Isolation of a novel alkaline-induced laccase from Flammulina velutipes and its application for hair coloring

Laccase is a member of the multi-copper oxidase family and a promising for hair coloring. In this study, we isolated a novel alkaline-induced laccase from the white-rot fungus Flammulina velutipes and studied the possibility to apply the enzyme for hair coloring. Laccase activity detected in the culture supernatant of F. velutipes was found to significantly increase when exchanging the medium to laccase inducing one whose pH was adjusted to 9.0. Three isozymes were detected by activity staining on non-denaturing SDS-PAGE. The major isozyme, Flac1, was purified from the culture supernatant after being induced at pH 9.0 by ion-exchange column chromatography. The N-terminal peptide sequence of Flac1 was determined, revealing clear homology with laccases from other white-rot fungi. Optimum pH of oxidation was found to be around pH 5.0-6.5 regardless of several different substrates used. Oxidation activities of Flac1 to several hair dye agents as substrate showed the higher activity at pH 6.5 than that at pH 9.0. Oxidation activity was also detected at pH 9.0 which was suitable for hair coloring. When the purified Flac1 was applied for hair coloring system without using hydrogen peroxide, effective coloring was observed at the protein amount of 0.25mg/1g of hair used. These results indicated that this alkaline-induced novel laccase isolated from the culture supernatant of F. velutipes might be a useful enzyme for hair color.     

Precise synthesis of asymmetric star-shaped polymers by coupling reactions of new specially designed polymer anions with chain-end-functionalized polystyrenes with benzyl bromide moieties

We have developed a novel methodology using polymer anions specially designed to comprise either of the three same or different polymer segments for the synthesis of well-defined regular and asymmetric star-shaped polymers. The polymer anion was prepared by the addition reaction of living anionic polymer to in-chain-functionalized polymer with 1,1-diphenylethylene (DPE) moiety and in situ coupled with chain-end-functionalized polystyrene with two or four benzyl bromide moieties. Although the anions located at the cores of 3-armed star polymers were believed to be highly sterically hindered, the coupling reactions proceeded virtually quantitatively under the conditions in THF at – 78 °C for 24 h. Regular 7-armed A₇ and 13-armed A₁₃ star-shaped polystyrenes as well as quite new asymmetric 7-armed A₂B₂C₂D and 13-armed A₄B₄C₄D star-shaped polymers were synthesized in ca. 100% yields. Four polymer segments, A, B, C, and D, were, poly(4-trimethylsilylstyrene), poly(4-methoxystyrene), poly(4-methylstyrene), and polystyrene prepared by sec-BuLi-initiated living anionic polymerization. Thus, three same or different polymer chains could be simultaneously introduced into either two or four benzyl bromide reaction sites only by one-step coupling reaction with the above-prepared polymer anion. The resulting star-shaped polymers all were well-defined in architecture and precisely controlled in-chain length and composition. Accordingly, they have a high molecular weight and structural homogeneity. Among them, asymmetric star-shaped polymers synthesized herein are the first successful examples comprised of four plural different polymer segments. As expected, new morphologies with nanoscopic ordered structures applicable to nanotechnology have been expressed.     

New gel-type polyolefin electrolyte film for rechargeable lithium batteries

A random poly(ethylene-co-acrylic acid) (PE-A) with an acrylic acid (AA) content of 5.0–20 mol% was functionalized by esterifying acrylic acid group with poly(ethylene glycol) monomethyl ether. Polyethylene oxide functional groups such as a pendant were introduced into the polyethylene backbone chain. The resulting polymer (PEGM-g-EAA) can be easily formed to a thin sheet and possesses the adhesion property such as gluing. Its thin film could absorb and hold a large quantity of the electrolyte solutions employed for the lithium batteries. The ionic conductivity of the PEGM-g-EAA gel electrolyte obtained with the starting PE-A with acrylic acid content of 9.0 mol% was a value of around 1.5 × 10⁻³ S cm⁻¹ at 20 °C. The ionic conductivity results obtained for the network type gel, which was entangled with the present PE-A-based polymer, were 1.1 × 10⁻³ S cm⁻¹ and 5.5 × 10⁻³ S cm⁻¹ at 0 °C and 80 °C, respectively. The characteristics of good thermostability, transparency and good adhesion to the electrodes have also been demonstrated. As an example, the test cell consisted of the proposed polyolefin gel electrolyte, a LiCoO₂ cathode and a lithium anode showed excellent charge/discharge characteristics.     

