Kiyoshi Nokihara

Structural Evaluation of Tandem Hairpin Pyrrole–Imidazole Polyamides Recognizing Human Telomeres

A polyamide containing N-methylpyrrole (Py) and N-methylimidazole (Im), designated PIPA, binds with high affinity and specificity to specific nucleotide sequences in the minor groove of double-helical DNA. Based on a recent report of the synthesis of PIPA for telomere visualization, the present paper focused on the size of the connecting part (hinge region) of two PIPA segments of the tandem hairpin PIPA, Dab(Im-Im-Py)-Py-Py-Py-Im-[Hinge]-Dab(Im-Im-Py)-Py-Py-Py-Im-βAla-NH(CH2)3N(CH3)-(CH2)3NH-[Dye]. The present paper also describes the characterization of binding by measuring the thermal melting temperature and surface plasmon resonance and by specific staining of telomeres (TTAGGG)n in human cells. Microheterogeneity was also investigated by high-resolution mass spectrometry. We found that the optimal compound as the hinge segment for telomere staining was [-NH(C2H4O)2(C2H4)CO-] with tetramethylrhodamine as the fluorescent dye.

Preparative scale isolation, purification and derivatization of mimosine, a non-proteinogenic amino acid

Focusing on drug discovery non-proteinogenic amino acids have often been used as important building blocks for construction of compound libraries in the filed of combinatorial chemistry and chemical biology. Highly homogeneous l-mimosine, α-amino-β-(3-hydoxy-4-oxo-1,4-dihydropyridin-1-yl)-propanoic acid, a non-proteinogenic amino acid, has been successfully isolated and purified on an industrial scale from wild leaves of Leucaena (Leucaena leucocephala de Wit) which is a widely distributed legume in Okinawa, a sub-tropical island in Japan. Optical purity determinations used for quality control have been established through diastereomer formation. Physico-chemical properties and biological properties of purified mimosine have been clarified. Mimosine is sparingly soluble in water and organic solvents but can be dissolved in aqueous alkaline solution. The tyrosinase pathway is of particular interest in the cosmetic field, since mimosine is an analog of tyrosine. Thus the present purified mimosine have been tested in tyrosinase inhibitory assays. The IC50 for tyrosinase inhibitory activity of purified Mim was compared with kojic acid. Mimosine shows significant inhibition of melanin production in murine melanoma cells. The derivatization of mimosine has been investigated with a focus on its use in conventional peptide syntheses to generate mimosyl peptides. N-(9-Fluorenylmethoxycarbonyloxy)-mimosine and resin-bound mimosine for solid-phase syntheses have also been performed. Highly homogeneous Mim is a useful material for the development of functional cosmetics or active pharmaceutical ingredients.

Novel assay with fluorescence-labelled PrP peptides for differentiating L-type atypical and classical BSEs, and scrapie

PrP‐peptides HPP01, 02, 03, 06, 11 physically interact with PrPSc of L‐type atypical and classical BSEs, and scrapie by pull down

Structural Conversion Rate Changes of Recombinant Bovine Prion by Designed Synthetic Peptides

An understanding of structural changes and self-assembly of proteins, which are thought to involve specific peptide–peptide interactions, will contribute to the development of therapeutic agents and diagnosis for the detection of conformational diseases. We hypothesize that certain peptides may contribute to the conformational change of prion proteins. The present paper describes the discovery of prion-related synthetic peptides which influence structural conversion of recombinant bovine prion protein. The peptides designed are prion-protein fragments containing core domains consisting of α-helical (human prion protein fragment 180–195) and known β-sheet (human prion protein fragment 169–175) structures. Additionally several reported known β-sheet breaker peptides and a conjugate consisting of β-sheet and α-helix segments based on the secondary structures of human prion protein, designated HPPSH, have been chemically synthesized by the conventional Fmoc solid-phase method and characterized by circular dichroism and the Thioflavin T fluorescence method. Our data indicated that the co-existence of peptides, HPPSH or other prion fragment peptides involving toxic core sequence (the fragment 106–126), influenced the kinetic rate of aggregation and the lag-time of fibril formation of recombinant bovine prion protein except the core sequence itself. The method will be used for discovery of responsible material from natural resources. And designed peptides can be also used for bio-detection.

Telomere Visualization in Tissue Sections using Pyrrole–Imidazole Polyamide Probes

Pyrrole–Imidazole (PI) polyamides bind to specific DNA sequences in the minor groove with high affinity. Specific DNA labeling by PI polyamides does not require DNA denaturation with harsh treatments of heat and formamide and has the advantages of rapid and less disruptive processing. Previously, we developed tandem hairpin PI polyamide probes (TH59 series), which label telomeres in cultured cell lines more efficiently than conventional methods, such as fluorescence in situ hybridization (FISH). Here, we demonstrate that a TH59 derivative, HPTH59-b, along with immunostaining for specifying cell types in the tissues, visualizes telomeres in mouse and human tissue sections. Quantitative measurements of telomere length with single-cell resolution suggested shorter telomeres in the proliferating cell fractions of tumor than in non-tumor tissues. Thus, PI polyamides are a promising alternative for telomere labeling in clinical research, as well as in cell biology.

