Synthetic Oligopeptides Research Articles

Assessment of the effect of using immunomodulatory drugs for emergency prevention of experimental plague caused by a virulent strain of the main subspecies Yersinia pestis

Introduction. Immunomodulatory drugs (IMD) have great potential to increase the nonspecific reactivity of the organism in a set of measures for emergency prevention of plague. The purpose of the work is to evaluate the protective effectiveness of the use of IMD of different groups in experiments on modeling infection with a highly virulent strain of the plague microbe. Materials and methods. IMD (rIFN-γ — recombinant interferon-gamma, PO — azoximer bromide, synthetic immunomodulatory oligopeptides O1, O2, O3) was administered to white mice and guinea pigs subcutaneously thrice by the schedule 3 days, 1 day, and 1 hour prior the infection with a virulent test strain of plague Yersinia pestis 231 (708) at dose from 1 to 625 CFU. In addition, the effect of IMD on the production of IFN-γ and interleukin-10 was investigated in white mice before infection. Results. The study of the effect of IMD on the survival of unvaccinated biomodels made it possible to establish that only rIFN-γ and PO increase the survival of two types of laboratory animals by 20–50% and significantly increase the LD50. All tested IMD contribute to an increase in the average life expectancy of biomodels by at least one day. An increase in spontaneous and mitogen-induced cytokine production was found only in white mice receiving rIFN-γ and PO, which correlates with animal survival rates. Conclusion. The obtained data indicate the effectiveness of the use of IMD, especially rIFN-γ and PO in protecting the macroorganisms from infection with Y. pestis, which determines the prospects for research on the further improvement of emergency prevention of plague.

P1' specificity of the S219V/R203G mutant tobacco etch virus protease.

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.

Functional oligo- and polypeptide assemblies for photochemical, optical and electronic applications.

The primary and secondary structures of peptides are useful as scaffolds to sequentially arrange functional groups of molecules. In this review, we review self-assembled functional peptides, whereby peptides with appropriate amino acid sequences can assemble using functional groups on their side chains. First, we apply our design strategies for the synthesis of peptide-based materials with sequenced side chains with polar moieties, organic dyes and metal complexes. The synthetic oligopeptides thus obtained exhibit inherent photoinduced charge separation and electrochemical redox activities, as well as responses to bio-sequences. Next, catalytic and photocatalytic oxidation reduction reactions and hydrogen evolution reactions are shown by utilizing the peptides with separated functionalities on both sides of β-sheets by hybridizing with electro- and photoactive graphene oxide and metal nanoparticles. Finally, the self-assembled natural proteins that form micrometre-scale spherical geometry and fibres are utilized for optical and electronic applications. The silk fibroin forms well-defined microspheres with smooth surface morphology, leading to properties suitable for use in optical resonators, which can sense external humidity because of the hygroscopic nature of silk fibroin. Dragline silk fibres can act as optical waveguides that can perform intermediate natural polymer-based optical logic operations. These functional peptides are utilizable for various applications in catalysis, optics and electronics.

Conformation Dependent Architectures of Assembled Antimicrobial Peptides with Enhanced Antimicrobial Ability.

Antimicrobial peptides (AMPs) are a developing class of natural and synthetic oligopeptides with host defense mechanisms against a broad spectrum of microorganisms. With in-depth research on the structural conformations of AMPs, synthesis or modification of peptides has shown great potential in effectively obtaining new therapeutic agents with improved physicochemical and biological properties. Notably, AMPs with self-assembled properties have gradually become a hot research topic for various biomedical applications. Compared to monomeric peptides, these peptides can exist in diverse forms (e.g., nanoparticles, nanorods, and nanofibers) and possess several advantages, such as high stability, good biocompatibility, and potent biological functions, after forming aggregates under specific conditions. In particular, the stability and antibacterial property of these AMPs can be modulated by rationally regulating the peptide sequences to promote self-assembly, leading to the reconstruction of molecular structure and spatial orientation while introducing some peptide fragments into the scaffolds. In this work, four self-assembled AMPs are developed, and the relationship between their chemical structures and antibacterial activity is explored extensively through different experiments. Importantly, the evaluation of antibacterial performance in both in vitro and in vivo studies has provided a general guide for using self-assembled AMPs in subsequent treatments for combating bacterial infections.

Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates.

Proteases control plant growth and development by limited proteolysis of regulatory proteins at highly specific sites. This includes the processing of peptide hormone precursors to release the bioactive peptides as signaling molecules. The proteases involved in this process have long remained elusive. Confirmation of a candidate protease as a peptide precursor-processing enzyme requires the demonstration of protease-mediated precursor cleavage in vitro. In vitro cleavage assays rely on the availability of suitable substrates and the candidate protease with high purity. Here, we provide a protocol for the expression, purification, and characterization of tomato (Solanum lycopersicum) phytaspases as candidate proteases for the processing of the phytosulfokine precursor. We also show how synthetic oligopeptide substrates can be used to demonstrate site-specific precursor cleavage. Graphical abstract.

ЭКСПЕРИМЕНТАЛЬНОЕ ИССЛЕДОВАНИЕ СПОСОБНОСТИ ОЛИГОПЕПТИДА TRP-ASP-PHE-ASP СВЯЗЫВАТЬ ИНТЕРЛЕЙКИН-8

Background. One of the perspectives of anticytokine therapy is the development of synthetic oligopeptides, which can bind and inhibit the activity of cytokines. The peptide, which is a structural analogue of the cytokine-binding region of the chemokine receptor, is of interest as a ligand for interaction with interleukin-8 (IL-8). The aim of the research was to evaluate the interaction of the Trp-Asp-Phe-Asp with IL-8. Material and methods. The interaction between the peptide and cytokine was evaluated by the change in the IL-8 concentration, which was assessed by enzyme immunoassay. Oligopeptide was used in free form adsorbed on the bottom of the plate well and immobilized in a polyacrylamide gel at a concentration of 1 µM/ml. Results. The results of the study showed that Trp-Asp-Phe-Asp, both in free and immobilized form, has the ability to bind IL-8. The maximum concentration of IL-8 bound by the free peptide is 22.13 (14.09; 30.17) pM/ml, for the adsorbed peptide – 4.22 (3.69; 4.75) pM/ml. Oligopeptide immobilized in the gel reduces the IL-8 concentration in blood plasma by 25.39 (21.34; 29.44) pM/ml. Conclusions. The results obtained are the basis for the development of medical devices for hemosorption in order to extract IL-8 from human blood plasma.

Synthetic Oligopeptides Mimicking γ-Core Regions of Cysteine-Rich Peptides of Solanum lycopersicum Possess Antimicrobial Activity against Human and Plant Pathogens.

Plant cysteine-rich peptides (CRPs) represent a diverse group of molecules involved in different aspects of plant physiology. Antimicrobial peptides, which directly suppress the growth of pathogens, are regarded as promising templates for the development of next-generation pharmaceuticals and ecologically friendly plant disease control agents. Their oligopeptide fragments are even more promising because of their low production costs. The goal of this work was to explore the antimicrobial activity of nine short peptides derived from the γ-core-containing regions of tomato CRPs against important plant and human pathogens. We discovered antimicrobial activity in peptides derived from the defensin-like peptides, snakins, and MEG, which demonstrates the direct involvement of these CRPs in defense reactions in tomato. The CRP-derived short peptides appeared particularly active against the gram-positive bacterium Clavibacter michiganensis, which causes bacterial wilt—opening up new possibilities for their use in agriculture to control this dangerous disease. Furthermore, high inhibitory potency of short oligopeptides was demonstrated against the yeast Cryptococcus neoformans, which causes serious diseases in humans, making these peptide molecules promising candidates for the development of next-generation pharmaceuticals. Studies of the mode of action of the two most active peptides indicate fungal membrane permeabilization as a mechanism of antimicrobial action.

Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein.

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

Involvement of Huntingtin in Development and Ciliary Beating Regulation of Larvae of the Sea Urchin, Hemicentrotus pulcherrimus.

The multiple functions of the wild type Huntington’s disease protein of the sea urchin Hemicentrotus pulcherrimus (Hp-Htt) have been examined using the anti-Hp-Htt antibody (Ab) raised against synthetic oligopeptides. According to immunoblotting, Hp-Htt was detected as a single band at around the 350 kDa region at the swimming blastula stage to the prism larva stage. From the 2-arm pluteus stage (2aPL), however, an additional smaller band at the 165 kDa region appeared. Immunohistochemically, Hp-Htt was detected in the nuclei and the nearby cytoplasm of the ectodermal cells from the swimming blastula stage, and the blastocoelar cells from the mid-gastrula stage. The Ab-positive signal was converged to the ciliary band-associated strand (CBAS). There, it was accompanied by several CBAS-marker proteins in the cytoplasm, such as glutamate decarboxylase. Application of Hp-Htt morpholino (Hp-Htt-MO) has resulted in shortened larval arms, accompanied by decreased 5-bromo-2-deoxyuridin (BrdU) incorporation by the ectodermal cells of the larval arms. Hp-Htt-MO also resulted in lowered ciliary beating activity, accompanied by a disordered swirling pattern formation around the body. These Hp-Htt-MO-induced deficiencies took place after the onset of CBAS system formation at the larval arms. Thus, Hp-Htt is involved in cell proliferation and the ciliary beating pattern regulation signaling system in pluteus larvae.

Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases.

The human T-lymphotropic viruses (HTLVs) are causative agents of severe diseases including adult T-cell leukemia. Similar to human immunodeficiency viruses (HIVs), the viral protease (PR) plays a crucial role in the viral life-cycle via the processing of the viral polyproteins. Thus, it is a potential target of anti-retroviral therapies. In this study, we performed in vitro comparative analysis of human T-cell leukemia virus type 1, 2, and 3 (HTLV-1, -2, and -3) proteases. Amino acid preferences of S4 to S1′ subsites were studied by using a series of synthetic oligopeptide substrates representing the natural and modified cleavage site sequences of the proteases. Biochemical characteristics of the different PRs were also determined, including catalytic efficiencies and dependence of activity on pH, temperature, and ionic strength. We investigated the effects of different HIV-1 PR inhibitors (atazanavir, darunavir, DMP-323, indinavir, ritonavir, and saquinavir) on enzyme activities, and inhibitory potentials of IB-268 and IB-269 inhibitors that were previously designed against HTLV-1 PR. Comparative biochemical analysis of HTLV-1, -2, and -3 PRs may help understand the characteristic similarities and differences between these enzymes in order to estimate the potential of the appearance of drug-resistance against specific HTLV-1 PR inhibitors.

Development and Characterization of Polymeric Peptides for Antibody Tagging of Bacterial Targets.

