Biomedical subjects

Naoto Yagi

Publications and source records attributed to Naoto Yagi.

At least 19 recordsLinked to original sources

PubMed2006

High-resolution powder diffraction study of purple membrane with a large Guinier-type camera.

X-ray diffraction patterns from a film of oriented purple membranes, which comprise two-dimensional crystals of bacteriorhodopsin (BR) trimers, were recorded with a 1 m-pathlength Guinier-type camera at SPring-8 BL40B2. A well focused X-ray beam and a camera with a high angular resolution of 0.024 degrees enabled a powder diffraction profile with very sharp and well separated peaks to be obtained up to a resolution of 2.3 A. Using integrated diffraction intensities up to a Bragg spacing of 4.2 A, a cluster of bulky amino acid residues and the head group of the BR chromophore are apparent in the electron density map projected along the membrane normal. Thus, a combination of synchrotron X-rays and large Guinier camera can be used for analyzing the conformational changes of BR in the intact state. In addition, the method might be extended to the structural analysis of film materials composed of two-dimensional arrays of nanoparticles.

Equipment Design↗

PubMed2006

Evolution of long-range myofibrillar crystallinity in insect flight muscle as examined by X-ray cryomicrodiffraction.

Insect flight muscle is known for its crystal-quality regularity of contractile protein arrangement within a sarcomere. We have previously shown by X-ray microdiffraction that the crystal-quality regularity in bumble-bee flight muscle is not confined within a sarcomere, but extends over the entire length of a myofibril (>1000 sarcomeres connected in series). Because of this, the whole myofibril may be regarded as a millimetre-long, natural single protein crystal. Using bright X-ray beams from a synchrotron radiation source, we examined how this long-range crystallinity has evolved among winged insects. We analysed >4600 microdiffraction patterns of quick-frozen myofibrils from 50 insect species, covering all the major winged insect orders. The results show that the occurrence of such long-range crystallinity largely coincides with insect orders with asynchronous muscle operation. However, a few of the more skilled fliers among lower-order insects apparently have developed various degrees of structural regularity, suggesting that the demand for skillful flight has driven the lattice structure towards increased regularity.

Animals↗

PubMed2006

Naturally engineered glycolipid biosurfactants leading to distinctive self-assembled structures.

Self-assembling properties of "natural" glycolipid biosurfactants, mannosyl-erythritol lipids A and B (MEL-A, MEL-B), which are abundantly produced from yeast strains, were investigated by using the fluorescence-probe method, dynamic light-scattering (DLS) analysis, freeze-fracture transmission electron microscopy (FF-TEM), and synchrotron small/wide-angle X-ray scattering (SAXS/WAXS) analysis, among other methods. Both MEL-A and MEL-B exhibit excellent self-assembly properties at extremely low concentrations; they self-assemble into large unilamellar vesicles (LUV) just above their critical-aggregation concentration (CAC). The CAC(I) value was found to be 4.0x10(-6) M for MEL-A and 6.0x10(-6) M for MEL-B. Moreover, the self-assembled structure of MEL-A above a CAC(II) value of 2.0x10(-5) M was found to drastically change into sponge structures (L3) composed of a network of randomly connected bilayers that are usually obtained from a complicated multicomponent "synthetic" surfactant system. Interestingly, the average water-channel diameter of the sponge structure was 100 nm. This is relatively large compared with those obtained from "synthetic" surfactant systems. In addition, MEL-B, which has a hydroxyl group at the C-4' position on mannose instead of an acetyl group, gives only one CAC; the self-assembled structure of MEL-B seems to gradually move from LUV to multilamellar vesicles (MLV) with lattice constants of 4.4 nm, depending on the concentration. Furthermore, the lyotropic-liquid-crystal-phase observation at high concentrations demonstrates the formation of an inverted hexagonal phase (H2) for MEL-A, together with a lamella phase (L(alpha)) for MEL-B, indicating a difference between MEL-A and MEL-B molecules in the spontaneous curvature of the assemblies. These results clearly show that the difference in spontaneous curvature caused by the single acetyl group on the head group probably decides the direction of self-assembly of glycolipid biosurfactants. The unique and complex molecular structures with several chiral centers that are molecularly engineered by microorganisms must have led to the sophisticated self-assembling properties of the glycolipid biosurfactants.

