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ABSTRACT:DNA-chitosan bilayer membranes were designed and prepared as a biomedical adhesive for therapeutic application. Various DNA-chitosan bilayer membranes were prepared by applying chitosan solution (2.0 mg cm −2 ) on UV-irradiated DNA membranes (0.2-0.5 mg cm −2 ). Tensile strengths of the DNA-chitosan bilayer membranes were approximately 4.0 N similar to that of chitosan alone membrane 2.0 mg cm −2 . These results indicate that the bilayer membranes have enough tensile strength as a surgical tape. When the surface of the DNA-chitosan membranes was analyzed using a scanning electron microscope (SEM), fiber-like structure was observed on the DNA-side of the DNA-chitosan membranes, prepared from more than 0.3 mg cm −2 of UV-irradiated DNA membranes. In contrast, the chitosan-side of the DNA-chitosan membranes showed a smooth surface similar to that of the chitosan alone membrane. These results indicated that a DNA-chitosan bilayer membrane was successfully prepared using UV-irradiated DNA membranes (0.3-0.5 mg cm −2 ) and chitosan (2.0 mg cm −2 ). Next, we observed bonding strength of the membranes to rabbit peritoneum.The bonding strength of the chitosan-side was similar to that of fibrin glue. On the other hand, DNA side of bilayer membrane did not adhere to the peritoneum. The DNA-chitosan membrane is bi-functional and has a potential to serve as a bi-functional bio-adhesive. KEY WORDS DNA / Chitosan / Polyion / Complex / Bilayer Membrane / Bio-adhesive / Chitin is a mucopolysaccharide composed of Nacetyl-D-glucosamine by β(1→4) glycoside linkage and presents in the nature as outside skeleton of crustaceans, cell wall of bacteria, and insects.

Amphiphilic random copolymers, poly(R-HPMA-co-S-PAM) and poly(H-PMA-co-S-PAM), were prepared by radical copolymerization of N-methacryloyl-(S)-phenylalanine methyl ester (S-PAM) and N-[(R)-2-hydroxypropyl]methacrylamide (R-HPMA) or N-(2-hydroxypropyl)methacrylamide (HPMA) with various molar ratios of R-HPMA (or HPMA) (m) to S-PAM (n). Either aqueous solution of poly(R-HPMA-co-S-PAM) with the molar ratio of m : n ϭ 0.81 : 0.19 or poly(HPMA-co-S-PAM) with the molar ratio of m : n ϭ 0.79 : 0.21 exhibited the lower critical solution temperature (LCST) at 16°C. The LCST in the presence of (S)-(Ϫ)-phenylalanine (S-Phe) shifted from 16 to 20°C and 18°C for poly(R-HPMA-co-S-PAM) and poly(HPMA-co-S-PAM), respectively, whereas the LCST did not shift in the presence of (R)-(ϩ)-phenylalanine (R-Phe). Thermosensitive Gel(R-HPMA-co-S-PAM) and Gel(HPMA-co-S-PAM) were also prepared from radical copolymerization of S-PAM and R-HPMA or HPMA in the presence of N,NЈ-ethylenebisacrylamide (EBAAm) as a crosslinker. When the gels shrunk at 40°C, the release of dansyl-(R)-phenylalanine (Dans-R-Phe) from the gel in which loaded Dans-R-Phe occurred was more easily done than that of Dans-S-Phe from the gel that loaded Dans-S-Phe. Thus, these thermosensitive copolymers and gels were found to exhibit chiral recognition for phenylalanine derivatives.

Background:Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. Objectives:We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. Methods:We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. Results: cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. Conclusions:We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis.

Protease activities in allergen sources are thought to be involved in triggering allergic inflammation through the disruption of epithelial barrier or the induction of proinflammatory cytokines. Protease allergens may also work as type 2 helper T cell (TH2) adjuvants through the cleavage of cell surface receptors. Here, we report molecular cloning and immunochemical characterization of a new Japanese cedar (Cryptomeria japonica) pollen allergen (CPA9) homologous to serine protease, which is initially found as a high IgE-binding spot on our two-dimensional (2-D) IgE immunoblotting map. The cpa9 cDNA encoded a 757 amino acid polypeptide showing a significant sequence identity with plant subtilisin-like serine protease family members including melon major allergen Cuc m 1. We found that native CPA9 purified from C. japonica pollen showed a high IgE-binding frequency and IgE cross-reactivity with melon extract.