Yasutaka Morita

Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp.

A novel keratin-degrading bacterium Stenotrophomonas sp. strain D-1, isolated from deer fur, produced two types of extracellular proteins: proteolytic and disulfide bond-reducing. The results on the biochemical properties suggest that this protease belongs to the serine protease, and the disulfide bond-reducing protein could be the disulfide reductase type. None of these enzymes showed keratinolytic activity independently. However, after mixing of the two enzymes, the keratinolytic activity was increased tremendously (more than 50-fold) over that of the protease only. This keratinolytic activity was more than 2-fold higher than that of the combination with proteinase K (also known for its high keratinolytic activity). Since the two enzymes discovered in this study acted cooperatively and resulted in higher keratinolytic activity, a new mechanism of keratin degradation has been revealed. To our knowledge, this is the first report on the cooperative action of two enzymes resulting in the effective degradation of keratin.

Localized surface plasmon resonance based optical biosensor using surface modified nanoparticle layer for label-free monitoring of antigen–antibody reaction

Abstract In recent years, label-free biosensors not requiring external modifications have been receiving intense attention. A label-free optical biosensor, which retains many of the desirable features of conventional surface plasmon resonance (SPR) reflectometry, namely, the ability to monitor the kinetics of biomolecular interactions in real-time without a label has been developed with several important advantages: the biosensor device is easy to fabricate, and simple to implement, requiring only an UV–Vis spectrophotometer or flatbed scanner. Importantly, the label-free optical biosensor can be easily multiplexed to enable high-throughput monitoring of biomolecular interactions in an arraybased format. In this research,the development of a localized surface plasmon resonance (LSPR)-based label-free optical biosensor using a surface modified nanoparticle layer is aimed. This optical detection method promises to offer a massively parallel detection capability in a highly miniaturized package. The two-dimensional nanoparticle layer was formed by the surface modified silica nanoparticles. The optical properties and surface analysis of nanoparticle layer substrate were characterized through transmissionmeasurements and atomic force microscopy (AFM). Simultaneously, the nanoparticle layer substrate was applied to the optical LSPR-based biosensor for label-free monitoring of the antigen–antibody reaction. The anti-fibrinogen antibody wasimmobilized onto the nanoparticle layer substrate surface. Different concentrations of fibrinogen were introduced to the anti-fibrinogen antibody immobilized nanoparticle layer substrate surface, and the change in the absorption spectrum, caused by the antigen–antibody reaction, was observed. By using this anti-fibrinogen antibody immobilized nanoparticle layer substrate; the detection limit of this optical LSPR-based biosensor was 10 ng/ml.

Characterization of a new keratin-degrading bacterium isolated from deer fur

A keratin-degrading bacterium was isolated from soil containing deer fur. An axenic culture of the keratin-degrading bacterium was obtained in liquid culture using a keratin enrichment technique. The isolated bacterium was gram negative and catalase- and oxidase-positive. Transmission electron microscopic observations showed that the bacterium was rod-shaped, 1.0-1.3 microm long and 0.7 microm in diameter. Phylogenetic analysis of 16S rDNA revealed that the new isolate has only 90.6% homology with Stenotrophomonas nitritireducens. Hence, this new bacterium was designated as Stenotrophomonas sp. D-1. The optimum temperature was determined to be 20 degrees C for maximum growth and keratinolytic enzyme production. Amino acid data, obtained after treating keratin powder with the supernatant culture, suggest that the major free amino acids resulting from keratin degradation are phenylalanine, tyrosine and valine. In addition, native chicken feather was degraded completely at 20 degrees C in 2.5 d by this bacterium.

Cell separation by an aqueous two-phase system in a microfluidic device

We generated an aqueous two-phase laminar flow in a microfluidic chip and used the system to isolate leukocyte and erythrocyte cells from whole blood cells. The microfluidic system reduced the effect of gravity in the aqueous two-phase system (ATPS). Poly(ethylene glycol) (PEG) and dextran (Dex) solutions were used as the two phases, and the independent flow rates of the solutions were both 2 microL/min. When hydrophobic and hydrophilic polystyrene beads were introduced into the microfluidic device, the hydrophilic beads moved to the Dex layer and the hydrophobic beads to the interface between the two phases. In the case of living cells, Jurkat cells and erythrocytes moved more efficiently to the PEG and Dex layers, respectively, than they move in a conventional ATPS. When whole blood cells were inserted into the microfluidic chip, leukocytes could be separated from erythrocytes because erythrocytes moved to the Dex layer while leukocytes remained outside of this layer in the microfluidic system. The reported microfluidic chip for the whole blood cell separation can effectively be integrated into a Micro Total Analysis System designed for cell-based clinical, forensic, and environmental analyses.

Multianalyte immunoassay with self-assembled addressable microparticle array on a chip.

This paper describes the random fluidic self-assembly of metallic particles into addressable two-dimensional microarrays and the use of these arrays as a platform for constructing a biochip useful for bioassays. The basic units in the assembly were the microfabricated particles carrying a straightforward visible code and the corresponding array template patterned on a glass substrate. The particles consisted of a hydrophobic and magnetic Ni-polytetrafluoroethylene (PTFE) composite layer on one face, and on the other face a gold layer that was modified for biomolecular attachment. An array template was photoresist-patterned with spatially discrete microwells in which an electrodeposited Ni-PTFE hydrophobic composite layer and a hydrophobic photo-adhesive coating were deposited. The particles, after biomaterial attachment and binding processes in bulk, were self-assembled randomly onto the lubricated bonding sites on the chip substrate, driven by a combination of magnetic, hydrophobic, and capillary interactions. The encoding symbol carried by the particles was used as the signature for the identification of each target/assay attached to the particle surface. We demonstrate here the utility of microfabricated-encoded particle arrays for conducting multianalyte immunoassays in a parallel fashion with the use of imaging detection.

