Design and activity of multifunctional fibrils using receptor-specific small peptides.

We have designed multifunctional peptide fibrils using bioactive laminin-derived peptides and evaluated their potential as a biomedical material for tissue engineering. The Leu-Arg-Gly-Asp-Asn (LRGDN) peptide derived from laminin-111, which contains an RGD sequence bound to integrin alphavbeta3, was added to the N-terminus of the four amyloidogenic cell-adhesive laminin-derived peptides (A119: LSNIDYILIKAS, AG97: SAKVDAIGLEIV, B133: DISTKYFQMSLE, and B160: VILQQSAADIAR). The RGD-conjugated peptides were stained with Congo red and exhibited amyloid-like fibril formation in the electron microscopic. The RGD-conjugated peptides promoted human dermal fibroblasts spreading with well-organized actin stress fibers and focal contacts. Human dermal fibroblast attachment to the RGD-conjugated peptides was inhibited by anti-alphav integrin antibody. Further, cell attachment to B133 was inhibited by anti-alpha2 and anti-beta1 integrin antibodies, whereas attachment to RGD-B133 was inhibited by anti-alphav and anti-beta1 integrin antibodies. These results suggest that the RGD-conjugated peptides interact with integrin alphavbeta3 and that RGD-B133 interacts with both integrin alphavbeta3 and integrin beta1. The RGD-conjugated peptide fibrils promoted neurite outgrowth in a peptide-dependent manner. These results support that biologically active sequence-conjugated peptide fibrils interact in a receptor-specific manner with cells and promote multifunctional activities. These fibrils may have use as biological supports for cell-specific tissue engineering.

DNA/polyvinyl alcohol interpenetrating polymer network as stationary phase for thin layer chromatography.

Natural DNA was introduced to thin layer chromatography (TLC) with an aim to separate chemicals like DNA-affinity compounds and enantiomers. By cross-linking polyvinyl alcohol (PVA) with glutaraldehyde (GA) and subsequent cross-linking DNA with a UV irradiation, a DNA/PVA interpenetrating polymer network (IPN) is formed and was used to coat the surface of the porous silica particles of the TLC. Three typical DNA-binding compounds and eight amino acid enantiomers were used as model chemicals to investigate the chromatographic behavior of the modified TLC, and high separation efficiency was observed in both classes of the chemicals. On the practical side, the DNA-modified TLC have high prospects in diverse applications, including efficacy evaluation of a medicine, toxicity assessment of a pollutant at the molecular level, as well as separation of enantiomers such as dyes, amino acids, peptides, proteins, nucleotides, and drugs.

Applied chemistry of natural DNA.

Recently, natural DNA has emerged as an appealing biomacromolecule for functional materials. It is abundant and renewable, and possesses the well known double helix structure that promises many unique properties difficult to find in other polymers. Natural DNA has been applied in electronic, optical and biomaterials, as a catalyst for enantioselective reactions, and as a material for cleaning the environment. Most of the applications are based on combining DNA with other chemicals or nanoparticles by electrostatic binding, intercalation or groove binding. In this critical review article, recent developments in utilizing natural DNA are reviewed by focusing on three basic properties of DNA: the electrostatic property as a polyelectrolyte, selective affinity for small molecules, and biocompatibility (128 references).

Carcinogen adsorbent prepared from DNA complex by gamma-ray irradiation.

The effects of gamma-ray irradiation on aqueous solutions of chub mackerel chromatin, salmon milt DNA with CoCl(2), mixtures of DNA with Type A gelatin, bovine serum albumin (BSA), CM-chitosan, carboxymethyl cellulose (CMC) and Catinal (hydroxyethyl-cellulose, O-[2-hydroxy-3-(trimethyl ammonio)-propyl], chloride) and DNA in the presence of polyfunctional monomers with the aim to insolubilize DNA for preparing a novel carcinogen adsorbent have been studied. Among those, precipitates or inhomogeneous gel consisting of cross-linked DNA were prepared from the samples of aqueous DNA in the presence of CoCl(2) at low irradiation dose, around 10 Gy, and bulk homogeneous gels were successfully prepared from aqueous mixtures of DNA with gelatin, BSA, CMC and Catinal in a limited range of irradiation doses. Gel fraction and swelling ratio of the gels were measured. Adsorption of a carcinogen, acridine orange, was also examined for the gels. From the experimental results, the optimum conditions for preparing insolubilized homogeneous DNA gels were determined.

Identification of multiple amyloidogenic sequences in laminin-1.