A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter

The ability to monitor the development of a given cell lineage in a non-invasive manner by fluorescent markers both in vivo and in vitro provides a great advantage for the analysis of the lineage of interest. To date, a number of transgenic or knock-in mouse strains, in which developing germ cells are marked with fluorescent reporters, have been generated. We here describe a novel double transgenic reporter mouse strain that expresses membrane-targeted Venus (mVenus), a brighter variant of yellow fluorescent protein (YFP), under the control of Prdm1 (Blimp1) regulatory elements and enhanced cyan fluorescent protein (ECFP) under the control of Dppa3 (Stella/Pgc7). The double transgenic strain unambiguously marked Prdm1 expression in the lineage-restricted precursors of primordial germ cells (PGCs) in the proximal epiblast at embryonic day (E) 6.25 and specifically illuminated Prdm1- and Dppa3-positive migrating PGCs after E8.5. The double transgenic reporter also precisely recapitulated dynamic embryonic expression of Prdm1 outside the germ cell lineage. Moreover, we derived ES cells that bore both transgenes. These cells made a robust contribution both to the germ and somatic cell lineages in chimeras with accurate Prdm1-mVenus and Dppa3-ECFP expression. The transgenic strain and the ES cells will serve as valuable experimental materials not only for analyzing the origin and properties of the germ cell lineage in vivo, but also for establishing a culture system to efficiently induce proper germ cells with temporally coordinated Prdm1 and Dppa3 expression in vitro.     

Liposome and lipid bilayer arrays towards biosensing applications

Sensitive and selective biosensors for high-throughput screening are having an increasing impact in modern medical care. The establishment of robust protein biosensing platforms however remains challenging, especially when membrane proteins are involved. Although this type of proteins is of enormous relevance since they are considered in >60% of the pharmaceutical drug targets, their fragile nature (i.e., the requirement to preserve their natural lipid environment to avoid denaturation and loss of function) puts strong additional prerequisites onto a successful biochip. In this review, the leading approaches to create lipid membrane-based arrays towards the creation of membrane protein biosensing platforms are described. Liposomes assembled in micro- and nanoarrays and the successful set-ups containing functional membrane proteins, as well as the use of liposomes in networks, are discussed in the first part. Then, the complementary approaches to create cell-mimicking supported membrane patches on a substrate in an array format will be addressed. Finally, the progress in assembling free-standing (functional) lipid bilayers over nanopore arrays for ion channel sensing will be reported. This review illustrates the rapid pace by which advances are being made towards the creation of a heterogeneous biochip for the high-throughput screening of membrane proteins for diagnostics, drug screening, or drug discovery purposes.     

Measurement of space charge distribution in epoxy resin

Internal space charge behavior of insulating materials has recently attracted attention of many researchers, and a large number of experimental studies were carried out by using the materials for dc cables, such as XLPE, LDPE, and HDPE. Epoxy resins are used for insulation under strong electric fields in power apparatus and in electronic devices, and we investigated the behavior of internal space charge using the pulsed electroacoustic method. Two types of epoxy resins were studied: insulation-grade and craft-grade. When dc electric fields were applied to the craft resins treated by immersing them into room-temperature water for 8 and 24 h, positive and negative charges accumulated near the anode and the cathode, respectively, and the charge distribution changed with the immersion time. On the other hand, no charge was observed in the insulation-grade epoxy resin. Next, we treated the samples by immersing them into 100 °C water for 8 h. When the sample was treated for 8 h, hetero charge distribution, which means positive charges near the cathode and negative charges near the anode, was observed. This result is consistent with a previous paper reporting that by chemical analysis, secondary decomposition had occurred. These results show that water and temperature influence the internal space charge behavior of epoxy resins. © 1999 Scripta Technica, Electr Eng Jpn, 129(3): 9–16, 1999     

