens at 673K resulted in the precipitation of Ni-rich silicide.

Focused ion beam (FIB) irradiation of a thin Ni 2 Si and NiSi 2 layers deposited on a Si substrate was carried out and studied using an in-situ transmission electron microscope (TEM). A set of 3×3- μ m squares was patterned at room temperature (RT) with a 25-keV Ga + -FIB attached to the TEM. FIB patterning to Ni 2 Si/Si sample causes loss of silicide crystallinity. Annealing at 673 K resulted in the precipitation of Ni rich silicide. FIB patterning to NiSi 2 /Si sample causes fragmentation and produces defects near the irradiated edge. Annealing at 673 K improved silicide film quality.

The aging phenomena of porous silicon (PS) over a 192 day time span have been studied using photoluminescence (PL) spectroscopy, Fourier transform infrared spectroscopy, and transmission electron microscopy. As-prepared PS exhibits red PL peaking near 650 nm. The PL intensity increases with time to some equilibrium value. The PL peak shows no blueshift during aging. Infrared analysis reveals that the Si–O bond content in PS increases with time and correlates to the PL intensity. The PL lifetime increases with aging and its values range from 3 to 37 μs. Transmission electron microscopic observations indicate the presence of Si nanoparticles and amorphous substances in the PS layer. These results suggest that the PL from aged PS might be attributed to the combined effects of quantum confinement in Si nanoparticles and some defect in Si oxide covering the nanoparticles.

Focused ion beam (FIB) irradiation of a thin Ni 2 Si layer deposited on a Si substrate was carried out and studied using an in-situ transmission electron microscope (in-situ TEM). Square areas on sides of 4.5 by 4.5 and 9 by 9 μm were patterned at room temperature with a 25 keV Ga + -FIB attached to the TEM. The structural changes of the films indicate a uniform milling; sputtering of the Ni 2 Si layer and the damage introducing to the Si substrate. Annealing at 673 K results in the change of the Ni 2 Si layer into an epitaxial NiSi 2 layer outside the FIB irradiated area, but many dislocations and precipitates appear inside the treated area. Ion beam mixing of Ni into the substrate was shown by energy dispersive X-ray spectroscopy (EDS). A considerable amount of Ni and a small amount of Ga were found in precipitates, while only Si was detected outside the precipitates in the irradiated area. Selected area diffraction (SAD) patterns of the precipitates and the corresponding dark field images imply the formation of a Ni rich silicide which contains a small amount of Ga.

GC-mass spectrometric analysis of 280 samples of Conocephalum conicum, collected in various places showed the presence of three chemo-types. One elaborated (−)-sabinene (type I) as the major compound the second produced (+)-bornyl acetate (type II) and the third contained methyl cinnamate (type III) as the major constituent. The structures of three new monoterpenic esters were established by chemical and spectral means.

Ovalbumin (OVA) was chemically modified with poly(ethylene glycol) (PEG) derivatives: activated PEG2, 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine and activated PM13, copolymer of poly(oxyethylene) allyl methyl diether and maleic anhydride. Pretreatment of BALB/c mice with PEG2- or PM13-OVA suppressed the production of both IgG- and IgE-class anti-OVA antibodies induced by subsequent immunizations with unmodified OVA. PEG2-OVA had higher potency to suppress OVA-specific immune response than PM13-OVA. Although extensive modification (62%) of amino groups in the OVA molecule with activated PEG2 was required to diminish most of its immunoreactivity towards anti-OVA antibodies, only a low degree of modification (27%) was sufficient to induce tolerogenicity to OVA. Thus, the loss of immunoreactivity of PEG2-OVA was not prerequisite for its tolerogenic capacity. Moreover, the use of completely denatured PEG2-OVA immunogen lead to the loss of its ability to induce immune tolerance to native OVA. These observations are discussed in relation to the regulatory mechanism of immune response.