Amyloid fibril formation is associated with several pathologies, including Alzheimer's disease, Parkinson's disease, type II diabetes, and prion diseases. Recently, a relationship between basement membrane components and amyloid deposits has been reported. The basement membrane protein, laminin, may be involved in amyloid-related diseases, since laminin is present in amyloid plaques in Alzheimer's disease and binds to amyloid precursor protein. Recently, we showed that peptide A208 (AASIKVAVSADR), the IKVAV-containing peptide, formed amyloid-like fibrils. We previously identified 60 cell adhesive sequences in laminin-1 using a total of 673 12-mer synthetic peptides. Here, we screened for additional amyloidogenic sequences among 60 cell adhesive peptides derived from laminin-1. We first examined amyloid-like fibril formation by the 60 active peptides with Congo red, a histological dye binding to many amyloid-like proteins. Thirteen peptides were stained with Congo red. Four of the 13 peptides promoted cell attachment and neurite outgrowth like the IKVAV-containing peptide. The four peptides also showed amyloid-like fibril formation in both X-ray diffraction and electron microscopic analyses. The amyloidogenic peptides contain consensus amino acid components, including both basic and acidic amino acids and Ser and Ile residues. These results indicate that at least five laminin-derived peptides can form amyloid-like fibrils. We conclude that the laminin-derived amyloidogenic peptides have the potential to form amyloid-like fibrils in vivo, possibly when laminin-1 is degraded.

DNA-based gels for oral delivery of probiotic bacteria.

A single-stranded DNA, readily extracted from industrial discarded salmon milt, was used to prepare hydrogels and complex gels by cross-linking with gelatin and kappa-carrageenan, for the oral delivery of probiotic bacteria. The complex gels showed a higher protective capability over the hydrogels for approximately one log scale. However, the hydrogels were more stable during storage at 4 degrees C. The Lactobacillus and Lactococcus due to protection of the hydrogels could better tolerate to acid than the Bifidobacterium. Furthermore, food-graded hydrogels were prepared and optimized to a similar protective capability for future applications.

DNA-collagen complex as a carrier for Ag+ delivery.

The possibility of DNA-collagen complex as a drug carrier was investigated. The interaction between DNA and silver ions was proved by CD spectra. The release property of the complex of DNA-Ag+ was measured through turbidity of PBS solution to indicate that silver ions could coordinate with base pairs of DNA, and be released slowly from the complex of DNA-Ag+. Collagen film, collagen-Ag+ film, DNA-collagen film and DNA-collagen-Ag+ film were prepared, and studied through SEM. Particles were found present in DNA-collagen-Ag+ film by SEM. These show that silver ions may be enclosed inside these particles, which led to the slow release of Ag+ to the environments. Two bacteria, Escherichia coli and Staphylococcus aureus, were used to study the antibiotic properties of the complex films. The growth of E. coli and S. aureus could be inhibited by these films. It indicates that DNA-collagen may be a good drug carrier for the drug-controlled release.

Bifunctional peptides derived from homologous loop regions in the laminin alpha chain LG4 modules interact with both alpha 2 beta 1 integrin and syndecan-2.

Laminin alpha chains show diverse biological functions in a chain-specific fashion. The laminin G-like modules (LG modules) of the laminin alpha chains consist of a 14-stranded beta-sheet sandwich structure with biologically active sequences found in the connecting loops. Previously, we reported that connecting loop regions between beta-strands E and F in the mouse laminin alpha chain LG4 modules exhibited chain-specific activities. In this study, we focus on the homologous loop regions in human laminin alpha chain LG4 modules using five synthetic peptides (hEF-1-hEF-5). These homologous peptides induced chain-specific cellular responses in various cell types. Next, to examine the dual-receptor recognition model, we synthesized chimeras (cEF13A-cEF13E) derived from peptides hEF-1 and hEF-3. All of the chimeric peptides promoted fibroblast attachment as well as the parental peptides. Attachment of fibroblasts to cEF13A and cEF13B was inhibited by anti-integrin alpha2 and beta1 antibodies and by heparin, while cell adhesion to cEF13C, cEF13D, and cEF13E was blocked only by heparin. Actin organization of fibroblasts on cEF13C was not different from that on hEF-3, but cEF13B induced membrane ruffling at the tips of the actin stress fibers. These results suggest that cEF13B had bifunctional effects on cellular behaviors through alpha2beta1 integrin and heparin/heparan sulfate proteoglycan. We conclude that the approach utilizing chimeric peptides is useful for examining cellular mechanisms in dual-receptor systems.

Identification of neurite outgrowth active sites on the laminin alpha4 chain G domain.

