Kousaku Ohkawa

Preparation and characterization of DNA films induced by UV irradiation.

Large amounts of DNA-enriched materials, such as salmon milts and shellfish gonads, are discarded as industrial waste. We have been able to convert the discarded DNA to a useful material by preparing novel DNA films by UV irradiation. When DNA films were irradiated with UV light, the molecular weight of DNA was greatly increased. The reaction was inhibited by addition of the radical scavenger galvinoxyl suggesting that the DNA polymerization with UV irradiation proceeded by a radical reaction. Although this UV-irradiated DNA film was water-insoluble and resistant to hydrolysis by nuclease, the structure of the DNA film in water was similar to non-irradiated DNA and maintained B-form structure. In addition, the UV-irradiated DNA film could effectively accumulate and condense harmful DNA-intercalating compounds, such as ethidium bromide and acridine orange, from diluted aqueous solutions. The binding constant and exclusion number of ethidium bromide for UV-irradiated DNA were determined to be 6.8 +/- 0.3 x 10(4) M(-1) and 1.6 +/- 0.2, respectively; these values are consisted with reported results for non-irradiated DNA. The UV-irradiated DNA films have potential uses as a biomaterial filter for the removal of harmful DNA intercalating compounds.

UV-irradiation-induced DNA immobilization and functional utilization of DNA on nonwoven cellulose fabric

Immobilization of double-stranded DNA onto nonwoven cellulose fabric by UV irradiation and utilization of DNA-immobilized cloth were examined. The immobilized DNA was found to be stable in water, with the maximum amount of fabric-immobilized DNA being approximately 20 mg/g of nonwoven fabric. The DNA-immobilized cloth could effectively accumulate endocrine disruptors and harmful DNA intercalating pollutants, such as dibenzo-p-dioxin, dibenzofuran, biphenyl, benzo[a]pyrene and ethidium bromide. Additionally, DNA-immobilized cloth was found to bind metal ions, such as Ag+, Cu2+, and Zn2+. The maximum amounts of bound Ag+, Cu2+, and Zn2+ onto DNA-immobilized cloth (1 g) were approximately 5, 2, and 1 mg, respectively. DNA-immobilized cloth containing Ag+ showed antibacterial activity against Escherichia coli and Staphylococcus aureus. DNA-immobilized cloth without metal ion and with Cu2+ or Zn2+ did not show antibacterial activity. These results suggest that immobilized DNA imparts useful functionality to cloth. DNA-immobilized cloth prepared by UV irradiation has potential to serve as a useful biomaterial for medical, engineering, and environmental application.

Preparation of Pure Cellulose Nanofiber via Electrospinning

We describe the preparation of cellulose nanofibrous material directly from cellulose solution via electrospinning. The resulting nanofiber is composed of pure cellulose. The spinning procedure can be performed under ambient conditions at room temperature without post-spun treatment. By mixing drugs with the pre-spun cellulose solution, followed by electrospinning, the drug-loaded nanofiber was prepared and the releasing properties were examined with respect to biomedical applications.

A Glycosylated Byssal Precursor Protein from the Green Mussel Perna viridis with Modified Dopa Side-chains

Foot tissue of the green mussel Perna viridis contains a variety of byssal precursor proteins with the unusual redox-active amino acid, Dopa (L-β-3,4-dihydroxyphenyl-α-alanine). Eight proteins were detectable in acidic extracts of the Perna foot by a redox cycling assay with nitroblue tetrazolium. In one of these, however, P. viridis foot protein-1 (Pvfp-1), activity was not due to Dopa, but to another redox-active derivative. Based on specific colorimetric derivatization with Arnows reagent, ninhydrin and phenylisothiocyanate (Edman), mass spectrometry, the redox-active derivative in Pvfp-1 is not consistent with any known modification. Another uncommon modification of Pvfp-1 involves O-glycosylation of threonine by mannose, glucose or fucose. As in previously characterized fp-1s, the primary sequence of the Pvfp-1 (apparent mass 89 kDa) has two consensus decapeptide motifs; one is APPKPX1TAX2K and the other is APPPAX1TAX2K, where P is Pro/Hyp, and X1 and X2 are difucosylated threonine and a redox sensitive derivative of tyrosine or Dopa, respectively. Of these two unusual residues, X2 is unique to Pvfp-1, whereas O-glycosylated Thr has been previously detected in freshwater mussel fp-1. The sequence homology of Pvfp-1 with the common structural motifs of the fp-1 protein family strongly suggests that the Pvfp-1 functions as the byssal coating (lacquer) protein.

Studies on fouling by the freshwater mussel limnoperna fortunei and the antifouling effects of low energy surfaces

Attachment of the freshwater mussel, Limnoperna fortunei, was tested using non‐treated surfaces, viz. glass, nylon, rubber, silicone and Teflon, together with glass surfaces modified with nine kinds of silane coupling agents. Among the surfaces tested, the mussel avoided attaching to Teflon, silicone, and glass modified with 3‐bromopropyltrimethoxysilane or 3,3,3‐(trifluo‐ropropyl)‐trimethoxysilane. With respect to the relationship between the percentage attachment and the surface free energy (sfe) of the substrates, it was found that attachment was considerably reduced on the substrates which exhibited relatively low sfe, as above. The mean number of secreted byssuses per attaching mussel also decreased with decreasing substrate sfe. Furthermore, when the sfe was divided into the dispersion and polar components, the percentage mussel attachment was related to the polar component. These results suggest that effective antifouling towards L. fortunei is achieved on substrates with a low sfe polar component.