Chemistry Techniques, Analytical↗

PubMed2005

An X-Ray diffraction study on mouse cardiac cross-bridge function in vivo: effects of adrenergic {beta}-stimulation.

To investigate how beta-stimulation affects the contractility of cardiac muscle, x-ray diffraction from cardiac muscle in the left ventricular free wall of a mouse heart was recorded in vivo. To our knowledge, this is the first x-ray diffraction study on a heart in a living body. After the R wave in electrocardiograms, the ratio of the intensities of the equatorial (1,0) and (1,1) reflections decreased for approximately 50 ms from a diastolic value of 2.1 to a minimum of 0.8, and then recovered. The spacing of the (1,0) lattice planes increased for approximately 90 ms from a diastolic value of 37.2 nm to a maximum of 39.1 nm, and then returned to the diastolic level, corresponding to approximately 10% stretch of sarcomere. Stimulation of beta-adrenergic receptor by dobutamine (20 microg/kg/min) accelerated both the decrease in the intensity ratio, which reached a smaller systolic value, and the increase in the lattice spacing. However, the intensity ratio and spacing at the end-diastole were unchanged. The recovery of the lattice spacing during relaxation was also accelerated. The mass transfer to the thin filaments at systole in a beta-stimulated heart was close to the peak value in twitch of frog skeletal muscle at 4 degrees C, showing that the majority of cross-bridges have been recruited with few in reserve.

Actins↗

PubMed2005

Crystal structure of M-Ras reveals a GTP-bound "off" state conformation of Ras family small GTPases.

Although some members of Ras family small GTPases, including M-Ras, share the primary structure of their effector regions with Ras, they exhibit vastly different binding properties to Ras effectors such as c-Raf-1. We have solved the crystal structure of M-Ras in the GDP-bound and guanosine 5'-(beta,gamma-imido)triphosphate (Gpp(NH)p)-bound forms. The overall structure of M-Ras resembles those of H-Ras and Rap2A, except that M-Ras-Gpp(NH)p exhibits a distinctive switch I conformation, which is caused by impaired intramolecular interactions between Thr-45 (corresponding to Thr-35 of H-Ras) of the effector region and the gamma-phosphate of Gpp(NH)p. Previous 31P NMR studies showed that H-Ras-Gpp(NH)p exists in two interconverting conformations, states 1 and 2. Whereas state 2 is a predominant form of H-Ras and corresponds to the "on" conformation found in the complex with effectors, state 1 is thought to represent the "off" conformation, whose tertiary structure remains unknown. 31P NMR analysis shows that free M-Ras-Gpp(NH)p predominantly assumes the state 1 conformation, which undergoes conformational transition to state 2 upon association with c-Raf-1. These results indicate that the solved structure of M-Ras-Gp-p(NH)p corresponds to the state 1 conformation. The predominance of state 1 in M-Ras is likely to account for its weak binding ability to the Ras effectors, suggesting the importance of the tertiary structure factor in small GTPase-effector interaction. Further, the first determination of the state 1 structure provides a molecular basis for developing novel anti-cancer drugs as compounds that hold Ras in the state 1 "off" conformation.

Amino Acid Sequence↗

PubMed2005

X-ray microdiffraction and conventional diffraction from frozen-hydrated biological specimens.

A system for recording microdiffraction patterns from micrometer-sized quick-frozen hydrated biological specimens at the high-flux beamline BL40XU of SPring-8 is described. The optics consists of a pair of pinholes drilled into tantalum substratum, with a defining aperture of diameter 2 microm. The frozen specimens are placed in an in-vacuum cryochamber mounted on a three-axis goniometer, where the specimens are stably held at a liquid-nitrogen temperature ( approximately 74 K). A beam size of 1.5 microm (full width at half-maximum) is attained at the sample position. By using this system, diffraction patterns have been recorded from an isolated single myofibril (diameter approximately 3 microm) of an insect flight muscle in an area equivalent to a single sarcomere (length approximately 3 microm). The technique is potentially applicable to other micrometer-sized hydrated biological specimens, which are more susceptible to radiation damage than dry synthetic polymers or biopolymers. The quick-freezing of biological specimens has also been proven useful in reducing the specimen volume in the beam in conventional diffraction recordings.

Animals↗

PubMed2005

Structural changes of actin-bound myosin heads after a quick length change in frog skeletal muscle.