The laminin alpha4 chain is widely distributed in various mesodermal tissues, including the perineurium of peripheral nerves, dorsal root ganglion (DRG), skeletal muscle, and capillaries, and plays important roles in synaptic specialization at the neuromuscular junction and in microvascular formation. The C-terminal globular domain (G domain) of the laminin alpha4 chain was previously found to be critical for heparin binding and cell attachment activity. Here, we focused on neurite outgrowth activity of the laminin alpha4 chain G domain. We found that the recombinant alpha4 chain G domain protein (rec-alpha4G) promoted neurite outgrowth of rat pheochromocytoma PC12 cells. When 114 overlapping synthetic peptides that covered the entire G domain were tested for neurite outgrowth activity, nine peptides were active, but the 105 remaining peptides did not exhibit activity. Three of the nine active peptides, A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), and A4G107 (VIRDSNVVQLDV), strongly promoted neurite outgrowth of PC12 cells. A4G107 was found to form amyloid-like fibrils in Congo red, X-ray, and electron microscopy analyses. We also synthesized cyclic peptides to evaluate their conformational requirements. Cyclic peptide A4G82X (cyc-A4G82X;TLFLAHGRLVFX, where X is norleucine) significantly enhanced neurite outgrowth activity, but the rest of the cyclic peptides eliminated the activity. The A4G82 sequence is located on the loop region, suggesting that the activity of A4G82 is required for a loop conformation. These peptides also exhibited neurite outgrowth activity with dorsal root ganglion (DRG) explants and with DRG cells from E14.5 mouse embryos, indicating that they are active in both neuronal cell lines and native neuronal cells. Taken together, the data suggest that the peptides from the laminin alpha4 chain G domain promote neurite outgrowth activity via a specific conformation.

Preparation and characterization of DNA hydrogel bead as selective adsorbent of dioxins.

Salmon milt DNA hydrogel beads were synthesized by an inverse suspension polymerization of acrylamide in the continuous phase of cyclohexane. These DNA hydrogel beads in water medium are stable, more than 82% (w/w) of the DNA can be retained in the hydrogel after a sufficient soaking in water. Comparing with normal adsorbents such as activated carbon and alumina, this DNA matrix showed a selective adsorptivity for the dioxin derivatives with planar structure such as dibenzo-p-dioxin (DD), dibenzofuran (DF) and biphenyl (BP). Rinsing with hexane can regenerate the DNA beads after adsorption by the dioxin derivatives, even the adsorption-regeneration process repeated four times, no significant decrease in the dioxin removal capacity was observed.

Effect of DNA structure on the formation of collagen-DNA complex.

Using various types of DNAs prepared from plasmid DNA, complete double-stranded DNA (ds.DNA) with linear and cyclic forms and double-stranded DNA coexisting with single-stranded DNA (ss.DNA), the structure and fibrillogenesis of the collagen-DNA complex were investigated by means of turbidity, transmission electron microscopy, and confocal laser-scanning microscopy. The rate of fibrillogenesis of the collagen-DNA complex significantly depends on the DNA structure. The structure of the fibrils formed in the complexes showed a marked difference between the ds.DNA and ss.DNA complexes with collagen. Spatial distribution of the DNA and collagen in the complexes suggests that the characteristic collagen-DNA interaction depends on the DNA forms.

Molecularly imprinted polyethersulfone microspheres for the binding and recognition of bisphenol A.

BPA-imprinted polyethersulfone (PES) microspheres for the binding and recognition of bisphenol A (BPA) were fabricated by means of a liquid-liquid phase separation technique. The imprinted novel PES microspheres had a porous structure with a skin layer, under which was followed by a finger-like structure. The recognition experiments with the BPA-imprinted microspheres were carried out by applying the microspheres to various BPA solutions. In water, high binding amounts of BPA were observed in the range of 19-42µmol/g capacity, but the recognition was low in the BPA water solution. With the increase of the concentration in BPA solution, the binding amounts and the recognition coefficient increased. However, 1,4-butylene glycol/water media showed high recognition of the imprinted microspheres with a low binding capacity of BPA. In addtion, with the increase of the BPA amounts in the PES solution used to prepare the imprinted microspheres, the specific recognition sites increased, and the recognition ability increased. Evidence revealed that microsphere recognition was effective for BPA due to the binding to specific recognition sites [S](sites). The imprinted microspheres showed the selectivity for BPA in the wine including BPA and other organic compounds. Charge transfer and special cavities could be employed to explain the mechanism.

Cyclic peptides from the loop region of the laminin alpha 4 chain LG4 module show enhanced biological activity over linear peptides.

