Wako Pure Chemical Industries Research Articles

Determination of Nitrate Ion Concentration in Fresh Vegetable Juices Using Ion-Pair Ultra Performance Liquid Chromatography (IP-UPLC)

Determination of Nitrate Ion Concentration in Fresh Vegetable Juices Using Ion-Pair Ultra Performance Liquid Chromatography (IP-UPLC)

Investigation of Solvent and Carbon Particles Behavior during Drying Process from Catalyst Ink to Catalyst Layer

Mass transport properties of catalyst layers (CLs) for proton exchange membrane fuel cells (PEMFCs) strongly depends on its structure. Structure control, therefore, is important to achieve higher cell performance. However, the CLs have fabricated by trial and error approach. Understanding mechanism and key parameters of the structure determination during the fabrication process is necessary. In this study, we especially focused on drying process from a catalytic slurry named catalyst ink to CL. Coating catalyst ink on a membrane or a substrate is one of the well-known fabrication methods of the CLs. Catalyst ink consists of components of the CLs, which is catalyst-supported carbon and ionomer, and solvents such as water and alcohol. During drying process from catalyst ink to CL, the solvent evaporates and then density of the components increase in the slurry. The components agglomerate or deposit, and finally the porous structure of the CL is formed. The porous structure affects cell performance [1]. Therefore, controlling behavior of components during the drying process has potential to form a further well-established porous structure. For the fundamental understanding, there are several attempt to characterize catalyst ink [2,3]. However, there are few studies about the drying process, although there is some simulation of the process [4]. To determine drying fabrication process and control the resultant structure of the CLs, revealing transport phenomena and dynamics of solvent, carbon and ionomer in the catalyst ink during drying fabrication process is necessary. For the fundamental understanding of the process, solvent evaporation and carbon agglomeration were detected in this study. The solvent evaporates during the drying process, thus the weight of the coated catalyst ink decreases as time goes along. Therefore, weight variation during the drying process was detected by using electric balance. According to evaporation, a density of carbon particles in the coated catalyst ink increases and the particles gradually form a network with each other. Carbon is prime electric conductive material in the catalyst ink. Therefore, electric conductivity was measured during the drying process by using microelectrode. Catalyst ink is coated on the silicon substrate with gold terminals. Four-terminal sensing was conducted by means of high-frequency resistance. For the simple evaluation, carbon black (ketjenblack, Lion Corp.) and Nafion® ionomer (DE2020, Wako Pure Chemical Industries) were blended in the solvent which consist of water and propanol to form catalyst ink. Three types of the catalyst ink which differ in carbon density (1.8, 3.6 and 5.4wt.%) was synthesized. Ionomer to carbon ratio was 1.0. Although the weight measurement and resistance measurement were conducted independently, the same type of silicon substrate with SiO2layer was used as a substrate of the coating. Drying condition was room temperature and humidity (ca. 20°C and 30%RH). Aimed thickness of the CLs was 10 μm in all kinds of catalyst ink according to the previous work [5]. Figure 1 shows weight variation during the drying process. Lower carbon content ink takes a longer time to dry completely. This is because the ink contains a larger amount of solvent to form the CL with aimed thickness. Figure 2 shows resistance variation during the drying process. The result indicates that lower carbon content ink takes a longer time to form carbon network. It is showed that the carbon content in the catalyst ink affects both drying and agglomeration behaviors. As a comparison of the two results of weight and resistance variation, catalyst ink with 1.8wt.% carbon shows much faster agglomeration time of less than 15 min than drying time of about 50 to 60 min. A low-carbon content ink can show independent agglomeration behavior from drying behavior although high-carbon content inks show the relatively correlated behavior of evaporation and carbon agglomeration.