Microbe-Binding Peptides (MBPs) are currently being investigated to address the problem of antimicrobial resistance. Strategies enhancing their antimicrobial activity have been developed, including peptide dimerization. Here, we present an alternative approach based on peptide polymerization, yielding hapten-labelled polymeric MBPs that mediate tagging of bacteria with anti-hapten antibodies, for enhanced immune recognition by host phagocytes. C-terminally amidated analogs of the bacterial-binding peptide IIGGR were synthesized, with or without addition of cysteine residues at both N- and C-termini. Peptides were subjected to oxidizing conditions in a dimethyl-sulfoxide/water solvent system, and polymerization was demonstrated using SDS-PAGE. Peptides were then N-terminally labelled with a trinitrophenyl (TNP) group using trinitrobenzene sulfonate (TNBS). Binding to representative bacteria was demonstrated by ELISA using anti-TNP antibodies and was quantified as half-maximal effective concentration (EC50). Minimum Inhibitory Concentration (MIC) and concentration yielding 50% hemolysis (H50) were estimated. Neutrophil phagocytic index was determined for TNP-labelled polymeric bacterial- binding peptide (Pbac) with anti-TNP antibodies and/or serum complement. Polydisperse Pbac was synthesized. EC50 was lower for Pbac than for the corresponding monomeric form (Mbac), for both Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. MIC and H50 were >250μg/mL for both Pbac and Mbac. A complement-independent increase in neutrophil phagocytic index was observed for E. coli treated with TNP-labelled Pbac in conjunction with anti-TNP antibodies. Our data suggest that hapten-labelled polymeric bacterial-binding peptides may easily be produced from even crude synthetic oligopeptide precursors, and that such bacterial-binding peptides in conjunction with cognate anti-hapten antibodies can enhance immune recognition of bacteria by host phagocytes.

Enhanced angiogenic effects of RGD, GHK peptides and copper (II) compositions in synthetic cryogel ECM model

Synthetic oligopeptides are a promising alternative to natural full-length growth factors and extracellular matrix (ECM) proteins in tissue regeneration and therapeutic angiogenesis applications. In this work, angiogenic properties of dual and triple compositions containing RGD, GHK peptides and copper (II) ions (Cu2+) were for the first time studied. To reveal specific in vitro effects of these compositions in three-dimensional scaffold, adamantyl group bearing peptides, namely Ada-Ahx-GGRGD (1) and Ada-Ahx-GGGHK (2), were effectively immobilized in bioinert pHEMA macroporous cryogel via host-guest β-cyclodextrin-adamantane interaction. The cryogels were additionally functionalized with Cu2+ via the formation of GHK-Cu complex. Angiogenic responses of HUVECs grown within the cryogel ECM model were analyzed. The results demonstrate that the combination of RGD with GHK and further with Cu2+ dramatically increases cell proliferation, differentiation, and production of a series of angiogenesis related cytokines and growth factors. Furthermore, the level of glutathione, a key cellular antioxidant and redox regulator, was altered in relation to the angiogenic effects. These results are of particular interest for establishing the role of multiple peptide signals on regeneration related processes and for developing improved tissue engineering materials.

Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1).

The human retroviral-like aspartic protease 1 (ASPRV1) is a mammalian retroviral-like enzyme that catalyzes a critical proteolytic step during epidermal differentiation; therefore, it is also referred to as skin-specific aspartic protease (SASPase). Neutrophil granulocytes were also found recently to express ASPRV1 that is involved in the progression of acute chronic inflammation of the central nervous system, especially in autoimmune encephalomyelitis. Thus, investigation of ASPRV1 is important due to its therapeutic or diagnostic potential. We investigated the structural characteristics of ASPRV1 by homology modeling; analysis of the proposed structure was used for interpretation of in vitro specificity studies. For in-vitro characterization, activities of SASP28 and SASP14 enzyme forms were measured using synthetic oligopeptide substrates. We demonstrated that self-processing of SASP28 precursor causes autoactivation of the protease. The highest activity was measured for GST-SASP14 at neutral pH and at high ionic strength, and we proved that pepstatin A and acetyl-pepstatin can also inhibit the protease. In agreement with the structural characteristics, the relatively lower urea dissociation constant implied lower dimer stability of SASP14 compared to that of HIV-1 protease. The obtained structural and biochemical characteristics support better understanding of ASPRV1 function in the skin and central nervous system.

Biochemical characterization of Ty1 retrotransposon protease.

