Effects of nicotine and lipopolysaccharide stimulation on adhesion molecules in human gingival endothelial cells.

Smoking is a risk factor for periodontitis, and the immune response of periodontal tissues in patients with periodontitis may be strongly affected by smoking. The purpose of this study was to elucidate the bioactivity and signal transduction of human gingival endothelial cells (HGECs) due to nicotinic stimulation using a cultured medium supplemented with lipopolysaccharide (LPS) as a model of periodontitis. HGECs were cultured in medium supplemented with LPS, nicotine, nicotine + LPS, and medium supplemented without nicotine or LPS (control). Cell proliferation was assessed using Alamar blue. Cytotoxicity was assessed by lactate dehydrogenase leakage. The expression of adhesion molecule-1 (ICAM-1, VCAM-1) was assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay. The expression of nicotinic acetylcholine receptor (nAChR) subunits (α3, α5, α7, ß2 and ß4) was evaluated by RT-PCR. The involvement of p38 mitogen-activated protein kinase (p38MAPK) and protein kinase C (PKC) cell signaling pathways in ICAM-1 and VCAM-1 expression was investigated by RT-qPCR with specific inhibitors. HGECs stimulated with LPS, nicotine and nicotine + LPS showed inhibition of cell proliferation, increase of cell death, and increase of gene and protein expression of ICAM-1. Moreover, HGECs showed the presence of α5 and α7 nAChR subunits. The expression of ICAM-1 in HGECs stimulated with LPS, nicotine, and nicotine + LPS was significantly suppressed by p38MAPK inhibitor, but not by a PKC inhibitor. The nAChR subunits of HGECs are α5 and α7, and that HGECs stimulated with nicotine and LPS express ICAM-1 via p38MAPK pathway.

An Aqueous Redox Flow Battery Using CO2 as an Active Material with a Homogeneous Ir Catalyst.

For the application of CO2 as an energy storage material, a H2 storage system has been proposed based on the interconversion of CO2 and formic acid (or formate). However, energy losses are inevitable in the conversion of electrical energy to H2 as chemical energy (≈70 % electrical efficiency) and H2 to electrical energy (≈40 % electrical efficiency). To overcome these significant energy losses, we developed a system based on the interconversion of CO2 and formate for the direct storage and generation of electricity. In this paper, we report an aqueous redox flow battery system using homogeneous Ir catalysts with CO2 -formate redox pair. The system exhibited a maximum discharge capacity of 10.5 mAh (1.5 Ah L-1 ), capacity decay of 0.2 % per cycle, and total turnover number of 2550 after 50 cycles. During charging-discharging, in situ fluorescence X-ray absorption fine structure spectroscopy based on an online setup indicated that the active species was in a high valence state of IrIV .

Effects of Rice Straw Supplemented with Urea and Molasses on Intermediary Metabolism of Plasma Glucose and Leucine in Sheep.

An isotope dilution method using [U-(13)C]glucose and [1-(13)C]leucine (Leu) was conducted to evaluate the effects of rice straw supplemented with urea and molasses (RSUM-diet) on plasma glucose and Leu turnover rates in sheep. Nitrogen (N) balance, rumen fermentation characteristics and blood metabolite concentrations were also determined. Four sheep were fed either mixed hay (MH-diet), or a RSUM-diet with a crossover design for two 21 days period. Feed allowance was computed on the basis of metabolizable energy at maintenance level. The isotope dilution method was performed as the primed-continuous infusion on day 21 of each dietary period. Nitrogen intake was lower (p = 0.01) for the RSUM-diet and N digestibility did not differ (p = 0.57) between diets. Concentrations of rumen total volatile fatty acids tended to be higher (p = 0.09) for the RSUM-diet than the MH-diet. Acetate concentration in the rumen did not differ (p = 0.38) between diets, whereas propionate concentration was higher (p = 0.01) for the RSUM-diet compared to the MH-diet. Turnover rates as well as concentrations of plasma glucose and Leu did not differ between diets. It can be concluded that kinetics of plasma glucose and Leu metabolism were comparable between the RSUM-diet and the MH-diet, and rumen fermentation characteristics were improved in sheep fed the RSUM-diet compared to the MH-diet.

[Human Resource Development for Thyroid Ultrasound Examination Program in Fukushima].