Stable Carbon Isotope Ratio for Sugar, Amino Acid, and Caffeine by Liquid Chromatography/Isotope Ratio Mass Spectrometry

In 2004, liquid chromatography coupled with isotope ratio mass spectrometry (LC/IRMS) has been developed. Today, LC/IRMS system has been used for various compounds. Stable isotope analysis has proved to be a powerful tool for source apportionment of various compounds. We analysed δ13C of 6 sugar, 10 amino acid and 2 caffeine samples using direct injection mode and online LC/IRMS mode. In this study, the ion chromatography system was coupled with IRMS instrument.On direct injection mode, the standard deviation for all samples were <0.26‰. The difference between EA/IRMS and direct injection mode was 0.41‰ (sugar), 2.01‰ (amino acid), 5.09‰ (caffeine, Wako Pure Chemical Industries), and 7.41‰ (caffeine, IAEA). The differences for amino acid and caffeine samples were bigger than sugar samples. The reason of the difference is still unclear.In the case of online LC/IRMS mode, the differences between the EA/IRMS and online LC/IRMS without phosphoric acid for amino acid were from 2.54‰ to 9.93‰. In contrast, when 1% phosphoric acid was added, the differences between the EA/IRMS and online LC/IRMS was ranged from 0.09‰ to 2.09‰. The eluent for LC was 5mM sodium tetraborate decahydrate (pH=9.51). When the 1% phosphoric acid was not added, the oxidation potential of sodium peroxodisulfate was decreasing. Thus, when the eluent of alkalinity eluent was used, it is necessary to add phosphoric acid. However, the difference of 2 caffeine samples between EA/IRMS and online LC/IRMS without phosphoric acid and with phosphoric acid were 5.30‰ and 5.82‰, and 5.68‰ to 7.32‰, respectively. For caffeine samples, the δ13C did not change with or without phosphoric acid. Other reasons were considered e.g. incomplete combustion.

Amorphous Niobium Sulfides as Novel Positive-Electrode Materials

Experimental We mechanochemically synthesized the a-NbSx at room tempera- ture. Appropriate amounts of sulfur (S8, 99.9%, Wako Pure Chemical Industries) and niobium disulfide (NbS2, 99%, High Purity Chemi- cals) were weighed and mixed, and the 1.5-g mixture and 500 4-mm- diameter zirconia balls were placed into a 45-mL zirconia pot in a planetary ball mill (P-7, Fritsch GmbH) in an Ar-filled glove box. The mixture was ball-milled at 510 rpm until the amorphous powder was obtained. The X-Ray diffraction (XRD) patterns for the synthesized sam- ples were recorded using an X-ray diffractometer (Rotaflex RU- 200B/RINT, Rigaku). Prior to the measurements, the samples were covered with Kapton film in a glove box to prevent exposure to air. The conductivities of the prepared samples were measured using a two-electrodecell.Thepelletsusedfortheconductivitymeasurements were prepared by pressing powder samples under 360 MPa at 25 ◦ C. A TITAN3 G2 60-300 electron microscope (FEI, Co., Ltd.) was used for the transmission electron microscopy (TEM) measurements. The a-NbS5 powder was directly dispersed on a Cu-mesh-supported carbon film for the TEM measurements. The working electrodes were prepared from a-NbSx (10 mg), acetylene black (1 mg), and polytetrafluoroethylene (PTFE) powder (0.7 mg). A 1 M solution of LiPF6 dissolved in a 1:1 volumet- ric mixture of ethylene carbonate (EC) and dimethyl carbonate (DMC) (Tomiyama Pure Chemical Industries, Ltd.) was used as the electrolyte. The counter electrode consisted of a 15-mm-diameter, 0.2-mm-thick Li foil disk. A charge-discharge unit (TOSCAT-3100, Toyo System) was used to perform the electrochemical measurements at 30 ◦ C and 100 μ Ac m −2 between 1.5 and 3.0 V.