Ty1 is one of the many transposons in the budding yeast Saccharomyces cerevisiae. The life-cycle of Ty1 shows numerous similarities with that of retroviruses, e.g. the initially synthesized polyprotein precursor undergoes proteolytic processing by the protease. The retroviral proteases have become important targets of current antiretroviral therapies due to the critical role of the limited proteolysis of Gag-Pol polyprotein in the replication cycle and they therefore belong to the most well-studied enzymes. Comparative analyses of retroviral and retroviral-like proteases can help to explore the key similarities and differences which may help understanding how resistance is developed against protease inhibitors, but the available information about the structural and biochemical characteristics of retroviral-like, and especially retrotransposon, proteases is limited. To investigate the main characteristics of Ty1 retrotransposon protease of Saccharomyces cerevisiae, untagged and His6-tagged forms of Ty1 protease were expressed in E. coli. After purification of the recombinant proteins, activity measurements were performed using synthetic oligopeptide and fluorescent recombinant protein substrates, which represented the wild-type and the modified forms of naturally occurring cleavage sites of the protease. We investigated the dependence of enzyme activity on different reaction conditions (pH, temperature, ionic strength, and urea concentration), and determined enzyme kinetic parameters for the studied substrates. Inhibitory potentials of 10 different protease inhibitors were also tested. Ty1 protease was not inhibited by the inhibitors which have been designed against human immunodeficiency virus type 1 protease and are approved as antiretroviral therapeutics. A quaternary structure of homodimeric Ty1 protease was proposed based on homology modeling, and this structure was used to support interpretation of experimental results and to correlate some structural and biochemical characteristics with that of other retroviral proteases.

A modular approach for organizing dimeric coiled coils on peptoid oligomer scaffolds.

We report a general approach to promote the folding of synthetic oligopeptides capable of forming homodimeric coiled coil assemblies. By pre-organizing the peptides on macrocyclic oligomer scaffolds, the stability of the coiled coils is enhanced with an observed increase in the melting temperature of 30 °C to 40 °C. Molecular dynamics simulations substantiate the hypothesis that the enhanced stability is established by constraining motion at the peptide termini and by pre-organizing intramolecular helix-helix contacts. We demonstrate the modularity of this approach by using a family of peptoid scaffolds to promote the folding of a dimeric coiled coil. Importantly, this strategy for templating coiled coils allows preservation of native amino acid sequences. Comparing a macrocyclic peptoid scaffold to its linear counterparts indicates that both types of assemblies are effective for organizing stable coiled coils. These results will guide future designs of coiled coil peptides for biomedical applications and as building blocks for more complex supramolecular assemblies.

A Polysaccharide-based Hydrogel and PLGA Microspheres for Sustained P24 Peptide Delivery: An In vitro and In vivo Study Based on Osteogenic Capability

A bone morphogenetic protein-2(BMP-2) derived synthetic oligopeptide, S[PO4]KIPKASSVPTELSAI-STLYLDDD(P24), has shown great potential for facilitating bone regeneration. However, P24 cannot be directly used onto bone defects, while a continuous sustained delivery of P24 may lead to a better formation of bone tissue. Based on this issue, we have developed a sustained delivery system incorporating P24-loaded poly(lactide-co-glycolide) (PLGA) microspheres and nano-hydroxyapatite(n-HA) into the composite hydrogel. The P24-contained compound material was characterized with NMR, FTIR and SEM to demonstrate the formation of compound structure containing P24, PLGA and n-HA. A continuous drug release of P24 was observed for over 60 d that evidently enhanced the efficiency in promoting the proliferation of MC3T3-E1 cells and the secrete of alkaline phosphatase(ALP) in vitro. Moreover, the osteoinduction effect of the hydrogel system with P24 peptide microspheres was demonstrated in vivo and manifested by the result of immunohistochemistry. This novel injectable composite hydrogel is expected to be applied to improving the bone defect treatment in bone tissue engineering.

In Situ Photophysical Characterization of π-Conjugated Oligopeptides Assembled via Continuous Flow Processing.