As a response to the health effects associated with the Fukushima Daiichi Nuclear Power Plant accident that occurred after the Great East Japan Earthquake, the Thyroid Ultrasound Examination program as a part of the Fukushima Health Management Survey was started on Oct. 9th, 2011. Since this project required a large-scale cohort comprising all residents aged ≤ 18 years living in Fukushima when the earthquake happened, the nurturing of many ultrasound examiners was a matter of great urgency. Moreover, the standardization of examination procedures and skills of examiners were also important issues. Therefore, educational projects were established to develop ultrasound skills for medical doctors and technicians in Fukushima Prefecture. Marked efforts for this project resulted in increases in ultrasound examiners and institutions taking part in the Thyroid Ultrasound Examination project. Medical technicians specialized in ultrasound examinations are actively involved in these educational projects. We report the details of human resource development projects from the point of view of such medical technicians.

Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites.

The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.

Factors associated with the leftover rate of side dishes in Japanese school lunches.

This study investigated the leftover rate of side dishes in school lunches provided by communal kitchens in Japan's Chugoku region, with a focus on vegetable dishes supplied in containers and three types of menu items served daily to 20 elementary and junior high schools in communal kitchen A for 116 days. First, the leftovers in the containers that were returned to the communal kitchen were weighed and combined. The study then compared outside temperature, distance from communal kitchen A, school type, number of students per class, assignment of nutrition teachers, and time elapsed after cooking. Finally, we examined the relationship between these factors and the leftover rate using multiple regression analysis. The median leftover rate was 20.1% (0-96.9) for 250 side dishes with a high leftover rate; however, this was widely distributed. The number of students per class, assignment of nutrition teachers, and time elapsed after cooking were strongly related to the leftover rate; the adjusted coefficient of determination, R2, was 0.236. The regression results indicated that regarding the side dish leftover rate, the standardized coefficient, ß, was 0.414, 0.215, 0.107, 0.093, and 0.094 for the number of students per class, assignment of nutrition teacher, the time elapsed after the end of cooking, distance from communal kitchen A, and presence of seaweed, respectively (p<0.001). Dietary education by homeroom and nutrition teachers and reducing the time elapsed after cooking impacts students' awareness and preferences, which may decrease the leftover rate.

Yeast transformation mediated by Agrobacterium strains harboring an Ri plasmid: comparative study between GALLS of an Ri plasmid and virE of a Ti plasmid.

Agrobacterium strains containing a Ti plasmid can transfer T-DNA not only to plants but also to fungi, including the yeast Saccharomyces cerevisiae. However, no Agrobacterium strain harboring an Ri plasmid has been evaluated in fungal transformation. Some Ri plasmids have GALLS , instead of virE1 and virE2. GALLS protein can functionally substitute in plant transformation for a structurally different protein VirE2. In this study, we compared the yeast transformation ability among Agrobacterium donors: a strain containing a Ti plasmid, strains harboring either an agropine-type or a mikimopine-type Ri plasmid, and a strain having a modified Ri plasmid supplemented with a Ti plasmid type virE operon. Agrobacterium strains possessing GALLS transformed yeast cells far less efficiently than the strain containing virE operon. Production of GALLS in recipient yeast cells improved the yeast transformation mediated by an Agrobacterium strain lacking neither GALLS nor virE operon. A reporter assay to detect mobilization of the proteins fused with Cre recombinase revealed that VirE2 protein is much more abundant in yeast cells than GALLS. Based on these results, we concluded that the low yeast transformability mediated by Agrobacterium strains having the Ri plasmid is because of low amount of mobilized GALLS in yeast cells.

Electrochemistry in bicontinuous microemulsions derived from two immiscible electrolyte solutions for a membrane-free redox flow battery.