Polymyxin B-immobilized fiber column hemoperfusion removes endotoxin throughout a 24-hour treatment period

PurposeThe purpose of this study was to evaluate the extent of endotoxin adsorption by polymyxin B-immobilized fiber column hemoperfusion (PMX) performed for a 24-hour treatment period in patients with septic shock. Materials and methodsNineteen patients with septic shock were retrospectively studied. The plasma endotoxin concentrations of blood drawn from the radial artery and from the outlet circuit of the PMX column were measured by kinetic turbidimetric limulus assay using an MT-358 Toxinometer (Wako Pure Chemical Industries, Ltd, Osaka, Japan) after 24 hours of PMX treatment. The endotoxin removal rate was defined by the following equation: ([radial artery endotoxin concentration − outlet circuit of PMX column endotoxin concentration]/radial artery endotoxin concentration) × 100%. ResultsThe patients had a median Acute Physiology and Chronic Health Evaluation II score of 29 at intensive care unit admission and a 28-day mortality of 47%. Before the start of the PMX treatment, the median radial arterial plasma endotoxin concentration was 16.48 pg/mL. After 24 hours of PMX treatment, the median radial plasma endotoxin concentration had decreased to 1.857 pg/mL, and the concentration at the outlet circuit of the PMX column was further decreased to 0.779 pg/mL. The median endotoxin removal rate was 74.4%. ConclusionThese findings suggest that 24-hour PMX treatment was effective in removing endotoxin continuously throughout the entire treatment period.

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.

新PTドライ試薬の基礎的検討

The prothrombin time (PT) is used for detecting abnormalities in the extrinsic pathway of blood coagulation and monitoring the effects of warfarin administration. A new PT dry reagent has been developed in Japan with an International Sensitivity Index (ISI) of approximately 1.0 by the COAG 2 N System (supplied by Wako Pure Chemical Industries Ltd). In this study, we investigated the basic performance of the new reagent using the COAG 2 N System. The new reagent showed good reproducibility, especially when citrated plasma or whole blood was used as a sample, yielding interference results similar to those of bilirubin-F, bilirubin-C, hemolytic hemoglobin, and intralipid. It provided a level of performance that would satisfy the needs of users. Using specimens of patients who were prescribed a warfarin agent, good correlations were obtained in comparison to current reagents with an ISI of approximately 1.0. These findings suggest that this new PT dry reagent may be useful in testing routine coagulation and monitoring the effects of warfarin administration.

Analytical performance and accuracy verification of four domestic and two imported enzymatic creatinine assay kits

Objective To assessed the analytical performance of six commercial enzymatic creatinine (Cr) reagents and compared the accuracy among them.Four reagents came from domestic manufactures (Maker Biotechnology,Beijing Strong Biotechnologies,Biosino Bio-technology & Science,BaiDing Biological Engineering,labeled as A,B,C,D respectively) and the other two reagents came from oversea manufactures (Sekisui Medical Technology,Japan and Wako Pure Chemical Industries,Japan,labeled as E,F respectively). Methods Performance verification test.70 serum samples were obtained from chronic kidney disease outpatients and medical center healthy volunteers in Peking Union Medical College Hospital from May 2010.Among 30 patients ureh CKD (15 moles,15 females) aged 18 to 80 years old;40 healthy volunteers (20 males,20 females) aged 20 to 60 years old.To evaluate the within-run imprecision and total imprecision according to the CLSI EP15-A protocol,two level control materials were measured with six reagents in five days,with a frequency of four times a day.To verify the linear ranges according to the EP6-A protocol,a series of samples that mixed by high and low level serum or urine pools with specific ratio were tested.To evaluate the anti-interference ability,we tested the sera added specific concentration of substances (bilirubin,hemoglobin,chyle).To verify the reasonable dilution ratio,diluted sera in different ratio were tested.Correlations and deviation among the detection results of six reagents were evaluated by testing 50 fresh patient samples.Accuracy was evaluated by measuring the NIST SRM 967a and NIST SRM 909b standard substances. Results There were no significant differences between the domestic and imported Cr reagents,with the within-run CV ranging from 0.5% to 1.2% and the total CV ranging from 0.5% to 1.9%,all of which were lower than the allowed biological variation for imprecision (3.0%).Linear ranges of the six Cr reagents were all relatively wide.No significant interference (bias%≤±10%) was found in the test of six reagents when conjugated bilirubin≤332μmol/L,unconjugated bilirubin≤327μmol/L,or chyle turbidity≤1530 FTU; no significant interference was found for the test of C,D,F reagents when Hb≤4.83 g/L; and for A,B,E when Hb≤3.381,3.864,4.347g/L,respectively.Percent deviation of the measured results of dulited serum samples diluted by 2 to 16 times compared to the results of the original samples ranged from 95.96% to 108.62%.Fresh patient samples comparison tests exhibited good correlation among the six reagents (r=1.000),although there were some differences (-4.91% to -4.75%).For NIST 967a,the percent bias ranged from -1.85% to 2.81% (level 1,66.5μmol/L),and from -2.28% to 2.05% (level 2,346.2μmol/L),none of the six reagents exceeded the allowed Cr bias derived from biological variation (4.0%).For NIST 909b,the percent bias ranged from -13.4% to 1.01% for level 1 (56.18μmol/L),while its value ranged from -6.49% to -1.79% for level 2 (467.4μmol/L),and these biases all exceeded the allowable range of target value. Conclusions Assessment of four domestic enzymatic Cr reagents and two imported reagents show high precision,wide linear range,and their dilution accuracy can meet the clinical needs.Moreover,the six regents show good anti-interference capability (bilirubin,hemoglobin,chyle) and high accuracy.No significant differences have been found in the above assessed properties among these reagents.(Chin J Lab Med,2013,36:806-811) Key words: Creatinine; Indicators and reagents; Reagent kits; diagnostic