Properties of a cold-active protease from psychrotrophic Flavobacterium balustinum P104

Abstract Protease activity was detected in the culture medium of Flavobacterium balustinum P104 grown at 10 °C, which was isolated from salmon (Oncorhynchus keta) intestine. The enzyme, designated as CP-70 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographyies. The molecular mass of the protease was 70 kDa, and its isoelectric point was close to 3.5. Maximal activity toward azocasein was observed at 40 °C and from pH 7.0 to 9.0. The activity was strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that the enzyme is a serine protease. The n-terminal amino acid sequence was Asp-Thr-Arg-Gln-Leu-Leu-Asn-Ala-Asn-Ser-Asp-Leu-Leu-Asn-Thr-Thr-Gly-Asn-Val-Thr-Gly-Leu-Thr-Gly-Ala-Phe-Asn-Gly-Glu-Asn. A search through the database for sequence homology yielded no significant match. The initial cleavage sites for oxidized insulin B-chain were found to be the Glu13-Ala14 and Phe24-Phe25 bonds. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-70 protease has a broader specificity than the other cold-active proteases against the peptide substrate.

Effects of bis(2-ethylhexyl) phthalate and benzo[a]pyrene on the embryos of Japanese medaka (Oryzias latipes)

Effects of two widely found chemical pollutants, bis(2-ethylhexyl) phthalate (DEHP) and benzo[a]pyrene (BaP), on the embryos of Japanese medaka were investigated. The embryos were exposed to different concentrations (0.01, 0.1, 1, and 10μg/l) of DEHP and BaP. The following were investigated: (1) hatching time and hatching rate in embryos, (2) mortality, sex ratio, body weight and gonadosomatic index (GSI) in adulthood. These two chemicals delayed the hatching time without dose-dependence, but these chemicals had no effect on hatching rate. Mortality was raised and body weight was reduced by DEHP and BaP-treatment; distortion of sex ratio appeared at the lowest concentration of DEHP tested. GSI was decreased because of the BaP-treatment. DEHP and BaP negatively affected Japanease medaka embryos, and the influences of the effects continued into adult stage. Moreover, the effects did not appear to be necessarily dose-dependent.

Effects of tamoxifen, 17α-ethynylestradiol, flutamide, and methyltestosterone on plasma vitellogenin levels of male and female Japanese medaka (Oryzias latipes)

The effects of oral administration of tamoxifen, 17α-ethynylestradiol (EE2), flutamide, and methyltestosterone (MT), on plasma vitellogenin levels of male and female medaka were investigated. Medaka were fed diets containing different concentrations of these chemicals for 7 days, and these plasma vitellogenin levels were measured. Tamoxifen increased significantly the vitellogenin levels in male, but inhibited the normal vitellogenin induction in female in the high concentration groups. EE2 increased significantly vitellogenin levels in both sexes. Flutamide increased significantly the vitellogenin levels in female, but gave no effects on male. MT inhibited the normal vitellogenin induction in female, but increased slightly vitellogenin levels in male without a clear tendency. Administration of tamoxifen, EE2, flutamide, and MT showed the different pattern in vitellogenin levels in both sexes.

Peptide nucleic acid-modified carbon nanotube field-effect transistor for ultra-sensitive real-time detection of DNA hybridization

We have developed a nanosensor array composed of carbon nanotube field-effect transistors (CNTFETs) on SiO2/Si substrates. Unlike previously reported CNTFETs where the recognition event occurred directly on the CNT, in this case, the reverse surface of the substrate was utilized for the biomolecular functionalization. A self-assembled monolayer (SAM) of peptide nucleic acid (PNA) probes associated with the tumor necrosis factor-α gene (TNF-α) was attached onto the gold electrode on the reverse side of the CNTFET device. A time-dependent conductance increase was monitored upon sequential introduction of wild-type (WT) DNA samples through a microfluidic channel of the poly(dimethylsiloxane) (PDMS) chip. High selectivity of PNA probes only toward the full-complementary WT DNA samples enabled rapid and simple discrimination against single-nucleotide polymorphism (SNP) or non-complementary (NC) DNA. Concentration-dependent measurements indicated a limit-of-detection (LOD) of 6.8 fM WT DNA. Our CNTFET-based biochip is a promising candidate for the development of an integrated, high-throughput, portable device for nucleic acid-based diagnostics.

Effect of alkylphenols on adult male medaka: plasma vitellogenin goes up to the level of estrous female

The effect that oral administration of four alkylphenols, (1) bisphenol A (BPA), (2) p-t-octylphenol (OP), (3) p-nonylphenol (NP) and (4) p-n-nonylphenol (n-NP), as well as 17α-ethynylestradiol (EE2) had on male medaka fish vitellogenin was investigated. The male medaka was fed diets containing different concentrations of these chemicals for 7 days, after which their plasma vitellogenin levels were measured. Vitellogenin levels up to ≈10(7) ng/ml were found. This value is close to that of the normal estrous female medaka. The median effective concentration (EC(50)) values resulting from BPA, OP, NP and EE2 in the diet were calculated as 1600, 2600, 940 and 0.37 μg/g diet, respectively.