HYPOTHESES: Bicontinuous microemulsions (BMEs) have attracted attention as unique heterogeneous mixture for electrochemistry. An interface between two immiscible electrolyte solutions (ITIES) is an electrochemical system that straddles the interface between a saline and an organic solvent with a lipophilic electrolyte. Although most BMEs have been reported with nonpolar oils, such as toluene and fatty acids, it should be possible to construct a sponge-like three-dimensionally expanded ITIES comprising a BME phase. EXPERIMENTS: Dichloromethane (DCM)-water microemulsions stabilized by a surfactant were investigated in terms of the concentrations of co-surfactants and hydrophilic/lipophilic salts. A Winsor III microemulsion three-layer system, consisting of an upper saline phase, a middle BME phase, and a lower DCM phase, was prepared, and electrochemistry was conducted in each phase. FINDINGS: We found the conditions for ITIES-BME phases. Regardless of where the three electrodes were placed in the macroscopically heterogeneous three-layer system, electrochemistry was possible, as in a homogeneous electrolyte solution. This indicates that the anodic and cathodic reactions can be divided into two immiscible solution phases. A redox flow battery comprising a three-layer system with a BME as the middle phase was demonstrated, paving the way for applications such as electrolysis synthesis and secondary batteries.

Redox Reaction in Ti-Mn Redox Flow Battery Studied by X-ray Absorption Spectroscopy.

We performed X-ray absorption studies for the electrolytes of a Ti-Mn redox flow battery (RFB) to understand the redox reaction of the Ti/Mn ions and formation of precipitates in charged catholyte, because suppression of the disproportionation reaction is a key to improve the cyclability of Ti-Mn RFB and enhance the energy density. Hard X-ray absorption spectroscopy with a high transmittance and soft X-ray absorption spectroscopy to directly observe the 3d orbitals were complementarily employed. Moreover, the Ti/Mn 3d electronic structure for each precipitate and solution in the charged catholyte was investigated by using scanning transmission X-ray microscopy: the valence of Mn in the precipitate is mostly attributed to 4+, and the solution includes only Mn2+ . This charge disproportionation reaction should occur after the Mn ions in the catholyte should be oxidized from Mn2+ to Mn3+ by charge.

Design of biomolecular interface for detecting carbohydrate and lectin weak interactions.

A hybrid functional biomolecular interface designed at a molecular size level is very effective at capturing an analyte with high sensitivity even if the interaction is very weak, as when detecting proteins with carbohydrate. We designed and processed a protein (lectin) recognition molecular interface taking the following points into consideration: (1) the height (molecular length) difference between the capturing and spacer molecules; (2) the ratio of capturing molecules in the recognition interface. When the height difference between the maltoside part (Concanavalin A (Con A) recognition group) and the OH group terminated spacer molecules exceeded (>(CH(2))(6)), the association rate constant (k(a)) became larger (k(a)(1/Ms): ∼2.6 times) and the dissociation constant (K(D)) became much smaller (K(D)(M): 1.0 × 10(-6): ∼0.17 times) compared with the similar heights (lengths) of both molecular interfaces. With regard to maltoside density, a 100% maltoside monolayer was unsuitable for detecting Con A. We constructed a nanostructured recognition site with a maltoside part of 10%, which was the most suitable ratio for Con A detection. The binding interaction between Con A and the maltoside group was changed from monovalent binding to bivalent binding when the maltoside part was diluted in the recognition interface. From electrochemical measurements, even though there was a small amount of maltoside component on the suitable recognition monolayer, quality similar to that of 100% maltoside was observed.

Surface modification of GC and HOPG with diazonium, amine, azide, and olefin derivatives.

Surface modification of glassy carbon (GC) and highly oriented pyrolytic graphite (HOPG) was carried out with diazonium, amine, azide, and olefin derivatives bearing ferrocene as an electroactive moiety. Features of the modified surfaces were evaluated by surface concentrations of immobilized molecule, blocking effect of the modified surface against redox reaction, and surface observation using cyclic voltammetry and electrochemical scanning tunneling microscope (EC-STM). The measurement of surface concentrations of immobilized molecule revealed the following three aspects: (i) Diazonium and olefin derivatives could modify substrates with the dense-monolayer concentration. (ii) The surface concentration of immobilized amine derivative did not reach to the dense-monolayer concentration reflecting their low reactivity. (iii) The surface modification with the dense-monolayer concentration was also possible with azide derivative, but the modified surface contained some oligomers produced by the photoreaction of azides. Besides, the blocking effect against redox reaction was observed for GC modified with diazonium derivative and for HOPG modified with diazonium and azide derivatives, suggesting fabrication of a densely modified surface. Finally, the surface observation for HOPG modified with diazonium derivative by EC-STM showed a typical monolayer structure, in which the ferrocene moieties were packed densely at random. On the basis of those results, it was demonstrated that surface modification of carbon substrates with diazonium could afford a dense monolayer similar to the self-assembled monolayer (SAM) formation.