Linfuranone A, a new polyketide from plant-derived Microbispora sp. GMKU 363

Actinomycetes are well-known producers of an enormous variety of secondary metabolites, many of which have beneficial applications in the field of medicine and agriculture. More recently, endophytic actinomycetes residing in plants revealed the potential sources of biodiversity carrying a variety of bioactive metabolites and acting as potential biocontrol agents.1–3 Particularly, endophytic actinomycetes isolated from tropical plants have been examined to possess significant biosynthetic potential, particularly polyketide synthase and nonribosomal peptide synthetase genes.4 We have recently reported a new polyketide compound from an endophytic actinomycete isolated from a Thai medicinal plant collected at the Eastern Botanical Garden (Khao Hin Son), Chachoengsao province, Thailand.5 Here, we now report a new furanone-containing polyketide, linfuranone A (Figure 1), produced by an endophytic actinomycete isolated from a Thai medicinal plant collected at the same location. An endophytic Microbispora sp. GMKU363 was isolated from a root of Thai medicinal plant ‘Lin Ngu Hao’ (Clinacanthus siamensis Bremek.) according to the reported protocol.6 The strain was identified as a member of the genus Microbispora on the basis of 99.9% 16S ribosomal RNA gene sequence identity (1387 nucleotides; GenBank accession number GU459171) with the Microbispora mesophila JCM 3151T type strain (accession number AF002266). Strain GMKU363 was cultured on Bn-2 slant agar medium consisting of soluble starch 0.5%, glucose 0.5%, meat extract (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) 0.1%, yeast extract (Difco Laboratories, Detroit, MI, USA) 0.1%, NZ-case (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 0.2%, NaCl 0.2%, CaCO3 0.1%, agar 1.5% and was inoculated into 500-ml K-1 flasks each containing 100ml of the V-22 seed medium (pH 7.0) consisting of soluble starch 1%, glucose 0.5%, NZ-case 0.3%, yeast extract 0.2%, triptone (Difco Laboratories) 0.5%, K2HPO4 0.1%, MgSO4 � 7H2O 0.05% and CaCO3 0.3%. The cultures were cultivated on a rotary shaker (200 r.p.m.) at 30 1C for 4 days. The seed culture (3ml) was transferred into 500-ml K-1 flasks each containing 100ml of the A-11M production medium (pH 7.0) consisting of soluble starch 2.5%, glucose 0.2%, yeast extract 0.5%, polypeptone (Wako Pure Chemical Industries, Ltd.) 0.5%, NZ-amine 0.5%, CaCO3 0.3% and Diaion HP-20 (Mitsubishi Chemical Co.) 1%. The cultures inoculated in flasks were cultured on a rotary shaker (200 r.p.m.) at 30 1C for 6 days, and the whole culture broth was extracted with 100ml of 1butanol on each flask by shaking for an additional hour. The organic layer was evaporated to give 3.0 g of crude extract from 1.5 l of culture. The crude extract (3.0 g) was subjected to silica gel column chromatography with a step gradient of CHCl3-MeOH (1:0, 20:1, 10:1, 4:1, 2:1, 1:1 and 0:1 v/v). The concentration of the fraction eluted with a 2:1 mixture of CHCl3-MeOH provided 0.23 g of dark viscous oil, which was further purified by preparative HPLC (Cosmosil AR-II, San Diego, CA, USA, 250� 10mm2) using 30%MeCN in distilled water at 4mlmin�1 to give linfuranone A (2.0mg, tR1⁄4 12.1min). Linfuranone A (Figure 1) was obtained as an optically active ([a]D �9.9 (c 0.16, MeOH)), colorless and amorphous compound that gave an [MþNa]þ peak at m/z 417.2257 in the high resolution electrospray ionization time-of-flight mass spectrometry appropriate for a molecular formula of C22H34O6, (calculated for C22H34O6Na, 417.2248), which was consistent with the 1H and 13C NMR data (Table 1). The IR spectrum of linfuranone A showed absorption bands for hydroxyl (3333 cm�1) and carbonyl (1691 cm�1) functionalities. The UV spectrum showed absorption maxima at 282 (e 23600) and 232 (e 75300) in MeOH. 13C NMR and HMQC spectral data confirmed the presence of 22 carbons, which were assigned to two oxygenated-quaternary sp2 carbons, seven olefinic carbons (five are proton-bearing), one quaternary sp3 carbon, four sp3 methines (three are oxygen-bearing), three sp3 methylenes and five methyl carbons. Analysis of the COSY spectrum led to the identification of four proton-bearing fragments, H-17–H-19, H-15/H-14/H-21, H-12/H-13 and H-6–H-11 (Figure 1). HMBC correlations were detected from the

