Toshikazu Majima

Oriented immobilization of bacterial photosynthetic membrane.

SummaryWe have examined a method for oriented immobilization of photosynthetic membrane fragments on a solid surface by specific avidin-biotin interaction. Photosynthetic membrane fragments from the purple non-sulphur photosynthetic bacterium Rhodopseudomonas viridis, of which the H-subunit of the photosynthetic reaction centre was biotinylated, was immobilized on an avidin-adsorbed plate. Orientation of the immobilized membrane on the plastic plate was checked by an antisera binding assay that could react to the respective sides of the membrane: the H-subunit side was selectively adsorbed on the plate. Light-induced potential and current responses could be measured when the membrane immobilized on the SnO2-coated glass plate was dried and sandwiched with a counter electrode of Hg. The electrical response in the immobilized membrane was much improved in comparison with the control (membranes were simply adsorbed on the plate), supporting the idea that the membranes have an orientation on solid surfaces.

Soft X-ray imaging using CR-39 plastics with AFM readout

Abstract Soft X-ray microscopy using CR-39 plastic track detectors was demonstrated with an atomic force microscope (AFM) readout for the first time. A transmission X-ray image of the biological cells was recorded on the CR-39 surface, and revealed as relief after etching process. The resolution of the X-ray imaging is expected to be 100 nm or less, therefore imaging of biological cells is possible at an intracellular structure level. Using this technique, the high resolution image of both biological cells and charged particle tracks can be obtained simultaneously on a CR-39 with the AFM. In boron neutron capture therapy, this new technique provides the information on the distribution of boron compounds and radiation dose by charged particles mapping inside a cell.

Thermal stability of dried photosynthetic membrane film for photoelectrodes

Abstract Thermal stability was examined on a photoelectrode made from dried film of a photosynthetic membrane (chromatophore) isolated from a photosynthetic bacterium Rhodopseudomonas viridis . Below 60°C, neither the optical absorbance of the film nor its photoelectric response were affected when heated for 1 h. When the temperature was raised over 120 °C, both the absorbance and the response were lost. The temperature of half-denaturation of the chromatophore was 90 °C. X-ray diffraction of the film showed that heating did not cause the complete denaturation of the proteins of chromatophores. Heating did not destroy the secondary structure ( α -helix) of the proteins but the bundles of α -helices were distorted.

Light-induced electrical responses of dried chromatophore film: effect of the addition of cytochrome c

Chromatophores, photosynthetic membranes of photosynthetic bacteria, were isolated and cast on a transparent electrode to form films. The absorption spectrum of the film was similar to that of the native chromatophore. This indicated that the chromophore-protein structures were maintained through drying. Electrical transients of the film on the light pulsed excitation were examined. The amplitude and decay of the electrical transient of the film were altered by the addition of horse heart cytochrome c to the chromatophore of Rhodobacter sphaeroides R-26, which lacks the reaction centre-bound cytochrome c. The addition of cytochrome c into the film resulted in an increase in the light-induced current and a decrease in current decay.

Characterization of silica-coated hematite and application to the formation of composite particles including egg yolk PC liposomes

According to the method of Ohmori et al. (J. Colloid Interface Sci. 150 (1992) 594), a procedure is examined for the buildup of uniform silica layers on monodispersed hematite particles. It appears that the silica layer resulting is homogeneous and the layer thickness is controlled by the concentration of tetraethylorthosilicate (TEOS) in the medium. Further, egg PC liposomes, a typical biocolloid, are introduced onto the silica-coated hematite particle. The formation was proceeded by two types of processes: (1) heterocoagulation between the silica-coated hematite and egg PC liposomes by controlling the concentration of LaCl(3) in the medium, or (2) buildup using two proteins (lysozyme or cytochrome C) as binder molecules. These results were analyzed by zeta-potential measurements and a contact-type X-ray microscope, which is a unique technique for obtaining X-ray images of biological specimens in water with high resolution.

Soft X-ray imaging of living cells in water: flash contact soft X-ray microscope

Abstract Flash contact soft X-ray microscopy (FCSXRM) is an imaging technology for observing living cells in aqueous conditions with a spatial resolution of several tens of nm. The principle of soft X-ray imaging for living cells is carbon imaging, which provides us with a carbon-density-distribution map of the specimens. Carbon and oxygen have absorption edges in the soft X-ray wavelength range at 4.4 and 2.3 nm, respectively. Between these edges, the photoabsorption cross-section of carbon is about 10 times greater than that of oxygen. Thus, one can obtain soft X-ray images of living cells in water using this wavelength range. Laser-produced plasma is used as a flash X-ray source. A table-top FCSXRM, named FCSXRM ETL Mark 3, was developed in 1996 for convenient laboratory use of SXRM. The X-ray image of a specimen is recorded on a photoresist polymethyl methacrylate (PMMA) membrane supported on a silicon wafer. The X-ray image is then enlarged by an atomic force microscope (AFM). The practical resolution achieved by the system is about 40 nm, including the tip effect of the AFM. In this article, I discuss the possible roles of the FCSXRM for nanometer-scale imaging of living cells under physiological conditions.