Synthesis and galectin-binding activities of mercaptododecyl glycosides containing a terminal ß-galactosyl group.

Mercaptododecyl glycosides containing a terminal ß-galactosyl group were prepared from D-galactose or from D-lactose via hexa-O-acetyl-lactal (10) as a key intermediate. Interactions of these glycolipids (5 kinds) and galectins (ß-galactoside binding lectins, 6 species) were evaluated by surface plasmon resonance (SPR) method. High binding responses were observed for the lactoside, 2-deoxy-lactoside, and lactosaminide with some galectins (Gal-3, -4, -8), whereas the galactoside and 2,3-dideoxy-lactoside showed low binding activities.

Surface plasmon resonance analysis of interactions between diacylglycerol acyltransferase and its interacting molecules.

To measure the interactions of diacylglycerol acyltransferase (DGAT) by surface plasmon resonance (SPR), we immobilized Saccharomyces cerevisiae DGAT2 encoded by DGA1 on a BIACORE sensor chip surface. We used N-terminally truncated Dga1p with a FLAG tag at the C-terminus, which was purified to apparent homogeneity, maintaining significant DGAT activity (Kamisaka et al., Appl. Microbiol. Biotechnol., 88, 105-115 (2010)). Truncated Dga1p with a FLAG tag was immobilized with an anti-FLAG antibody that had been coupled with an L1 chip surface consisting of a carboxymethyl dextran matrix with additional hydrophobic alkane groups. The Dga1p-immobilized chip surface was analyzed for interactions of Dga1p with oleoyl-CoA, its substrate, and anti-Dga1p IgG, its interacting protein, by SPR. The binding of these analytes with the Dga1p-immobilized chip surface was specific, because butyryl-CoA, which cannot be used as a substrate for DGAT, and anti-glyceraldehyde-3-phosphate dehydrogenase IgG, did not induce any signals on SPR. Furthermore, injection of organic compounds such as xanthohumol, a DGAT inhibitor, into the Dga1p-immobilized chip surface induced significant SPR signals, probably due to interaction with DGAT. Another DGAT inhibitor, piperine, did not induce SPR signals on application, but induced them due to piperine on application together with oleoyl-CoA, in which piperine can be incorporated into the micelles of oleoyl-CoA. The results indicate that the Dga1p-immobilized L1 chip surface recognized DGAT inhibitors. Taking all this together, SPR measurement using the Dga1p-immobilized L1 chip surface provided a useful system to elucidate the structure-function relationships of DGAT and screen DGAT inhibitors.

Successful anesthetic management of separation surgery for pygopagus conjoined twins: a case report.

BACKGROUND: Conjoined twins are an extremely rare congenital occurrence, and anesthetic management for surgical separation presents unique challenges for anesthesiologists. CASE PRESENTATION: Five-month-old male pygopagus conjoined twins underwent separation surgery. We performed anesthesia induction in the supine position and surgery in the prone position. This presented a challenge because the transition from supine to prone position reversed the positional relationship between the two babies, resulting in crossing of the respiratory circuits and monitors. To solve the problem, we used anesthesia machines and monitors on the opposite side of each baby during anesthesia induction. The positional relationship between the twins and anesthesia machines and monitors normalized after the change to the prone position. Following the separation surgery, the twins were discharged without any complications. CONCLUSIONS: Our method of using opposite side anesthetic machines and monitors for anesthesia induction was useful for the safe anesthetic management of pygopagus conjoined twins.

Development of an automated transportable continuous system to measure the total alkalinity of seawater.

Anthropogenic CO2 emissions are contributing to global warming and ocean acidification. Rapid and accurate measurements of seawater carbonate chemistry are critical to understand current changes in the ocean and to predict future effects of such changes on marine organisms and ecosystems. Total alkalinity (AT) measurements can be used to directly determine the calcification rate, but they are time-consuming and require large sample volumes. Herein, we describe an automated and transportable flow-through system that can conduct continuous AT measurement using an ion sensitive field effect transistor (ISFET) - Ag/AgCl sensor and three different reference materials. The response time, stability, and uncertainty of our system were evaluated by comparing AT values of calibrated reference materials to those calculated by our system. Our system requires only small amounts of seawater (<10 mL) and a short time per sample (<5 min) to produce results with a relative uncertainty of less than 0.1% (approx. 2.2 µmol kg-1). This system is expected to facilitate easy and rapid in-situ measurement of AT. Continuous AT measurements would enable us to determine short-term calcification responses to changes in light or temperature and improve our understanding of the metabolic mechanisms of creatures such as corals.