Bioinspired materials have been developed with the aim of harnessing natural self-assembly for precisely engineered functionality. Microfluidics is poised to play a key role in the directed assembly of advanced materials with ordered nano and mesoscale features. More importantly, there is a strong need for understanding the kinetics of continuous assembly processes. In this work, we describe a continuous microfluidic system for the assembly and alignment of synthetic oligopeptides with π-conjugated cores using a three-dimensional (3D) flow focusing of inlet reactant streams. This system facilitates in situ confocal fluorescence microscopy and in situ fluorescence lifetime imaging microscopy (FLIM), which can be used in unprecedented capacity to characterize the integrity of peptides during the assembly process. To achieve continuous assembly, we integrate chevron patterns in the ceiling and floor of the microdevice to generate a 3D-focused sheath flow of the reactant peptide. Consequently, the peptide stream is directed toward an acidic triggering stream in a cross-slot geometry which mediates assembly into higher-order fiber-like structures. Using this approach, the focused peptide stream is assembled using a planar extensional flow, which ensures high degrees of microstructural alignment within the assembled material. We demonstrate the efficacy of this approach using three different synthetic oligopeptides, and in all cases, we observe the efficient and continuous assembly of oligopeptides. In addition, finite element simulations are used to guide device design and to validate 3D focusing. Overall, this approach presents an efficient and effective method for the continuous assembly and alignment of ordered materials using microfluidics.

Optimizing the Conditions and Use of Synthetic Matrix for Three-Dimensional In Vitro Retinal Differentiation from Mouse Pluripotent Cells.

The development of retinal regenerative therapies relies on the reproducible and renewable source of retinal neurons for drug discovery and cell transplantation. Three-dimensional approach for retinal differentiation from pluripotent cells recently emerged as the robust strategy for retinal tissue differentiation. In this work, we present the combination of optimized conditions and techniques for three-dimensional retinal differentiation from mouse embryonic cells that improves reproducibility and efficiency of retinal differentiation in organoid cultures. We also show that the retinal induction can be achieved with the synthetic oligopeptide instead of Matrigel that allows to approach xeno-free conditions for cell production.

Engineering of Supramolecular β-Sheet and Nontoxic Amyloid Fibrils from Synthetic Oligopeptides Containing γ-Aminobutyric Acid as the N-Terminal Residue.

Here we demonstrate that three synthetic tripeptides containing conformationally flexible γ-aminobutyric acid (γ-Abu) as the N-terminal residue form supramolecular β-sheet and nanofibrillar aggregates upon self-association in aqueous medium. Congo red and thioflavin T binding study establish that these nanofibrillar aggregates are amyloidogenic in nature. The MTT cell survival assay suggests that these amyloid-like nanofibrillar aggregates are nontoxic toward cultured Neuro 2A cells. Interestingly, none of these tripeptides bear sequence identity with any amyloid forming proteins or peptides; however upon self-association, they form supramolecular β-sheet and amyloid-like nanofibrils those are nontoxic in nature. The results highlight the self-assembling nature of the conformationally flexible peptides in aqueous environment and support the hypothesis that amyloid formation is the intrinsic property of the polypeptide chain. Also the cytotoxicity is not predictive from amyloid fibril formation alone. Such nontoxic amyloid fibrils can be exploited in future to design functional biomaterials for various biomedical applications.

Charge/polarity-dependent 2D separation: a new chromatography for monitoring biomolecules

Thin-layer chromatography is a fast and easy tool for analyzing biomolecules, but opportunities for its use have up to now been limited due to its low resolution of separation. To overcome this problem, we have designed a new two-dimensional biomolecule analysis tool, in which analytes are first subjected to isoelectric focusing in a charge-specific-separation step, which is then followed by thin-layer chromatography in a polarity-specific-separation step. This new technique enables the separation of mixed large proteins as well as that of mixed synthetic oligopeptides that have similar physical properties. We propose here a newly developed thin-layer chromatographic technique that can be used to analyze various combinations of biomolecules, such as proteins, amino acids, and oligopeptides, within a single chip. The entire system is designed to be as rapid and easily applied as classic thin-layer chromatography to permit its use for high-throughput testing.