Kozue Kato

Laminin-1 peptide-conjugated chitosan membranes as a novel approach for cell engineering

Laminin, a major component of the basement membrane, has diverse biological activities. Recently, we identified various biologically active sequences on laminin‐1 by using a large set of synthetic peptides. Chitosan, a polysaccharide, is biodegradable and has been used as a biomaterial. Here, we conjugated several biologically active laminin peptides onto chitosan membranes and measured the cell attachment activity of peptide‐conjugated chitosan membranes with various cell types. The active laminin peptide‐conjugated chitosan membranes promoted cell attachment with cell type specificity. A99 (AGTFALRGDNPQG)‐chitosan membrane promoted cell attachment with well‐organized actin stress fibers. This adhesion was inhibited by EDTA but not by heparin. AG73 (RKRLQVQLSIRT)‐chitosan membrane promoted cell attachment with filopodia formation, and this adhesion was inhibited by heparin but not by EDTA. These data suggest that the A99‐chitosan membrane interacted with an integrin cellular receptor and that the AG73‐chitosan membrane promoted proteoglycan‐mediated cell attachment, as previously reported. Furthermore, both AG73‐chitosan and A99‐chitosan membranes effectively promoted neurite outgrowth with PC12 rat pheochromocytoma cells. We conclude that conjugation on a chitosan membrane is applicable for testing quantitatively the biological activity of synthetic peptides and that these constructs have a potential ability to serve as bioadhesive materials for tissue regeneration and engineering.

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.

Ile-Lys-Val-Ala-Val (IKVAV)-containing laminin α1 chain peptides form amyloid-like fibrils

The Ile‐Lys‐Val‐Ala‐Val (IKVAV) sequence derived from laminin‐1 promotes cell adhesion, neurite outgrowth, and tumor growth and metastasis. Here, we examined amyloid formation of an IKVAV‐containing peptide (LAM‐L: AASIKVAVSADR, mouse laminin α1 chain 2097–2108). The LAM‐L peptide was stained with Congo red and exhibited fibrils in electron microscopy with a characteristic cross‐β X‐ray diffraction pattern. Further, infrared spectra of LAM‐L suggested a β‐sheet structure. These results indicate that LAM‐L forms amyloid‐like fibrils. We also examined amyloid‐like fibril formation of LAM‐L analogs. The neurite outgrowth activity of the LAM‐L analogs was closely related to their amyloid‐like fibril formation.

Cell Attachment and Neurite Outgrowth Activities of Laminin Peptide-Conjugated Chitosan Membrane

Basement membranes have been found to play a critical role in tissue development and repair. Laminin, a major cell adhesive protein of the basement membrane matrix, has multiple biological activities [1]. There are at least twelve isoforms of laminin (laminin-1 to-12), each consisting of three different chains α, β, and γ [1]. The most extensively characterized laminin, laminin-1 (Mr = 900,000), consists of α1, β1, and yl chains, which assemble into a triple-stranded coiled-coil structure at the long arm to form a cross-like structure [1], Laminin-1 has multiple biological activities including promotion of cell adhesion, spreading, proliferation, neurite outgrowth, angiogenesis, and tumor metastasis [1]. Recently, we demonstrated a systematic peptide screening for identification of cell binding sites from the laminin-1 molecule using 673 overlapping peptides [2–5]. Approximately twenty different cell-binding sequences with various biological functions were identified. Five peptides (A13, A99, A208, AG73, and CI6) showed unusually strong cell attachment activity as well as additional biological functions (Figure 1). Al3 and C16 inhibited laminin-mediated endothelial cell tube formation and promoted aortic sprouting and tumor metastasis suggesting it as a potent angiogenic sequence [6,7]. A99, located on the short arm of the ocl chain and containing the RGD sequence [8], was found to interact with integrins and to promote cell adhesion and migration [9]. A208, on the C-terminus of the coiled-coil region of the laminin ocl chain and containing the IKVAV sequence, promoted cell adhesion, neurite outgrowth, angiogenesis, and tumor metastasis [10].