A novel JNK2/SREBP-1c pathway involved in insulin-induced fatty acid synthesis in human adipocytes

Insulin plays important roles in apoptosis and lipid droplet (LD) formation, and it is one of the determinants involved in increasing fat mass. However, the mechanisms underlying insulin-induced enlargement of fat mass remain unclear. Our previous study suggested that insulin-induced increases in LDs are related to c-Jun N-terminal kinase (JNK)2-mediated upregulation of cell death-inducing DNA fragmentation factor-α-like effector (CIDE)C in human adipocytes. However, other genes involved in insulin/JNK2-induced LD formation are unknown. Here, we explored insulin/JNK2-regulated genes to clarify the mechanism of enlargement of LDs. Microarray analysis revealed that an insulin/JNK2 pathway mostly regulates expression of genes involved in lipid metabolism, including sterol regulatory element binding protein (SREBP)-1, a key transcription factor of lipogenesis. The JNK inhibitor SP600125 blocked insulin-induced upregulation of SREBP-1c expression. Small interfering RNA-mediated depletion of JNK2 suppressed insulin-induced nuclear accumulation of the active form of SREBP-1 protein and upregulation of SREBP-1c. Furthermore, depletion of JNK2 attenuated insulin-induced upregulation of SREBP-1c target lipogenic enzymes, leading to reduced de novo fatty acid synthesis. In addition, JNK2 coimmunoprecipitated with SREBP-1, reinforcing the correlation between JNK2 and SREBP-1. These results suggest that SREBP-1c is a novel insulin/JNK2-regulated gene and that the JNK2/SREBP-1c pathway mediates insulin-induced fatty acid synthesis, which may lead to enlargement of LDs in human adipocytes.