Crystallographic Data of a New Phase of Dicalcium Silicate

Unit-cell parameters and the space group of a new phase of dicalcium silicate (Ca₂SiO₄) were determined by using powder X-ray diffractometry and selected-area electron diffraction techniques. This phase could be synthesized via the dissociation of hydrothermally synthesized alpha-Ca₂(SiO₄H)OH at temperatures of ∼500°-920°C. Crystallographic data for the sample synthesized at 600°C were as follows: Ca₂SiO₄, monoclinic; P 2₁/ c space group; lattice parameters of a = 0.82147(9) nm, b = 0.9808(1) nm, c = 0.9741(1) nm, and β= 94.642(7)°; cell volume ( V ) of 0.7857(1) nm³; Z = 8 (where Z is the number of chemical formula units in a unit cell); and a density of 2.91 g/cm³. The crystallographic data for samples synthesized at 800°C had slightly different unit-cell parameters of a = 0.82124(6) nm, b = 0.97348(7) nm, c = 0.97935(7) nm, β= 94.831(5)°, and V = 0.7849(1) nm³. Structural relationships of the new phase with the other dicalcium silicates are discussed.     

Methotrexate Alters the Expression of microRNA in Fibroblast-like Synovial Cells in Rheumatoid Arthritis

We aimed to investigate the effect of methotrexate (MTX) on microRNA modulation in rheumatoid arthritis fibroblast-like synovial cells (RA-FLS). RA-FLS were treated with MTX for 48 h. We then performed miRNA array analysis to investigate differentially expressed miRNAs. Transfection with miR-877-3p precursor and inhibitor were used to investigate the functional role of miR-877-3p in RA-FLS. Gene ontology analysis was used to investigate the cellular processes involving miR-877-3p. The production of cytokines/chemokines was screened by multiplex cytokine/chemokine bead assay and confirmed by ELISA and quantitative real-time PCR. The migratory and proliferative activities of RA-FLS were analyzed by wound healing assay and MKI-67 expression. MTX treatment altered the expression of 13 miRNAs (seven were upregulated and six were downregulated). Among them, quantitative real-time PCR confirmed that miR-877-3p was upregulated in response to MTX (1.79 ± 0.46-fold, p < 0.05). The possible target genes of miR-877-3p in RA-FLS revealed by the microarray analysis were correlated with biological processes. The overexpression of miR-877-3p decreased the production of GM-CSF and CCL3, and the overexpression of miR-877-3p inhibited migratory and proliferative activity. MTX altered the miR-877-3p expression on RA-FLS, and this alteration of miR-877-3p attenuated the abundant production of cytokines/chemokines and proliferative property of RA-FLS.     

Stereoselective Synthesis of 24-Fluoro-25-Hydroxyvitamin D3 Analogues and Their Stability to hCYP24A1-Dependent Catabolism

Two 24-fluoro-25-hydroxyvitamin D₃ analogues (3,4) were synthesized in a convergent manner. The introduction of a stereocenter to the vitamin D₃ side-chain C24 position was achieved via Sharpless dihydroxylation, and a deoxyfluorination reaction was utilized for the fluorination step. Comparison between (24R)- and (24S)-24-fluoro-25-hydroxyvitamin D₃ revealed that the C24-R-configuration isomer 4 was more resistant to CYP24A1-dependent metabolism than its 24S-isomer 3. The new synthetic route of the CYP24A1 main metabolite (24R)-24,25-dihydroxyvitamin D₃ (6) and its 24S-isomer (5) was also studied using synthetic intermediates (30,31) in parallel.