Takashi Kanno

The role of disulfide bond in the amyloidogenic state of β2‐microglobulin studied by heteronuclear NMR

β2‐Microglobulin (β2‐m) is a major component of dialysis‐related amyloid fibrils. Although recombinant β2‐m forms needle‐like fibrils by in vitro extension reaction at pH 2.5, reduced β2‐m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of β2‐m in the acid‐denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. {1H}‐15N NOE at the regions between the two cysteine residues (Cys25–Cys80) revealed a marked difference in the pico‐ and nanosecond time scale dynamics between that the acid‐denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, ΔCα, ΔCO, and ΔHα, and 3JHNHα coupling constants indicated that both the oxidized and reduced β2‐m at pH 2.5 have marginal α‐helical propensity at regions close to the C‐terminal cysteine, although it is a β‐sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of β2‐m, and that the marginal helical propensity at the C‐terminal regions might play a role in modifying this potential.

Physicochemical and immunological characterization of hepatitis B virus envelope particles exclusively consisting of the entire L (pre-S1 + pre-S2 + S) protein.

The hepatitis B virus (HBV) envelope (env) protein is composed of three regions; the 108- or 119-residue pre-S1 region involved in the direct interaction with hepatocytes, the 55-residue pre-S2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Thus, to improve the immunogenic potency of conventional HB vaccines, development of a new vaccine containing the entire pre-S1 region in addition to pre-S2 and S is desired. We previously reported the efficient production of the HBV env L (pre-S1 + pre-S2 + S) protein in the recombinant yeast cells [J Biol Chem 267 (1992) 1953]. In this study, the HBV env L protein produced as nano-particles in yeast has been purified and characterized. By equilibrium sedimentation, an average molecular weight of L particle was estimated to be approximately 6.4 x 10(6), indicating that about 110 molecules of L proteins are assembled into an L particle. By atomic force microscopy in a moist atmosphere, the L particles were observed as large spherical particles with a diameter of 50-500 nm. The L particles were stable on short-time heating at a high temperature and long-time storage at a low temperature but rather unstable on repeated freezing and thawing and treatment with dithiothreitol. When immunized in mice, L particles elicited efficiently and simultaneously the anti-S, anti-pre-S2, and anti-pre-S1 antibodies. The ED(50) values in mice for the anti-S and anti-pre-S2 antibodies were similar to those elicited by the M (pre-S2 + S) particles. Furthermore, the anti-pre-S1 rabbit antibodies were found to recognize various segments of the pre-S1 region, including the pre-S1 (21-47) segment. These results show the high ability of L particles to induce all antibodies against HBV env proteins, hence promising the future application of L particles for the next generation HB vaccine.

High-resolution scanning tunneling microscopy imaging of DNA molecules on Cu(111) surfaces

Abstract Using the pulse injection method, single-stranded DNA and double-stranded plasmid DNA have been deposited on well-defined Cu(111) surfaces under ultrahigh vacuum (UHV) conditions to obtain high-resolution scanning tunneling microscope (STM) images. These particular UHV-STM images have revealed that DNA molecules are adsorbed directly onto a clean Cu(111) surface and exhibited the detailed structures of DNA, which has not been resolved previously. The single-stranded DNA oligomers have exhibited the images of individual internal base molecules and the helix structures made of complementary base sequences. For the double-stranded plasmid DNA, the images have shown the Watson–Crick double-helix structure.

Size distribution measurement of vesicles by atomic force microscopy

Vesicles have been utilized as nanoscale vehicles for reagents including potential drug delivery systems. When used to deliver drugs, vesicle size and the size distribution are important factors in the determination of the dosage, cell specificity, and rate of clearance from the body. Current size measurement techniques for vesicles are electron microscopy and dynamic light scattering, but their results are not equal. Therefore atomic force microscopy was attempted as another size measurement technique. After adsorption of the vesicles from a low-concentration solution of vesicles on mica substrate, each vesicle is generally found as a flattened structure. The diameters of vesicles in these solutions and their distribution have been successfully estimated from the surface area of the flattened structure of each vesicle. At higher concentrations, we have found a monolayer crammed with dome-shaped vesicles on the substrate. The diameters of vesicles in these solutions have also been successfully estimated from the surface area of the dome-shaped structure of each vesicle. Diameters of vesicles in solution estimated from two different vesicle concentrations are not close to those reported by electron microscope studies but are close to those reported by dynamic light scattering studies.

