Biomedical subjects

Mitsuhiro Hirai

Publications and source records attributed to Mitsuhiro Hirai.

7 recordsLinked to original sources

PubMed2004

Hierarchical map of protein unfolding and refolding at thermal equilibrium revealed by wide-angle X-ray scattering.

Hierarchical features of the thermal unfolding-refolding structural transition of hen egg white lysozyme (HEWL) have been studied in the temperature range from 13 to 84 degrees C by using high-resolution wide-angle X-ray scattering (WAXS) measurements at a third-generation synchrotron source. We have gathered high-statistic WAXS data of the reversible unfolding-refolding process of HEWL in the q range from approximately 0.05 to approximately 3 A(-1) [q = (4pi/lambda) sin(theta/2), where theta is the scattering angle and lambda the wavelength]. This measured q range corresponds to the spatial distance from approximately 2 to approximately 125 A, which covers all hierarchical structures of a small globular protein such as HEWL, namely, tertiary, domain, and secondary structures. Because of this, we have found that the pH dependence of the thermal structural transition of HEWL is well characterized by the various hierarchical levels and the transition concurrence among them. In this report, we present a new hierarchical map depiction of unfolding-refolding transitions. Using scattering with various ranges of q values, we determine the molar ratio of native-like protein structure defined by the data in each range, thus producing a map of the amount of native-like structure as a function of the hierarchical level or resolution. This map can visualize a detailed feature of the unfolding-refolding transition of a protein depending on various structural hierarchical levels; however, the exact meaning of the map will await sharpening by additional works.

Animals↗

PubMed2004

Glycolipid based cubic nanoparticles: preparation and structural aspects.

Kinetically stable cubic colloidal particle dispersion was produced from a glycolipid using a novel preparation strategy based on the dialysis principle. The use of synchrotron small-angle X-ray diffraction (SSAXD) permitted the identification of exact structure of these dispersed particles in the colloidal state. Dynamic light scattering methods were used to obtain size and size distributions. A glycoside, 1-O-phytanyl-beta-D-xyloside (beta-XP), that exhibits Pn3m cubic phase in an excess aqueous medium, was used as the lipid material. The dialysis technique includes controlled stirring action both inside and outside of the dialysis membrane tube. Initially, a mixed micellar system composed of beta-XP, n-octyl-beta-D-glucopyranoside (beta-OG) and a triblock copolymer, Pluronic F127 (PL) was prepared in the aqueous medium. About 10 wt.% of PL to lipid weight was found to be sufficient to produce stable colloidal dispersions. The mean volume diameter of these colloidal particles was found to be in the range of 0.85 +/-0.05 microm. The cubic phase structure of these colloidal particles is greatly depended on the final beta-OG concentration level in the system. Coexistence of Im3m and Pn3m cubic structures has been identified in these colloidal particles. This coexistence has the characteristics of Bonnet relation, which forms a compelling case for the infinite periodic minimal surface (IPMS) descriptions. These colloidal particles could restore pure Pn3m phase structure, but a longer dialysis time was needed. This work, in general, will open up new possibilities for membrane protein reconstitution and other relevant biological applications using colloidal cubic lipid particles.

Dialysis↗

PubMed2003

An assay of ganglioside using fluorescence image analysis on a thin-layer chromatography plate.

A new and simple fluorometric method for determine quantities of gangliosides ranging from pico- to nanomoles is reported. Spraying hydrochloric acid followed by a heating treatment, sugars (glucose, galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid (sialic acid)), gangliosides (G(M1), G(D1a), G(T1b)), and asialoganglioside (asialoG(M1)) on thin-layer chromatography plates produced fluorescence under 365-nm UV light. This fluorescence production of each sample was greatly dependent on the heating temperature. As sialic acid fluoresced readily at lower temperature (approximately 90 degrees C), we were able to distinguish sialic acid easily from other sugars tested. To determine gangliosides based on this sialic acid fluorescence, calibration curves for gangliosides were obtained by high-performance thin-layer chromatography and the image-analyzing system equipped with a CCD camera. The observed fluorescence images were analyzed using image-analyzing software packages and the determined calibration curves for ganglioside-bound sialic acids were reproducible and showed a high linearity in a wide range from 47 pmol to 4.5 nmol. Since the fluorescence from sialic acid is easily measurable on TLC plates and is sensitive over a wide range of sample concentration, the present method is applicable for quantitative determination of gangliosides.

Animals↗

PubMed2003

Determination of asymmetric structure of ganglioside-DPPC mixed vesicle using SANS, SAXS, and DLS.