Changes in the x-ray diffraction pattern from a frog skeletal muscle were recorded after a quick release or stretch, which was completed within one millisecond, at a time resolution of 0.53 ms using the high-flux beamline at the SPring-8 third-generation synchrotron radiation facility. Reversibility of the effects of the length changes was checked by quickly restoring the muscle length. Intensities of seven reflections were measured. A large, instantaneous intensity drop of a layer line at an axial spacing of 1/10.3 nm(-1) after a quick release and stretch, and its partial recovery by reversal of the length change, indicate a conformational change of myosin heads that are attached to actin. Intensity changes on the 14.5-nm myosin layer line suggest that the attached heads alter their radial mass distribution upon filament sliding. Intensity changes of the myosin reflections at 1/21.5 and 1/7.2 nm(-1) are not readily explained by a simple axial swing of cross-bridges. Intensity changes of the actin-based layer lines at 1/36 and 1/5.9 nm(-1) are not explained by it either, suggesting a structural change in actin molecules.

Actins↗

PubMed2005

Localized morphometric deformations of small airways and alveoli in intact mouse lungs under quasi-static inflation.

Localized morphometric deformations of small airways and alveoli during respiration have several biomechanical and physiological implications. We developed fast synchrotron radiation CT system to visualize the small airways and alveoli of an intact mouse lung without fixation and dehydration, and analyzed their localized morphometric deformations between functional residual capacity (FRC) and total lung capacity (TLC). The maximum resolution of 32.6lp/mm at the 5% modulation transfer function level can be achieved with 11.8-microm voxels and 7-min scanning. Compared with the values at FRC, the diameter and length for smaller airways (diameter at FRC <200 microm) increased by 68.8% and 29.5% (averaged value), and those for larger airways (diameter at FRC >400 microm) increased by 45.2 and 22.9% (averaged value), at TLC. Moreover we defined the volume behavior as the percentage of airway volume at FRC for TLC. The volume behavior for the small airways was not similar to that of the lung volume. These results indicated that all airways did not behave homogenously.

Animals↗

PubMed2005

Morphometric deformations of small airways and alveoli under quasi-static inflation process.

Localized morphometric deformations of small airways and alveoli during respiration have many biomedical and physiological implications. We developed fast synchrotron radiation CT system to visualize the small airways and alveoli of an intact mouse lung without fixation and dehydration, and analyzed their localized morphometric deformations between functional residual capacity (FRC) and total lung capacity (TLC). In the diameter behavior, the averaged and range values were significantly larger for smaller airways (68.8%, range: 0.36-0.89) than larger airways (45.2%, range: 0.40-0.57). These results indicated that the airway did not deformed in same manner and that these morphological differences characterized the heterogeneous lung function.

Air Pressure↗

PubMed2004

Structural transition of bacteriorhodopsin is preceded by deprotonation of Schiff base: microsecond time-resolved x-ray diffraction study of purple membrane.

The structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of 7 angstroms by a time-resolved x-ray diffraction experiment utilizing synchrotron x rays from an undulator of SPring-8. The x-ray diffraction measurement system, used in coupling with a pulsed YAG laser, enabled us to record a diffraction pattern from purple membrane film at a time-resolution of 6 micros over the time domain of 5 micros to 500 ms. In the time domain, the functionally most important M-intermediate appears. A series of time-resolved x-ray diffraction data after photo-excitation showed clear intensity changes caused by the conformational changes of helix G in the M-intermediate. The population of the reaction intermediate was prominently observed at approximately 5 ms after a photo-stimulus. In contrast, absorption measurement indicated the deprotonation of the Schiff base predominantly occurred at approximately 300 micros after a photo-stimulus. These results showed that the conformational changes characterizing structurally the M-intermediate predominantly occur at a later stage of the deprotonation of the Schiff base. Thus, the M-intermediate can be divided into two metastable stages with different physical characteristics.

Bacteriorhodopsins↗

PubMed2004

CCD-based X-ray area detector for time-resolved diffraction experiments.

A fast X-ray area detector for diffraction, scattering and imaging experiments at microsecond to millisecond time resolution has been developed. The key element of the detector is a fast (291 frames s(-1)) framing camera with three CCDs. A prism forms identical images on the CCDs and the frame rate is increased three times by reading them alternately. In order to convert X-rays into visible light that is detectable with the CCDs, an X-ray image intensifier is used. The camera can also be used with a high-resolution X-ray detector. In both cases it was found to be important to use a phosphor with a short decay time to fully make use of the high-speed framing capability of the camera. Preliminary results of a fibre diffraction experiment on a skeletal muscle and coronary angiography are presented.