We previously found that genetic polymorphism in cytochrome P450 2A6 (CYP2A6) is one of the potential determinants of tobacco-related lung cancer risk. It has been reported that the plasma concentration of cotinine, a major metabolite of nicotine, in carriers of wild-type alleles of CYP2A6 is considerably higher than that in carriers of null or reduced-function alleles of CYP2A6, raising the possibility that cotinine plays an important role in the development of lung cancer. As a novel mechanism of lung tumorigenesis mediated by CYP2A6, we investigated the effects of cotinine on the suppression of apoptosis and promotion of lung tumor growth. In human lung adenocarcinoma A549 cells, cotinine inhibited doxorubicin-induced cell death by suppressing caspase-mediated apoptosis. Enhanced phosphorylation of Akt, a key factor responsible for cell survival and inhibition of apoptosis, was detected after cotinine treatment. These data suggest that cotinine suppresses caspase-mediated apoptosis induced by doxorubicin through activation of the PI3K/Akt pathway. Furthermore, we clarified that cotinine significantly facilitated tumor growth in the Lewis lung cancer model and accelerated development of lung adenomas induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice. We herein propose that cotinine induces tumor promotion by inhibiting apoptosis and enhancing cellular proliferation, thus underlining the importance of CYP2A6 in tobacco-related lung tumorigenesis.

The purpose of this study was to assess the efficacy of tolvaptan, a vasopressin V2 receptor agonist, for the management of postoperative surgical fluid retention after heart valve surgery. This was a prospective observational study of 64 patients with heart valve disease who underwent valve surgery between 2013 and 2014. Those in the tolvaptan group received tolvaptan in addition to conventional diuretic therapy. The results were compared to the results of 55 patients who underwent heart valve surgery between 2007 and 2010 and received conventional postoperative diuretics alone. The time to return to the preoperative BW was significantly shorter in the patients who received tolvaptan (6.1 ± 3.8 vs. 8.7 ± 6.7 days, p

Axial spondylometaphyseal dysplasia (SMD) (OMIM 602271) is an uncommon skeletal dysplasia characterized by metaphyseal changes of truncal-juxtatruncal bones, including the proximal femora, and retinal abnormalities. The disorder has not attracted much attention since initially reported; however, it has been included in the nosology of genetic skeletal disorders [Warman et al. (2011); Am J Med Genet Part A 155A:943-968] in part because of a recent publication of two additional cases [Isidor et al. (2010); Am J Med Genet Part A 152A:1550-1554]. We report here on the clinical and radiological manifestations in seven affected individuals from five families (three sporadic cases and two familial cases). Based on our observations and Isidor's report, the clinical and radiological hallmarks of axial SMD can be defined: The main clinical findings are postnatal growth failure, rhizomelic short stature in early childhood evolving into short trunk in late childhood, and thoracic hypoplasia that may cause mild to moderate respiratory problems in the neonatal period and later susceptibility to airway infection. Impaired visual acuity comes to medical attention in early life and function rapidly deteriorates. Retinal changes are diagnosed as retinitis pigmentosa or pigmentary retinal degeneration on fundoscopic examination and cone-rod dystrophy on electroretinogram. The radiological hallmarks include short ribs with flared, cupped anterior ends, mild spondylar dysplasia, lacy iliac crests, and metaphyseal irregularities essentially confined to the proximal femora. Equally affected sibling pairs of opposite gender and parental consanguinity are strongly suggestive of autosomal recessive inheritance.

Transductin (Tα and Tβγ) is a GTP-binding protein involved in the visual transduction process in a rod outer segment. We have previously demonstrated that Tβγ is a mixture composed of two components, Tβγ-1 and Tβγ-2, with distinctive γ-subunits, Tγ-1 and Tγ-2, respectively (Fukada et al., 1989, J. Biol. Chem. , 264 , 5937–5943). To investigate the interaction between Tα and the two components of Tβγ, the effect of either Tβγ-1 or Tβγ-2 on the ADP-ribosylation of Tα catalyzed by pertussis toxin (IAP) was examined. Tβγ-2 stimulated the ADP-ribosylation of Tα by IAP, while Tβγ-1 displayed almost no enhancement of the ADP-ribosylation. Addition of Tβγ-1 to the mixture of Tα and Tβγ-2 had no effect on the ADP-ribosylation of Tα. These results indicate that Tα and Tβγ-2 form a complex that serves as a substrate of IAP in the ADP-ribosylation reaction, while Tβγ-1 has a little affinity for Tα. It was suggested that Tγ-2 is an essential subunit for Tβ to interact with Tα.