Assessment of prognosis in hepatocellular carcinoma (HCC) using biomarkers and serum measurements.

e15093 Background: Factors that influence prognosis in HCC are well-recognised. They include tumour size and number, the severity of liver dysfunction and the symptomology. Several staging systems combining a number of these factors offer an estimate of prognosis. Some concerned that these staging systems encompass factors that are inherently subjective, e.g. the extent of HCC in CLIP or the use of performance status in BCLC. This have led to the development BALAD score by Toyoda et al, using serum Bilirubin and Albumin and three biomarkers: alpha-fetoprotein-L3 (L3), Alpha-fetoprotein (AFP) and Des-gamma-carboxy prothrombin (DCP). This system was developed in a Japanese population which has mainly HCV-related HCC. Our aim was to develop an objective staging system using the three biomarkers for a western population and compared it to BALAD to assess its performance. Methods: 317 subjects with HCC were recruited from Birmingham, UK between 2007 and 2011. Blood samples were collected at the time of diagnosis. Liver, renal functions plus AFP, L3 and DCP were measured. L3 and DCP were measured by microchip capillary electrophoresis and liquid-phase binding assay on a uTASWako i30 auto analyzer (Wako Pure Chemical Industries Ltd, Japan). Results: Univariable and multivariable regression analyses were performed. Our scoring system (CRCTU score) was developed based on a flexible baseline hazard functions fitted using restricted cubic splines. Suitable fractional polynomials (FP) were used to explain relationship between the outcome and covariates. The level of discrimination achieved using the risk groups was assess by Harrell’s–C statistic. The CRCTU score permitted identification of 4 clearly distinct subgroups as indicated both graphically and by Harrell’s-C values. Both the CRCTU and BALAD scores return Harrell’s-C values of 0.7. Conclusions: We have shown that it is possible to use entirely objective markers to predict prognosis of HCC in a western population. Our model is comparable to the BALAD with the same variables included. However, our treatment of variables with FP is much more robust statistical technique for continuous variable as compared to having artificial categories that were seen in the BALAD system.

Rabex-5 Protein Regulates Dendritic Localization of Small GTPase Rab17 and Neurite Morphogenesis in Hippocampal Neurons

Small GTPase Rab17 has recently been shown to regulate dendritic morphogenesis of mouse hippocampal neurons; however, the exact molecular mechanism of Rab17-mediated dendritogenesis remained to be determined, because no guanine nucleotide exchange factor (GEF) for Rab17 had been identified. In this study we screened for the Rab17-GEF by performing yeast two-hybrid assays with a GDP-locked Rab17 mutant as bait and found that Rabex-5 and ALS2, both of which were originally described as Rab5-GEFs, interact with Rab17. We also found that expression of Rabex-5, but not of ALS2, promotes translocation of Rab17 from the cell body to the dendrites of developing mouse hippocampal neurons. The shRNA-mediated knockdown of Rabex-5 or its known downstream target Rab5 in hippocampal neurons inhibited morphogenesis of both axons and dendrites, whereas knockdown of Rab17 affected dendrite morphogenesis alone. Based on these findings, we propose that Rabex-5 regulates neurite morphogenesis of hippocampal neurons by activating at least two downstream targets, Rab5, which is localized in both axons and dendrites, and Rab17, which is localized in dendrites alone.

Deficiency of Growth Factor Midkine Exacerbates Necrotizing Glomerular Injuries in Progressive Glomerulonephritis

Inflammatory cell infiltration and fibrin deposition play important roles in the development of crescentic glomerulonephritis (GN). In particular, activation of coagulation is an indispensable factor in crescent formation. However, the mechanisms underlying the pathogenesis of crescent formation have not been completely elucidated. We identified the growth factor midkine (MK) as a novel key molecule in the progression of crescentic GN induced by anti-glomerular basement membrane antibody. Despite the lack of significant differences in autologous and heterologous reactions, MK-deficient (Mdk(-/-)) mice unexpectedly showed a greater number of necrotizing glomerular injuries than wild-type (Mdk(+/+)) mice. Likewise, more tubulointerstitial damage was observed in Mdk(-/-) mice, and this damage positively correlated with glomerular injury. Plasminogen activator inhibitor (PAI)-1 was strongly induced in the injured glomerulus of Mdk(-/-) mice, particularly in crescents and endothelial cells. This enhanced PAI-1 production was associated with an increase in inflammatory cell infiltration and matrix deposition in the glomerulus and the interstitium of Mdk(-/-) mice. In line with these invivo data, primary cultured endothelial cells derived from Mdk(-/-) mice exhibited higher PAI-1 mRNA expression on fibrin challenge and less fibrinolysis than Mdk(+/+) mice. In contrast, the expression of plasminogen activators was not affected. Our combined data suggest that MK leads to a blockade of PAI-1, which is closely associated with the suppression of crescentic GN.