Formation and control of two-dimensional deoxyribonucleic acid network

Recently, we have successfully fabricated large-scale deoxyribonucleic acid (DNA) networks on mica surfaces using a simple fabrication method. This report describes how we fabricated a variety of structures depending on the type of DNA, and controlled these structures using a post-treatment ethanol which we observed using atomic force microscopy. We found that the height of fiber in the DNA network depended on the type of DNA and its original length, and that the fiber height and mesh diameter could be shortened and widened, respectively, with ethanol treatment.

Real Space Observation of Double-Helix DNA Structure Using a Low Temperature Scanning Tunneling Microscopy

Reproducible, reliable and high resolution low temperature scanning tunneling microscopy (LT-STM) images of plasmid DNA (deoxyribonucleic acid) (2739 bp), deposited on a well-defined Cu(111) substrate using pulse injection method under ultra-high vacuum (UHV) condition, have been obtained by using a novel sample preparation technique. These particular low temperature LT-UHV-STM images have revealed the double-helix structures of the DNA that have not been resolved earlier.

A New Self-Fabrication of Large-Scale Deoxyribonucleic Acid Network on Mica Surfaces.

We have successfully fabricated large-scale deoxyribonucleic acid (DNA) networks on mica surfaces using a simple and easy fabrication method for the first time. Sample drops of poly(dA-dT)poly(dA-dT) which is a synthetic linear DNA were spotted on freshly cleaved mica and blown off with air. At low DNA concentrations, clusters of DNA molecules were separated from each other. However at high concentrations, substrates were covered with a two-dimensional DNA network measuring more than 12 mm laterally. The DNA network discovered in our study seems highly practical due to its simple and easy fabrication method and its length. We believe this DNA network has the potential to serve as a biomaterial for medical, engineering and environmental applications.

Electrochemical detection of nonlabeled oligonucleotide DNA using biotin-modified DNA(ss) on a streptavidin-modified gold electrode

In the electrochemical detection of nonlabeled DNA, it is important to control the bonding at the interface between the DNA and the electrode. Atomic force microscope (AFM) was taken for the commonly used thiol-modified DNA on a gold surface. It was found that the coverage of the DNA was very low. On the other hand, a streptavidin-modified gold electrode provided a much better alternative where DNA hybridization resulted in large changes in the electrochemical reaction responses. This work demonstrates that streptavidin-modified gold electrodes could be used in the development of a new electrochemical protocol for the detection of nonlabeled DNA.

Does the Macrostructure of Deoxyribonucleic Acid Molecules Adsorbed on Substrates by the Pulse Injection Method Reflect That in Solution

In order to investigate whether the concentration-dependent macrostructure of Deoxyribonucleic acid (DNA) molecules can be retained after pulse injection, as compared to that originally present in the aqueous solution, we performed scanning tunneling microscopy (STM) imaging of plasmid DNA pulse-injected from an aqueous solution containing different DNA concentrations. The observed STM images revealed the presence of a relaxed circular macrostructure at low concentrations (1.2 µg/ml of DNA and 0.3 mM of Na+) whereas an entangled circular macrostructure was observed at high concentration (60 µg/ml of DNA and 15 mM of Na+). We conclude that plasmid DNA molecules retain the macrostructures possessed in the aqueous solution even after pulse injection onto a solid surface under vacuum environment.

Base Sequence Dependence of Deoxyribonucleic Acid Studied by Scanning Tunneling Microscopy

In order to identify high-order structures and pitch length of deoxyribonucleic acid (DNA), poly(dA-dT)poly(dA-dT) and poly(dG-dC)poly(dG-dC) were adsorbed on Cu(111) substrates by the pulse injection method and were observed by ultra high vacuum (UHV) scanning tunneling microscopy (STM). In large-scale STM images, the high-order structure of poly(dG-dC)poly(dG-dC) has a higher divergence and shorter dimension compared with those of poly(dA-dT)poly(dA-dT). High-resolution STM images revealed that the pitch lengths of poly(dA-dT)poly(dA-dT) and poly(dG-dC)poly(dG-dC) adsorbed onto the surface were longer than those possessed in the aqueous solution.