The diethyl ether extract of Pallavicinia subciliata yielded unique skeletal diterpenoids in addition to the previously known compound. The structures of six new diterpenoids have been established by 2D NMR and/or X-ray crystallographic analysis. They are shown to be a modified labdane-type diterpenoid.

Polycyclic aromatic hydrocarbons (PAHs) have been known to induce atherosclerosis. It has been reported that the metabolic activation of PAHs by cytochrome P450 (CYP) is an important step for PAH-induced atherosclerosis. We recently reported that PAHs down-regulated the liver X receptor (LXR) alpha-regulated genes via aryl hydrocarbon receptor (AHR) as one of the causes responsible for atherosclerosis induced by PAHs. Thus, the aim of this study was to clarify the role of CYP1A1 in the suppression of LXR-mediated signal transductions by 3-methlychoranthrene (MC), one of the PAHs. We found that LXR-mediated transactivation was inhibited by the PAH, but not by halogenated aromatic hydrocarbon, which is scarcely metabolized by CYP1A1. The repression of LXR-mediated signal transductions by MC was restored by co-treatment of HepG2 cells with a CYP1A1 inhibitor, alpha-naphthoflavone, and by the transfection of short interference RNA for CYP1A1. Based on these lines of evidence, we propose that the metabolic activation of PAHs by CYP1A1, but not the activation of AHR by PAHs, is a direct mechanism for atherosclerosis via the suppression of LXR-mediated signal transductions.

Structural changes of epitaxial Ni disilicide thin films on Si(111) were investigated with an in situ transmission electron microscope (TEM) incorporated with a focused ion beam (FIB). Epitaxial growth of NiSi2 on a Si(111) TEM specimen was achieved by the combination of Ni deposition and subsequent annealing. Three different sized squares were microfabricated by 25 keV Ga+‐FIB onto this specimen during TEM observation at room temperature. FIB lithography caused the exfoliation of Ni silicide film along the microfabricated lines and the defects adjacent to the beam incident area. The beam affected area outside of the microfabricated line was about 2–3 μm.

Recent studies have shown that C-reactive protein (CRP) is not just a predictor of cardiovascular events but also acts directly as a proinflammatory stimulus in vascular cells. In this report, we studied the molecular mechanisms underlying vascular cellular adhesion molecule-1 (VCAM-1) induction by CRP. CRP-induced VCAM-1 mRNA expression and this induction was inhibited by protein kinase C (PKC) inhibitors, p38 mitogen-activated protein kinase (MAPK) inhibitor, and tyrosine kinase inhibitors. In addition, parthenolide, a nuclear factor kappaB (NF-kappaB) inhibitor, abolished VCAM-1 induction. Moreover, CRP increased VCAM-1 promoter activity, indicating that CRP induces VCAM-1 mRNA expression at the transcriptional level. Mutation of NF-kappaB-binding sites resulted in a loss of induction. Finally, an electrophoretic mobility shift assay confirmed binding of the p65 subunit of NF-kappaB to kappaB-binding sites. Taken together, our findings suggest that VCAM-1 induction by CRP is mediated by PKC, p38MAPK, tyrosine kinase and the NF-kappaB-dependent signaling pathways in vascular endothelial cells.

Trimethylaminuria (TMAU) is a metabolic disorder characterized by the inability to oxidize and convert dietary-derived trimethylamine (TMA) to trimethylamine N-oxide (TMAO). This disorder has been relatively well-documented in European and North American populations, but no reports have appeared regarding patients in Japan. We identified seven Japanese individuals that showed a low metabolic capacity to convert TMA to its odorless metabolite, TMAO. The metabolic capacity, as defined by the concentration of TMAO excreted in the urine divided by TMA concentration plus TMAO concentration, in these seven individuals ranged from 70 to 90%. In contrast, there were no healthy controls examined with less than 95% of the metabolic capacity to convert TMA to TMAO. The intake of dietary charcoal (total 1.5 g charcoal per day for 10 days) reduced the urinary free TMA concentration and increased the concentration of TMAO to normal values during charcoal administration. Copper chlorophyllin (total 180 mg per day for 3 weeks) was also effective at reducing free urinary TMA concentration and increasing TMAO to those of concentrations present in normal individuals. In the TMAU subjects examined, the effects of copper chlorophyllin appeared to last longer (i.e., several weeks) than those observed for activated charcoal. The results suggest that the daily intake of charcoal and/or copper chlorophyllin may be of significant use in improving the quality of life of individuals suffering from TMAU.