Laminins, heterotrimeric glycoproteins in the basement membrane, are involved in diverse biological activities. So far, five alpha, three beta, and three gamma chains have been identified, and at least 15 laminin isoforms exist composed of various combinations of the different three chains. The major cell-surface receptors for laminins are integrins and proteoglycans, such as dystroglycans and syndecans. Previously, we reported that synthetic peptide A4G82 (TLFLAHGRLVFM, mouse laminin alpha4 chain residues 1514-1525) showed strong cell attachment and syndecan binding activities. On the basis of the crystal structure of the LG module and sequence alignment, A4G82 is located in the connecting loop region between beta-strands E and F in the laminin alpha4 chain LG4 module. Here, we have focused on the structural importance of this E-F loop region for the biological activity of the alpha4 chain LG4 module. To determine the importance of the loop structure, we synthesized peptide A4G82X (cyclo-A4G82X, Cys-TLFLAHGRLVFX-Cys, X= norleucine), which was cyclized via disulfide bridges at both the N- and C-termini. The cyclic peptides derived from A4G82X inhibited the heparin binding activity of the alpha4 chain G domain and promoted HT-1080 cell attachment better than the corresponding linear peptides. We determined FLAHGRLVFX as a minimal sequence of cyclo-A4G82X important for cell adhesion and heparin binding using a series of truncated peptides. Moreover, HT-1080 cell attachment to the cyclic peptides was more efficiently blocked by heparin than cell attachment to the linear peptides. Furthermore, the cyclic peptides showed significantly enhanced syndecan-2-mediated cell attachment activity. These results indicate that the activity of A4G82 is highly conformation-dependent, suggesting that the E-F loop structure is crucial for its biological activity.

Preparation of DNA-loaded polysulfone microspheres by liquid-liquid phase separation and its functional utilization.

DNA-loaded polysulfone (PSf) microspheres were fabricated by means of a liquid-liquid phase separation technique. The porous microspheres were then used to remove DNA-binding intercalating materials--ethidium bromide, acridine orange, and endocrine disruptors. The DNA-loaded PSf microspheres are stable in water. The stability of the DNA-loaded microspheres and/or the release rate of DNA from the microspheres can be controlled by manipulating the microsphere structure. Increasing the polymer concentration, which causes lower porosity and smaller pores on the outer surface of the microspheres, led to increased stability of the microspheres and decreased release rate of DNA. Additionally, the drying temperature also affected the stability of the microspheres. The DNA-loaded PSf microspheres could effectively accumulate harmful DNA-intercalating pollutants and endocrine disruptors, such as ethidium bromide, acridine orange, biphenyl, dibenzofuran, and dibenzo-p-dioxin. The amount of pollutants removed by the microspheres is dependent on the amount of incorporated DNA and on the microsphere structure. The DNA-loaded microspheres have the potential to be used in environmental applications.

Encapsulation of multi-walled carbon nanotubes (MWCNTs) in Ba(2+)-alginate to form coated micro-beads and their application to the pre-concentration/elimination of dibenzo-p-dioxin, dibenzofuran, and biphenyl from contaminated water.

We report preliminary data on the first use of multi-walled carbon nanotubes as adsorbents for the pre-concentration/elimination of dibenzo-p-dioxin, dibenzofuran and biphenyl from contaminated water.

Multifunctional peptide fibrils for biomedical materials.

The Ile-Lys-Val-Ala-Val (IKVAV) containing peptide, A208 (AASIKVAVSADR, mouse laminin alpha1 chain 2097-2108), was recently found to form amyloid-like fibrils. Fibril formation is critical for its biological activities, including promotion of cell adhesion and neurite outgrowth. In the present study, we designed multifunctional peptide fibrils using the A208 peptide and an Arg-Gly-Asp (RGD)-containing fibronectin active sequence for biomedical applications. The fibronectin active sequence GRGDS (FN) or a scrambled sequence RSGGD (SC) were conjugated to either A208 or to A208S (AASVVIAKSADR), a scrambled peptide of A208, with a glycine as a spacer. The FN-A208 and SC-A208 peptides formed a gel and were stained with Congo red similar to that of A208, but FN-A208S and SC-A208S did not form a gel. These results indicate that FN-A208 and SC-A208 form amyloid-like fibrils similar to A208. A208 and SC-A208 promoted cell attachment with filopodia formation, and this adhesion was inhibited by the IKVAV-containing peptide, but not by EDTA or a GRGDS peptide. FN-A208 promoted cell attachment with well-organized actin stress fibers, and this adhesion was partially inhibited by either EDTA, GRGDS, or IKVAV. These data suggest that A208 binds to only IKVAV receptor(s) while the FN-A208 interacts with both integrins and the IKVAV receptor(s). We conclude that multifunctional peptide fibrils can be designed by conjugation of active peptides on A208 and that this construct has potential to serve as a bioadhesive for tissue regeneration and engineering.