Simultaneous visualization of contact microscopic image and energetic charged particle tracks and its application to medicine

Contact microscopy, enables us to visualize the detailed internal structure of biological cells. Exposure of biological specimen mounted on X-ray or UV sensitive materials to soft X-ray or UV introduces chemical damage to the materials, and the damage distribution due to the absorption of the X-rays or the UV by the specimen reveals as relief on the surface of the material after development process. The relief can be visualized with an AFM at high resolution of ~100 nm. We have applied the contact microscopy technique to high-resolution neutron-induced alpha-autoradiography for boron imaging in boron neutron capture therapy (BNCT). In BNCT, energetic alpha/lithium particles (range ~ single cell) from boron-neutron reactions introduce lethal damage to tumor cells selectively through thermal neutron irradiation with tumor-accumulating boron compounds. To understand the mechanism of drug delivery of those boron compounds is significant to evaluate the efficacy of BNCT. In the new technique, we can visualize those alpha/lithium particle tracks as etch pits and contact X-ray/UV microscopic image of tumor cells as relief on the surface of CR-39 plastic track detectors after etching process. Achievable resolution was ~100 nm with AFM readout, so that we can perform the boron imaging at subcellular scale.

High-Resolution Alpha-Autoradiography with Contact Microscopy Technique

We have been developing a novel method for high-resolution neutron-induced alpha-autoradiography (NIAR) using CR-39 plastic track detectors and an atomic force microscope (AFM) with contact microscopy technique. In this technique,sliced samples such as tissues including boron compounds are mounted on CR-39 plates, and then irradiated by thermal neutrons. The irradiated samples are exposed to soft X-rays, and then etched in NaOH solution for short time.Etch pits for alpha/lithium particle tracks and relief for transmission X-ray image of the specimen can be observed on the CR-39 surface with an AFM at about 100 nm resolution. In the NIAR, discrimination of proton background is required for quantification of boron concentration, and incident angle measurement is significant for accurate positioning of the alpha/lithium tracks on the specimen image. These properties were tested for the measurement of small etch pits observed with an AFM. It was confirmed that alpha/lithium particles and protons could be distinguished by etch pit size, and that incident angle of those charged particles could be measured at the accuracy of several degrees. These informations approve the reliability of the NIAR, especially for the subcellular measurement of boron compound distribution inside a cell in boron neutron capture therapy (BNCT).

Load-dependent sliding direction change of a myosin head on an actin molecule and its energetic aspects: Energy borrowing model of a cross-bridge cycle

A model of muscle contraction is proposed, assuming loose coupling between power strokes and ATP hydrolysis of a myosin head. The energy borrowing mechanism is introduced in a cross-bridge cycle that borrows energy from the environment to cover the necessary energy for enthalpy production during sliding movement. Important premises for modeling are as follows: 1) the interaction area where a myosin head slides is supposed to be on an actin molecule; 2) the actomyosin complex is assumed to generate force F(θ), which slides the myosin head M* in the interaction area; 3) the direction of the force F(θ) varies in proportion to the load P; 4) the energy supplied by ATP hydrolysis is used to retain the myosin head in the high-energy state M*, and is not used for enthalpy production; 5) the myosin head enters a hydration state and dehydration state repeatedly during the cross-bridge cycle. The dehydrated myosin head recovers its hydrated state by hydration in the surrounding medium; 6) the energy source for work and heat production liberated by the AM* complex is of external origin. On the basis of these premises, the model adequately explains the experimental results observed at various levels in muscular samples: 1) twist in actin filaments observed in shortening muscle fibers; 2) the load-velocity relationship in single muscle fiber; 3) energy balance among enthalpy production, the borrowed energy and the energy supplied by ATP hydrolysis during muscle contraction. Force F(θ) acting on the myosin head is depicted.

Photoelectrical Responses of Dried Purple Membrane Film in Blue Form and Acid Purple Form

The modification of photoelectrical property of dried purple membrane (PM) film by acid treatment and deionization method is reported. By acid treatment and deionization, blue and acid purple form of bacteriorhodopsin, which have been obtained only in suspension, are able to be obtained in dried PM films. Using these films, the photoelectrical responses for blue and acid purple form of bacteriorhodopsin in dried films are measured. The disappearance of the normal photocur rent and appearance of a photocurrent with an opposite polarity is observed for blue and acid purple form of dried PM. The photoelectrical property of bR in dried PM is able to be modified by acid treatment and deionization. These treatments are able to control the polarity of bacteriorhodopsin photoresponse without change of the orientation of the bacteriorhodopsin molecules.