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

Mechanism of Nanoparticle Formation from Ternary Coground Phenytoin and Its Derivatives

The mechanism of drug nanoparticle formation of phenytoin (DPH) and its derivatives monomethylphenytoin (MDPH) and dimethylphenytoin (DMDPH) was investigated. The drug, polyvinylpyrrolidone K17 (PVP), and sodium dodecyl sulfate were coground to obtain the ground mixture (GM). The DPH GM was amorphous; however, MDPH and DMDPH GMs contained drug crystals. Spectral changes in infrared and (13)C solid-state nuclear magnetic resonance were observed in the DPH GM, partially observed in the MDPH GM, and hardly observed in the DMDPH GM. Mean particle sizes of the DPH, MDPH, and DMDPH GM nanosuspension were almost the same; however, stability after storage differed in the order of DPH > MDPH > DMDPH. The intermolecular interaction between the drug and PVP reflected not only the crystallinity of the drug in the GM but also the stability of the GM suspension. The size and stiffness of drug nanoparticles were evaluated using atomic force microscopy. Crystallization of the amorphous GM and agglomeration of the primary nanocrystals were observed in the DPH GM suspension. In contrast, primary nanocrystals were observed in the DMDPH GM suspension. The size of the drug nanocrystals formed from the different molecular states of the drug in the GM reflects the agglomerated states in water and stability.

Effect of the Cellular-Type Artificial Oxygen Carrier Hemoglobin Vesicle as a Resuscitative Fluid for Prehospital Treatment

The hemoglobin vesicle (Hb-vesicle) is a cellular-type artificial oxygen carrier showing a resuscitative effect comparable to that of blood transfusion in several animal models. However, the efficacy of Hb-vesicles for resuscitation when the hemorrhage cannot be controlled remains unclear. Therefore, we used Hb-vesicles in a rat hemorrhagic shock model caused by continuous bleeding. For inducing uncontrolled hemorrhage, animals were heparinized and bled from the caudal artery. Fluid resuscitation was subsequently performed with five materials: Hb-vesicle suspension in a 5% albumin (Alb) solution (HbV), washed red blood cells (wRBC) in a 6% hydroxyethyl starch (HES) solution, 5% Alb, 6% HES, and saline (Sal). During the experiment, all animals in the HbV and wRBC groups survived, whereas all those in the Alb and HES groups died. In the Sal group, five of seven animals died. In the HbV and wRBC groups, the heart rate, mean arterial pressure, and blood lactic acid levels were stabilized during resuscitation. Meanwhile, the hematocrit levels of the HbV, Alb, and HES groups showed sharp decreases (HbV: 6.8% ± 1.7%, Alb: 6.8% ± 0.8%, HES: 5.5% ± 0.7% at 100% total circulated blood volume; final hematocrit of the HbV group: 1.5% ± 0.5%). These results suggest that shocked animals can survive longer when the Hb-vesicle supply is maintained and that HbV showed a similar effect to wRBC in maintaining the circulating volume and oxygen metabolism. Continuous infusion of Hb-vesicles may extend the survival of trauma victims with uncontrolled hemorrhage until they have reached a trauma center.

Epidermal Hyperplasia and Appendage Abnormalities in Mice Lacking CD109

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109(-/-)) mice. Although CD109(-/-) mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109(-/-) mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109(-/-) mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109(-/-) mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109(-/-) mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.