In response to oxidative stress, the pathogenesis of a number of cardiovascular events and several genes are stimulated by extracellular signal-regulated kinases (ERK1/2). Biphasic (early, 10 min; and delayed, 120 min) ERK1/2 activation by H(2)O(2), a reactive oxygen species, was observed in cultured neonatal rat cardiomyocytes. We investigated the hypothesis that the delayed activation of ERK1/2 depends on a factor secreted by oxidative stress (FSO). The delayed activation was inhibited by calphostin C, a protein kinase C inhibitor. Conditioned medium (CM) obtained from cells stimulated with H(2)O(2) induced rapid and monophasic ERK1/2 activation, which was not inhibited by calphostin C. In contrast, calphostin C-pretreated CM did not activate ERK1/2. Macrophage migration inhibitory factor (MIF) was one of the candidate FSOs activating ERK1/2. The existence of MIF in CM, the recombinant MIF-stimulated ERK1/2 rapid activation, and anti-MIF neutralizing antibody-induced inhibition of the delayed activation implied that MIF could be the FSO. Pretreatment of cardiomyocytes with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor did not suppress the MIF secretion, although it prevented the ERK1/2 activation by H(2)O(2). These results indicate that MIF is secreted from cardiomyocytes as a result of oxidative stress and activates ERK1/2 through a MEK1/2-dependent mechanism, although the secretion is not regulated by ERK1/2 but by protein kinase C.

Polymerization of methyl methacrylate (MMA) was conducted with a series of dimethylsilylene-bridged zirconocene complexes (rac-Me2ilnd2ZrMe2, 1; Me2SiCpIndZrMe2, 2; Me2SiCpFluZrMe2, 3: Cp, cyclopentadienyl; Ind, indenyl; Flu, fluorenyl) to investigate the effects of the cyclopentadienyl ligand. Thezirconocene complexes were activated by [Ph3C][B(C6F5)4] or [PhMe2NH][B(C6F5)4]in the presence of diethylzinc. It was found that 1 and 2 gave polymers but 3 did not at 0°C. The dependence of polymer yieldand molecular weight on polymerization time indicated that some chain transfer reaction occurred in both systems. 13C NMR analysis revealed that C1symmetric 2 as well as C 2-symmetric 1 gave isotactic rich polymer (mmmm = 84–87%) by enantiomorphic-site controlled mechanism. Polymerization of allyl methacrylate (ALMA) was also conducted with Cp2ZrMe2 and 1. These catalysts selectively polymerized the methacryl group to give syndiotactic-rich and isotactic polymers, respectively with pendant allyl group.

Focused ion beam (FIB) irradiation of a thin Ni2Si layer deposited on a Si substrate was carried out and studied using an in-situ transmission electron microscope (in-situ TEM). Square areas on sides of 4 by 4 and 9 by 9 μm were patterned at room temperature with a 25keV Ga+-FIB attached to the TEM. The structural changes of the films indicate a uniform milling; sputtering of the Ni2Si layer and the damage introducing to the Si substrate. Annealing at 673 K results in the change of the Ni2Si layer into an epitaxial NiSi2 layer outside the FIB irradiated area, but several precipitates appear around the treated area. Precipitates was analyzed by energy dispersive X-ray spectroscopy (EDS). Larger amount of Ni than the surrounding matrix was found in precipitates. Selected area diffraction (SAD) patterns of the precipitates and the corresponding dark field images imply the formation of a Ni rich silicide. The relation between the FIB tail and the precipitation is indicated.

The chemical constituents of the Japanese liverwort, Conocephalum conicum, belonging to chemotype I, have been investigated and 2-(3,4-dihydroxypheny)ethyl-β-allopyranoside has been isolated from the methanol extract.