Animals↗

PubMed2004

A large-area CMOS imager as an X-ray detector for synchrotron radiation experiments.

The performance of a CMOS flatpanel imager from Hamamatsu Photonics (C7942) has been tested in various synchrotron radiation experiments. This detector has a detection area of about 120 mm x 120 mm with 0.05 mm pixels, and a frame rate of 2 s(-1). The commercially available product was insensitive to X-rays with an energy lower than 15 keV, but slight modifications solved this problem. Images obtained in small-angle scattering, protein crystallography and medical imaging experiments were all of high quality. The fast readout and the large area are advantageous in real-time imaging. Although its noise level is higher than the area detectors that are currently used in synchrotron radiation experiments, it is particularly useful in experiments where other bulky detectors cannot be used. Its relatively low price (about 30,000 US dollars) makes it a unique option in the choice of detectors.

Animals↗

PubMed2004

In situ measurements of crossbridge dynamics and lattice spacing in rat hearts by x-ray diffraction: sensitivity to regional ischemia.

BACKGROUND: Synchrotron radiation has been used to analyze crossbridge dynamics in isolated papillary muscle and excised perfused hearts with the use of x-ray diffraction techniques. We showed that these techniques can detect regional changes in rat left ventricle contractility and myosin lattice spacing in in situ ejecting hearts in real time. Furthermore, we examined the sensitivity of these indexes to regional ischemia. METHODS AND RESULTS: The left ventricular free wall of spontaneously beating rat hearts (heart rate, 290 to 404 bpm) was directly exposed to brief high-flux, low-emittance x-ray beams provided at SPring-8. Myosin mass transfer to actin filaments was determined as the decrease in reflection intensity ratio (intensity of 1,0 plane over the 1,1 plane) between end-diastole and end-systole. The distance between 1,0 reflections was converted to a lattice spacing between myosin filaments. We found that mass transfer (mean, 1.71+/-0.09 SEM, n=13 hearts) preceded significant increases in lattice spacing (2 to 5 nm) during systole in nonischemic pericardium. Left coronary occlusion eliminated increases in lattice spacing and severely reduced mass transfer (P<0.01) in the ischemic region. CONCLUSIONS: Our results suggest that x-ray diffraction techniques permit real-time in situ analysis of regional crossbridge dynamics at molecular and fiber levels that might also facilitate investigations of ventricular output regulation by the Frank-Starling mechanism.

Actin Cytoskeleton↗

PubMed2004

Sarcomere-length dependence of lattice volume and radial mass transfer of myosin cross-bridges in rat papillary muscle.

We examined the sarcomere length-dependence of the spacing of the hexagonal lattice of the myofilaments and the mass transfer of myosin cross-bridges during contraction of right ventricular papillary muscle of the rat. The lattice spacing and mass transfer were measured by using X-ray diffraction, and the sarcomere length was monitored by laser diffraction at the same time. Although the lattice spacing and the sarcomere length were inversely related, their relationship was not exactly isovolumic. The cell volume decreased by about 15% when the sarcomere length was shortened from 2.3 micro m to 1.8 micro m. Twitch tension increased with sarcomere length (the Frank-Starling law). At the peak tension, the ratio of the intensity of the (1,0) equatorial reflection to that of the (1,1) reflection was smaller when the tension was greater, showing that the larger tension at a longer sarcomere length accompanies a larger amount of mass transfer of cross-bridges from the thick to the thin filament. The result suggests that the Frank-Starling law is due to an increase in the number of myosin heads attached to actin, not in the average force produced by each head.

Animals↗

PubMed2004

Intensity of X-ray reflections from skeletal muscle thin filaments partially occupied with myosin heads: effect of cooperative binding.