Vanadium isotopic composition of the sea squirt (Ciona savignyi)

Vanadium (V) in the sea squirt (Ciona savignyi) from Onagawa Bay, Miyagi, Japan, was isolated and purified through adsorption on a diamine resin and anion and cation exchanges after the dissolution of sea squirt samples with nitric acid and hydrogen peroxide. The 50V/51V isotope ratio of V thus obtained was mass-spectrometrically determined to be from 2.51×10−3 to 2.55×10−3 with the average of 2.53×10−3 by the thermal ionisation technique. This value agreed with those of vanadyl chloride and vanadyl nitrate both prepared from vanadyl sulphate (Wako Pure Chemical Industries, Ltd., Japan) and of V in coastal seawater (Shimokita Peninsula, Aomori, Japan) within experimental uncertainties (standard deviation of±0.04), which suggested that no appreciable V isotope fractionation occurs accompanying V uptake by the sea squirt from sea water.

Organ-specific Sulfation Patterns of Heparan Sulfate Generated by Extracellular Sulfatases Sulf1 and Sulf2 in Mice

Heparan sulfate endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate, thereby regulating cellular signaling. Previous studies have revealed that Sulfs act predominantly on UA2S-GlcNS6S disaccharides and weakly on UA-GlcNS6S disaccharides. However, the specificity of Sulfs and their role in sulfation patterning of heparan sulfate in vivo remained unknown. Here, we performed disaccharide analysis of heparan sulfate in Sulf1 and Sulf2 knock-out mice. Significant increases in ΔUA2S-GlcNS6S were observed in the brain, small intestine, lung, spleen, testis, and skeletal muscle of adult Sulf1(-/-) mice and in the brain, liver, kidney, spleen, and testis of adult Sulf2(-/-) mice. In addition, increases in ΔUA-GlcNS6S were seen in the Sulf1(-/-) lung and small intestine. In contrast, the disaccharide compositions of chondroitin sulfate were not primarily altered, indicating specificity of Sulfs for heparan sulfate. For Sulf1, but not for Sulf2, mRNA expression levels in eight organs of wild-type mice were highly correlated with increases in ΔUA2S-GlcNS6S in the corresponding organs of knock-out mice. Moreover, overall changes in heparan sulfate compositions were greater in Sulf1(-/-) mice than in Sulf2(-/-) mice despite lower levels of Sulf1 mRNA expression, suggesting predominant roles of Sulf1 in heparan sulfate desulfation and distinct regulation of Sulf activities in vivo. Sulf1 and Sulf2 mRNAs were differentially expressed in restricted types of cells in organs, and consequently, the sulfation patterns of heparan sulfate were locally and distinctly altered in Sulf1 and Sulf2 knock-out mice. These findings indicate that Sulf1 and Sulf2 differentially contribute to the generation of organ-specific sulfation patterns of heparan sulfate.

Maternal administration of busulfan before in utero transplantation of human hematopoietic stem cells enhances engraftments in sheep

In utero transplantation (IUT) of human hematopoietic stem cells has been conducted in sheep, which are used as large animal models of human hematopoietic reconstitution and models for clinical IUT; however, the levels of engraftment have generally been low. Busulfan (BU), a myeloablative agent, is often administered to patients before hematopoietic stem cells transplantation to improve the engraftment. In this study, hematopoietic activity was evaluated in adult sheep after administering BU at different doses. Next, pregnant ewes were administered BU, and dams as well as their fetuses were evaluated, as BU readily crosses the sheep placenta. Then, the BU dose with the desired outcomes was selected and administered to pregnant ewes at 2 or 6 days before performing IUT using human cord blood CD34(+) cells. The engraftment was evaluated in recipients that underwent IUT in the presence or absence of BU. As a result, hematopoietic activity was safely and transiently suppressed in adult sheep treated with 5 to 7.5 mg/kg BU. BU crossed the sheep placenta, and fetal sheep were indeed conditioned by administering 3 mg/kg BU to pregnant ewes. Engraftment of human CD34(+) cells in fetal recipients was enhanced when IUT was carried out 6 days post-BU. Up to 3.3% engraftment levels (in terms of bone marrow colony-forming units) were achieved with the IUT of 0.72 to 2.4 million CD34(+) cells when BU was used. BU can be administered to pregnant ewes to effectively condition the fetal recipient for IUT with enhanced engraftment of donor cells.