Differences in Action of PACAP-27 and PACAP-38 on Guinea Pig Gallbladder Smooth Muscle Using Synthetic C-terminally Modified PACAP Peptides

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two bioactive forms, PACAP-38 and PACAP-27 that have identical N-terminal sequences but differ by the presence of a C-terminal 11 residue elongation in the former. Although VIP and PACAP have several similar biological actions due to their amino acid sequence similarity, we have found that they evoke opposite responses in the guinea pig gallbladder smooth muscle, where PACAP induces contraction while VIP causes relaxation. In addition the response to PACAP-38 is four times lower than that of PACAP-27. In a previous study we have reported the role of the N-terminal α-helical regions of PACAP-27 which play a key role in gallbladder contraction. In the present study the biological action on the guinea pig gallbladder was investigated using a synthetic mini-library of C-terminally deleted peptides related to PACAP-38. The effects caused by residues within the C-terminus are not a result of a response via the M-receptor or Na+ channel, but most likely arise from a delicate balance between the differential effects of PACAP-38 on specific PAC1 and VPACs receptors.

Peptidoglycan microarray as a novel tool to explore protein–ligand recognition

Peptidoglycan is a giant bag‐shaped molecule essential for bacterial cell shape and resistance to osmotic stresses. The activity of a large number of bacterial surface proteins involved in cell growth and division requires binding to this macromolecule. Recognition of peptidoglycan by immune effectors is also crucial for the establishment of the immune response against pathogens. The availability of pure and chemically defined peptidoglycan fragments is a major technical bottleneck that has precluded systematic studies of the mechanisms underpinning protein‐mediated peptidoglycan recognition. Here, we report a microarray strategy suitable to carry out comprehensive studies to characterize proteins–peptidoglycan interactions. We describe a method to introduce a functional group on peptidoglycan fragments allowing their stable immobilization on amorphous carbon chip plates to minimize nonspecific binding. Such peptidoglycan microarrays were used with a model peptidoglycan binding protein—the human peptidoglycan recognition protein‐S (hPGRP‐S). We propose that this strategy could be implemented to carry out high‐throughput analyses to study peptidoglycan binding proteins.

SEPARATION OF PEPTIDES HAVING STRUCTURES AND DERIVATIVES OF MIMOSINE, A NONPROTEINOGENIC AMINO ACID, BY A NOVEL REVERSE-PHASE HPLC COLUMN PACKED WITH WIDE-PORE SILICA

To elucidate the mechanisms of the self-assembly and understand the molecular basis of the pathogenesis of conformational changes of proteins, β-amyloid or prion protein related peptides are indispensable materials. Hence, these peptides are known to have β-sheet rich structures that tend to cause difficulty in syntheses, as well as characterization by the conventional solid-phase assembly with reversed-phase columns. In general, reversed-phase HPLC combined with on-line mass spectrometry (LCMS) is routinely used for analyses and purification of resulting crude peptides and is carried out using preparative scale HPLC-columns. However, for such peptides, conventional C18 columns often give broad-peaks. We have improved separation procedures using a novel column packed with wide-pore silica having hydrophilic properties that gave separation of structural conformers with improved profiles. Thus, we have prepared several peptides for conformational studies. The same column also offers advantages for the chiral analysis of mimosine, a nonproteinogenic amino acid, which cannot be separated by the conventional C18 columns. Supplemental materials are available for this article. Go to the publishers online edition of the Journal of Liquid Chromatography and Related Technologies to view the supplemental file.

Screening of Peptides that Inhibit Bacterial Binding to Fibronectin using Combinatorial Peptide Libraries

Infective endocarditis is often caused by passage of oral endogenous bacteria into the blood stream. Such bacteremia occurs after tooth extraction, and occasionally even after brushing of the teeth. Abnormal or damaged heart valves, including artificial valves, show high risk of infection. Antibiotics are widely used to prevent this infection, however, frequent use of these have resulted in the generation of resistant mutants, which generate serious social problems. Thus, the development of more effective and safer drugs for the prevention of such infections is very desirable. The adhesion of bacteria to fibronectin, one of the major extracellular matrix (ECM) protein exposed on the wound endocardia, is considered critical for the infection. We have previously found a novel mode of interaction between endocarditis-causing bacteria and human fibronectin. The present study focuses on the discovery of candidate compounds that inhibit the association between microorganisms and fibronectin. Positional scanning libraries (PSL) with N-terminal biotinylated 6-mer peptides have been constructed and screened for binding to a monoclonal antibody for fibronectin that inhibits the bacterial fibronectin-binding. The consensus sequences derived from these experiments are expected to be structural mimetics of the local structure of fibronectin involved in the bacterial adhesion. Since individual synthetic 6-mer peptides did not show the desired action, discontinuous epitopes can be envisaged and therefore a 9-mer-PSL was constructed to reveal conformational epitopes. In the second library, several 9-mer peptides based on the screening were synthesized and gave improved results.

Total synthesis of maxadilan and its disulfide isomers, and structural requirements for binding to the PACAP type 1 receptor

Maxadilan (Maxa) was recently isolated from the salivary gland lysates of the bloodfeeding sand fly Lutzomyia longipalpis (a vector of leishmaniasis) [l]. The structure of Maxa is predicted to consist of 61 amino acids with two disulfide linkages. It is a potent and persistent vasodilator that acts as an agonist of the type I receptor for pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide with vascular activity, although there is no significant sequence similarity [2]. Previously we have synthesized numerous PACAP/VIP and their related peptides, elucidated their solution structures [3,4] and compared many aspects of their biological actions. The discrimination between two distinct PACAP receptor types (R1 and R2) was found to be in positions 4 and 5 of the N-terminal region [5,6]. It is interesting that Maxa is recognized only by R1 and hence the development of potent agonists from this source may have important clinical applications. The present paper describes the total chemical syntheses of Maxa and its disulfide isomers. Additionally, various partial fragments from the Nand Ctermini as well as the central region have been prepared by highly efficient SPPS using the Fmoc strategy [7] to investigate the active center.