For quantitative analysis of contractile proteins of muscle by means of X-ray diffraction, it is important to know how the intensities of individual reflections are related to the number of diffracting objects, i.e., the amount of constituent contractile protein in the muscle cell. Here we diffused various amounts of exogenous myosin subfragment-1 (S1) into overstretched skinned skeletal muscle fibers, either in the presence or absence of Ca2+ , and derived the relationship between the S1 content and the intensities of reflections arising from the S1. In theory, the intensities should be proportional to the square of the S1 content (square law). However, the intensity-content relation deviated systematically from the square law as the S1 content was lowered, and it was better described as a linear function at the lower end of the S1 contents (<20% of saturation level). Model calculations show that the way of deviation is explained by the cooperative manner of S1 binding to the regulated thin filament.

Actins↗

PubMed2004

X-ray diffraction from a left ventricular wall of rat heart.

We studied x-ray diffraction from the left ventricular wall of an excised, perfused whole heart of a rat using x rays from the third-generation synchrotron radiation facility, SPring-8. With the beam at right angles to the long axis of the left ventricle, well-oriented, strong equatorial reflections were observed from the epicardium surface. The reflections became vertically split arcs when the beam passed through myocardium deeper in the wall, and rings were observed when the beam passed into the inner myocardium of the wall. These diffraction patterns were explained by employing a layered-spiral model of the arrangement of muscle fibers in the heart. In a quiescent heart with an expanded left ventricle, the muscle fibers at the epicardium surface were found to have a (1,0) lattice spacing smaller than in the rest of the wall. The intensity ratio of the (1,0) and (1,1) equatorial reflections decreased on contraction with a similar time course in all parts of the wall. The results show that it is possible to assign the origin of reflections in a diffraction diagram from a whole heart. This study offers a basis for interpretation of x-ray diffraction from a beating heart under physiologically and pathologically different conditions.

Animals↗

PubMed2004

In vitro evaluation of normal and abnormal lungs with ultra-high-resolution CT.

Synchrotron radiation microtomography is a new technique with high directionality of a synchrotron radiation beam and a high-resolution detector array. In this study, we estimated the visualization of the peripheral human lung structure with an ultra-high-resolution computed tomography (CT) system, the synchrotron radiation CT. The synchrotron radiation CT system uses the bending magnet beamline emitted from the 8.0 GeV electron storage ring. Six lung specimens were obtained at autopsy: 3 normal, 1 cellular alveolitis, 1 diffuse alveolar damage, and 1 pulmonary hemorrhage. Each specimen was cut down to a cylindrical sample with 6-mm diameter and 15- to 25-mm height. The synchrotron radiation CT images of these lung samples were obtained using the ultra-high-resolution image detector system with a charge coupled device (CCD) array detector (1024 x 1024 pixels with 12 x 12 microm2 pixel size). After that, the sample specimens were sliced to 200 microm (micrometer) thickness, and were observed with a stereomicroscope and by contact radiography. Finally, approximately 10-microm thick microscopic images were obtained and compared with the synchrotron radiation CT images. The synchrotron radiation CT could depict the peripheral lung including peripheral airways, airspaces, and alveolar walls individually. Each finding in the 3 disease processes seen on the synchrotron radiation CT images correlated well with the microscopic findings. The synchrotron radiation CT allows microtomographic imaging of human lung specimens with ultra high-spatial resolution (12 microm) on a level approaching that of conventional histopathology, without sectioning.

Humans↗

PubMed2004

Structural transients of contractile proteins upon sudden ATP liberation in skeletal muscle fibers.

Structural changes of contractile proteins were examined by millisecond time-resolved two-dimensional x-ray diffraction recordings during relaxation of skinned skeletal muscle fibers from rigor after caged ATP photolysis. It is known that the initial dissociation of the rigor actomyosin complex is followed by a period of transient active contraction, which is markedly prolonged in the presence of ADP by a mechanism yet to be clarified. Both single-headed (overstretched muscle fibers with exogenous myosin subfragment-1) and two-headed (fibers with full filament overlap) preparations were used. Analyses of various actin-based layer line reflections from both specimens showed the following: 1), The dissociation of the rigor actomyosin complex was fast and only modestly decelerated by ADP and occurred in a single exponential manner without passing through any detectable transitory state. Its ADP sensitivity was greater in the two-headed preparation but fell short of explaining the large ADP effect on the transient active contraction. 2), The decay of the activated state of the thin filament followed the time course of tension more closely in an ADP-dependent manner. These results suggest that the interplay between the reattached active myosin heads and the thin filament is responsible for the prolonged active contraction in the presence of ADP.

Actomyosin↗