Functions of mammalian cell membrane microdomains being rich in glycosphingolipids, so-called rafts, are now one of the current hot topics in cell biology from the intimate relation to cell adhesion and signaling. However, little is known about the role of glycosphingolipids in the formation and stability of the domains. By the use of the inverse contrast variation method in small-angle neutron scattering (SANS), combined with small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS), we have determined an asymmetric internal structure of the bilayer of the small unilamellar vesicle (SUV) of monosialoganglioside (G(M1))-dipalmitoylphosphatidylcholine (DPPC) mixture ([G(M1)]:[DPPC] = 0.1:1). A direct method using a shell-model fitting with a size distribution function describes consistently all experimental results of SANS, SAXS, and DLS. We have found that G(M1) molecules predominantly localize at SUV outer surface to form a highly hydrophilic layer which is dehydrated with the rise of temperature from 25 degrees C to 55 degrees C accompanied by the conformational change of the oligosaccharide chains. The average SUV size determined is approximately 200 A, which is comparable to the reported value 260 +/- 130 A of glycosphingolipids microdomains. The present results suggest that the preferential asymmetric distribution of gangliosides is essential to define the size and stability of the domains.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

Structural hierarchy of several proteins observed by wide-angle solution scattering.

In the present study using a high-intensity X-ray beam from a third-generation synchrotron radiation source, it is demonstrated that a wide-angle X-ray scattering (WAXS) profile from several globular proteins in solution can reflect not only the overall structures (approximately 300 A distance resolution) but also intramolecular structures ranging to secondary structures (approximately 2.5 A distance resolution). The proteins treated in the present experiments are classified as different types of structure categories, namely, as all-alpha, all-beta and alpha + beta proteins. Here the full-range experimental scattering curves are compared with the theoretical curves, suggesting a further availability of the SR-WAXS method for studies of structure hierarchy and the function of proteins in solutions.

Animals↗

PubMed2002

Hydration and thermal reversibility of glycolipids depending on sugar chains.

To elucidate a relationship between the structural properties and hydration characteristic of gangliosides, time-resolved small-angle X-ray scattering measurements using synchrotron radiation have been performed on aqueous dispersions of various types of gangliosides (GM1, GD1a, GD1b and GM3) under a constant heating (5-65 degrees C) and cooling (65-5 degrees C) rate. In the case of GM3, they formed a vesicular aggregate with a high structural reversibility in the heating-and-cooling process. For the micelles of GM1, GD1a and GDlb, we found an evident thermal hysteresis in the structural changes of their headgroups and evaluated quantitatively the amounts of water molecules occluded in the micellar hydrophilic regions by using the shell modeling method reported previously. For all cases of GM1, GD1a and GD1b, the thickness of the hydrophilic region of the micelle shrunk after the heating process, and stayed mostly constant over the entire cooling range. On the other hand, the amounts of water molecules and the behavior of the GM1, GD1a and GD1b micelles in the heating-and-cooling process greatly depended on the number of sialic acid residues in the sugar chain, that is, the penetration of water molecules was much more reversible for the GM1 micelle compared with those for the GD1a and GD1b micelles. The observed clear hysteresis and the hydration characteristics of GD1 gangliosides would relate to their role in neuronal membranes, where GD1 gangliosides show the greatest concentrations.

Animals↗

PubMedDate not supplied

Polymer-dispersed bicontinuous cubic glycolipid nanoparticles.

We found that certain amphiphilic polymers such as PEO-PPO-PEO triblock copolymer (PL) can directly disperse a cubic glycolipid, 1-O-phytanyl-beta-D-xyloside (beta-XP), into bicontinuous cubic nanoparticles in water medium. The use of synchrotron small-angle X-ray diffraction (SSAXD) permitted the identification of the exact structure of these dispersed particles in the colloidal state. Dynamic light scattering method was used to obtain particle size distributions. The dispersion quality and the dispersion time can be improved by co-dissolving the lipid and the polymer in a common solvent. The mean volume diameter of these dispersed colloidal particles depends on the mixing time and polymer concentration. About 5 wt % (0.18 mol %) of polymer to lipid weight was found to be sufficient to produce stable colloidal dispersions. At this polymer content and at 3 h of stirring time, the mean volume diameter of cubic colloidal particles was found to be 1.0 microm. Increase of dispersion time to 6 h reduced the colloidal particle size from 1.0 microm to 660 nm. At 3 h of mixing time, the increase of polymer content, from approximately 5 to approximately 10 wt %, reduced the particle mean diameter from 1.0 microm to 675 nm. Irrespective of these dispersion times and polymer contents, the dispersed colloidal particles exhibit predominately the Pn3m cubic phase structure, the same as that of a beta-XP-water binary mixture, although a weak coexistence of Im3m cubic phase is identified in these colloidal particles. This coexistence is found to have the characteristics of a Bonnet relation, which forms convincing evidence for the infinite periodic minimal surface descriptions (IPMS). Considering the biotechnological significance, the preparation of these colloidal dispersions was carried out in a phosphate-buffered saline (PBS) system. These cubic colloidal dispersions exhibited good stability and the cubic phase structure remained intact in the PBS system.

Biotechnology↗