Polyion complex fiber and capsule formed by self-assembly of poly-L-lysine and gellan at solution interfaces

Different characteristic surface structures such as capsules, regularly spaced droplets, and fibers are formed by electrostatic interaction between poly-L-lysine (PLL) and gellan gum via polyion complex (PIC) formation. Spherical droplet PIC capsules of varying diameters form in solutions. Some dyes adsorb on the surface of the capsules, and other dyes penetrate into the capsules. The strong PIC fiber can be spinnable by gravity and by wet spinning in ethanol. This fiber possesses a counterion pairing structure and exhibits the nervation/veining pattern and hollow yarn. The tensile strength of the fiber is 27.8 kg/mm2 [1.40 g/denier (d)] and the knotting strength is 9.98 kg/mm2 (1.13 g/d). By using an organic crosslinking agent, epichlorohydrin, the tensile strength can be increased to 38.5 kg/mm2 (2.46 g/d) and the knotting strength can be increased to 12.2 kg/mm2 (1.99 g/d). The PIC fiber can be dyed by five different dyeing procedures such as direct and vat dyeings. The PLL PIC fiber is water insoluble and has potential as a new synthetic polypeptide fiber technology.

Biodegradation of ornithine-containing polylysine hydrogels

The degradation of the cross-linked cationic poly(amino acid)-glutaraldehyde (GA) hydrogels by two kinds of proteolytic enzymes, trypsin and Aspergillus Protease Type XXIII, and by seven species of soil filamentous fungi has been investigated using homo- and copolypeptides of lysine (Lys) and ornithine (Orn). Trypsin degraded the hydrogels prepared from poly(Lys) and copoly(Lys Orn)s but not poly(Orn), while Aspergillus protease degraded all of them. Degradation time of hydrogels by the two proteases became longer with increasing Orn content in the gel. Seven species of soil filamentous fungi were cultured with hydrogels on Czapeck medium to evaluate the degree of microbial degradation of the hydrogels, and the three species of the fungi, Aspergillus oryzae, Penicillium citrinum and Curvularia sp., were grown in culture with an accompanying degradation of the gel matrix, while the other four species, Mucor sp., Rhizopus sp., Cladosporium sp., and Trichoderma sp., were not. The degree of degradation of gel matrix with growth of the three fungi became lower with increasing Orn content in the gel matrix. The results might offer some clues to the applications for the controlled biodegradation of cationic poly(amino acid) hydrogel by introduction of Orn, suggesting that unnatural amino acid resists hydrolysis by proteases or microorganisms.

Nanofibers of Cellulose and Its Derivatives Fabricated Using Direct Electrospinning

A short review with 49 references describes the electrospinninng (ES) process for polysaccharides, cellulose and chitosan, and their derivatives, including cellulose acetate and hydroxypropyl cellulose. A majority of applied studies adopted a two step-process, in which the cellulose acetate was used for the first ES process, followed by acetyl group removal to regenerate cellulose thin fibers. The electrospun nonwoven fabrics (ESNW) of regenerated cellulose can be modified by introduction of aldehyde groups by oxidative cleavage of vicinal diols using periodates, and these aldehyde groups serve as acceptors of foreign substances, with various chemical/biological functions, to be immobilized on the fiber surfaces in the ESNW matrices. Direct electrospinning of cellulose from trifluroacetic acid solution was also developed and the applied studies were summarized to conclude the current trends of interests in the ES and related technologies.

Polyion complex fiber and capsule formed by self-assembly of chitosan and poly(α, L-glutamic acid) at solution interfaces

Different characteristic surface structures such as spherical capsules, regularly spaced droplets and fibers are formed by electrostatic polysaccharide interaction between chitosan and poly(α,L-glutamic acid) via polyion complex (PIC) formation. Spherical droplet-like PIC capsules of varying diameters form in solutions. Some dyes absorb on the surface of the capsules, while other dyes and benzoic acid derivatives penetrate into the capsules. The PIC fiber can be prepared by reactive-spinning at the solution interface, followed by gravity and by removing water in ethanol. The tensile strength of the fiber is 11.2 kg . mm -2 (1.48 g . denier -1 ) and the knotting stength is 2.15 kg . mm -2 (0.30 g . d -1 ). By reaction with an organic cross-linking agent, ethylene glycol diglydyl ether (EGDE), the tensile strength of the fiber can be increased to 18.1 kg . mm -2 (3.14 g . d -1 ), and by reacting with hexamethylene diisocyanate (HMDI), the knotting strength of the fiber can be increased to 7.58 kg . mm-2 (0.89 g . d -1 ). The interaction energies (work of absorption, W ads ) of PLG and chitosan in aqueous solution xere estimated by a surface chemical aaprroach, and a relationship between the W ads values and the tensile strength of the PIC fibers and the Wads indicated that the higher W ads system gives the higher tensile strength. The PIC fiber can be dyed by four different dyeing procedure such as direct and vat dyeings. When the cross-linked PIC fibers was incubated in an aqueous solution in the presence of endocrine disruptor related compounds such as dibenzofuran and biphenyls, these compounds were accumalated onto and/or into the fibers.

Tissue adhesive using synthetic model adhesive proteins inspired by the marine mussel

The surface free energy and its dispersion and polar components of pigskin were determined by wettability measurements. The contact angles and work of adhesion of solutions of the synthetic model adhesive sequence poly(Gly-Tyr-Lys) inspired by marine adhesive proteins were measured on the epidermis and the dermis of pigskin. Also the surface free energy of pigskin was determined using contact angles of certain probe liquids. When a poly(Gly-Tyr-Lys) buffer solution containing tyrosinase as a bioadhesion formulation was used to close an incision of a living pig, a good incision adhesion and reduced immunological response after 1 week were observed from photographs using an optical microscope and the amount of macrophages by image analysis.