One-step detection of galectins on hybrid monolayer surface with protruding lactoside.

Galectins, or beta-galactoside binding lectins, are detected deep in tumor tissue and are recognized as diagnostic and prognostic markers of cancer and other serious diseases. There is a need to develop a faster, easier, and simpler method for detecting galectins. We have succeeded in forming a mixed self-assembled monolayer (SAM) interface consisting of beta-galactoside terminated alkanethiol (lactoside protuberant dodecanethiol) and tri(ethylene glycol) (TEG) terminated short alkanethiol, which proved to be a superior protein resistant material, to enable us to develop a label-free, one-step, and highly sensitive system for detecting the expected biomarker, galectin. We successfully detected nanomolar level (~ 1 nM) galectin-4 and -8 on a 4% lactoside protrusive surface, even though the affinity between the galectins and lactoside was very weak (KD = 1 x 10(-3)~1 x 10(-6)). The combination of the suppression of background noise by filling with TEG terminated short alkanethiol and control of the ligand ratio in the interface contributed to the highly sensitive detection of galectin. We also detected galectin-4 at subten nanomolar levels even in a solution containing much higher concentrations of serum proteins (1800 times larger than the galectin concentration) without using molecule labeling or an immunological method.

Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family.

Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and α4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (ΔT m = -5 to 6 °C). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear ß-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties.

Crystallization and preliminary X-ray analysis of a novel haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94.

A novel enzyme, DbeA, belonging to the haloalkane dehalogenase family (EC 3.8.1.5) was isolated from Bradyrhizobium elkani USDA94. This haloalkane dehalogenase is closely related to the DbjA enzyme from B. japonicum USDA110 (71% sequence identity), but has different biochemical properties. DbeA is generally less active and has a higher specificity towards brominated and iodinated compounds than DbjA. In order to understand the altered activity and specificity of DbeA, its mutant variant DbeA1, which carries the unique fragment of DbjA, was also constructed. Both wild-type DbeA and DbeA1 were crystallized using the sitting-drop vapour-diffusion method. The crystals of DbeA belonged to the primitive orthorhombic space group P2(1)2(1)2(1), while the crystals of DbeA1 belonged to the monoclinic space group C2. Diffraction data were collected to 2.2 A resolution for both DbeA and DbeA1 crystals.

Recognition of lectin with a high signal to noise ratio: carbohydrate-tri(ethylene glycol)-alkanethiol co-adsorbed monolayer.

Densely packed co-adsorbed ultrathin mono molecular layers of short tri(ethylene glycol)-alkanethiolate (for repelling proteins) and maltoside terminated alkanethiolate (for capturing lectin) provided an extremely high signal to noise ratio surface: the repelling molecules, which had two different functions (highly flexible-hydrophilic arm and rigid packing tail group), worked as "nano barriers" in the recognition monolayer.

12-Mercaptododecyl beta-maltoside-modified gold nanoparticles: specific ligands for concanavalin A having long flexible hydrocarbon chains.

A simple and highly specific protein detection system using glycoconjugated gold nanoparticles was investigated. This system was based on the aggregation of gold nanoparticles coated with carbohydrate alkanethiols in the presence of corresponding proteins (lectins) that had specific recognition for certain carbohydrates. In order to construct an efficient specific recognition system, maltoside alkanethiol was adopted as an effective sensing modifier having a disaccharide group and a flexible long alkyl chain. The surface modification of gold nanoparticles with maltoside alkanethiol resulted in a shift and broadening (from 520 to 610 nm) of the absorption peak. Monodispersed maltoside-adsorbed gold nanoparticles aggregated with the specific lectin, concanavalin A (Con A). This phenomenon was used to detect the presence of Con A and to estimate concentrations of Con A in sample solutions. The precipitate of the maltoside-gold nanoparticle-Con A mixture was redispersed by addition of methyl alpha-D-mannopyranoside whose adsorption coefficient is larger than that of maltoside with Con A.