Characterization of a fasciclin I-like protein with cell attachment activity from sea urchin (Strongylocentrotus intermedius) ovaries.

Fasciclin I, a neuronal cell adhesion molecule, was first identified in the grasshopper. To date, various fasciclin I-like proteins have been identified but their biological functions have not been well characterized. Here, we have purified a fasciclin I-like protein with a molecular weight of 33kDa from sea urchin (Strongylocentrotus intermedius) ovaries using hydrophobic chromatography and gel filtration. The protein was not N-glycosylated. Partial amino acid sequences of cyanogen bromide (CNBr)-cleaved fragments were highly conserved to other sea urchin fasciclin I-like proteins identified previously. The circular dichroism (CD) spectrum analysis demonstrated that the 33kDa protein contained high content of alpha-helical structure. These results suggest that the 33kDa protein is a fasciclin I-like family. Additionally, the fasciclin I-like protein promoted HT1080 human fibrosarcoma cell attachment. Further, a synthetic peptide (P1: GLREAANIAEQVDLRQVLRDVDL) of the protein corresponding to a highly conserved region of the fasciclin I-like family promoted heparin-dependent HT1080 cell attachment. Moreover, the peptide inhibited HT1080 cell attachment to the fasciclin I-like protein. These results suggest that the 33kDa protein from sea urchin ovaries isolated here is a member of the fasciclin I family and that the N-terminal region of the protein is important for cell attachment activity. The protein has a potential to be involved in biological functions in sea urchin as a cell adhesive molecule.

Caged multiwalled carbon nanotubes as the adsorbents for affinity-based elimination of ionic dyes.

Multiwalled carbon nanotubes (MWCNTs) were used as the active elements for the first time for affinity-based elimination of ionic dyes. MWCNTs were encapsulated in cross-linked alginate (ALG) microvesicles using Ba2+ as the bridging ion. The Ba2+-alginate matrix constitutes a cage which holds the physically trapped MWCNTs. The cage carries negative charges on its surface. The cage restricts the access of anions of large molecular weight, such as humic acids, because of electrostatic repulsion. The cage also restricts the access of colloids of large size, because of size exclusion. Ionic dyes partition into the cage and then are captured by MWCNTs probably on the basis of van der Waals interactions occurring between the hexagonally arrayed carbon atoms in the graphite sheet of MWCNTs and the aromatic backbones of the dyes. As a result of these interactions the target species, namely, the ionic dyes, are eliminated efficiently by the MWCNTs of Ba2+-ALG/MWCNT composite adsorbents. The adsorptive capacities for elimination of acridine orange, ethidium bromide, eosin bluish, and orange G (the model species used for this study) were found as high as 0.44, 0.43, 0.33, and 0.31 micromol, respectively, for 1.0 mg of the caged MWCNTs. Adsorptive experiments with carbon nanofibers and activated carbons as the adsorbents were also performed. The MWCNT-based adsorbents provided the best capability for the affinity-based elimination of these targeted species. Biocompatibility experiments performed in vitro and in vivo provided promising results, suggesting potential applications of the caged MWCNTs in in situ environmental remediation.

Syndecan binding sites in the laminin alpha1 chain G domain.

The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K(D) = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC(50) = 147 microM) and AG75 (IC(50) = 206 microM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.

DNA aqueous solution used for dialytical removal and enrichment of dioxin derivatives.

In the present study, a dialytic method that uses a DNA aqueous solution to remove and enrich dioxins from polluted water was proposed. Circular dichroism (CD) and fluorescent spectra indicated that dibenzo-p-dioxin (DD), dibenzofuran (DF) and biphenyl (BP), which are dioxin derivatives, form complexes with DNA. Their experimental dialytic sorption coefficients were measured by quantifying the concentrations of DD, DF, and BP in aqueous solutions before and after dialysis of the DNA solution, and the values were 2.1x10(5), 1.3x10(5), and 1.5x10(7), respectively. As a simulated water treatment model, DNA solution was dialyzed in an aqueous mixture of DD, DF, and BP for 96h, the HPLC studies showed that the dioxin derivatives have been concentrated in the DNA solution about 200 times. The dialyzed DNA solution was reusable by an extraction with hexane.