Tatsuhito Matsuo

Evaluation of enamel crystallites in subsurface lesion by microbeam X-ray diffraction

Early caries lesion is a demineralization process that takes place in the top 0.1 mm layer of tooth enamel. In this study, X-ray microbeam diffraction was used to evaluate the hydroxyapatite crystallites in the subsurface lesion of a bovine enamel section and the results are compared with those obtained by transversal microradiography, a method commonly used for evaluation of tooth mineral. Synchrotron radiation from SPring-8 was used to obtain a microbeam with a diameter of 6 microm. Wide-angle X-ray diffraction reports the amount of hydroxyapatite crystals, and small-angle X-ray scattering reports that of voids in crystallites. All three methods showed a marked decrease in the enamel density in the subsurface region after demineralization. As these diffraction methods provide structural information in the nanometre range, they are useful for investigating the mechanism of the mineral loss in early caries lesion at a nanometre level.

Structure of amyloid fibrils of hen egg white lysozyme studied by microbeam X-ray diffraction

Structure of spherical aggregates formed by hen egg white lysozyme (HEWL) was studied with microbeam X-ray diffraction. Aggregates with a diameter of 50-100 microm were formed after incubation of HEWL at pH 1.6 and 60 degrees C up to 60 days. The scattering from the aggregate in solution showed a marked symmetry demonstrating it as a spherulite. A reflection at 1/0.46 nm(-1) along the fiber axis showed the presence of beta-sheets along the fiber. There were strong equatorial reflections at 1/2.4 and 1/1.2 nm(-1). The similarities to other amyloid fibers suggest that molecules are planar in the direction perpendicular to the fiber axis and beta-strands are making hydrogen bonds to neighboring molecules.

A compact intermediate state of calmodulin in the process of target binding.

Calmodulin undergoes characteristic conformational changes by binding Ca(2+), which allows it to bind to more than 300 target proteins and regulate numerous intracellular processes in all eukaryotic cells. We measured the conformational changes of calmodulin upon Ca(2+) and mastoparan binding using the time-resolved small-angle X-ray scattering technique combined with flash photolysis of caged calcium. This measurement system covers the time range of 0.5-180 ms. Within 10 ms of the stepwise increase in Ca(2+) concentration, we identified a distinct compact conformational state with a drastically different molecular dimension. This process is too fast to study with a conventional stopped-flow apparatus. The compact conformational state was also observed without mastoparan, indicating that the calmodulin forms a compact globular conformation by itself upon Ca(2+) binding. This new conformational state of calmodulin seems to regulate Ca(2+) binding and conformational changes in the N-terminal domain. On the basis of this finding, an allosteric mechanism, which may have implications in intracellular signal transduction, is proposed.

Structural changes in the muscle thin filament during contractions caused by single and double electrical pulses.

In order to investigate the structural changes of the myofilaments involved in the phenomenon of summation in skeletal muscle contraction, we studied small-angle x-ray intensity changes during twitches of frog skeletal muscle elicited by either a single or a double stimulus at 16 degrees C. The separation of the pulses in the double-pulse stimulation was either 15 or 30 ms. The peak tension was more than doubled by the second stimulus. The equatorial (1,0) intensity, which decreased upon the first stimulus, further decreased with the second stimulus, indicating that more cross-bridges are formed. The meridional reflections from troponin at 1/38.5 and 1/19.2 nm(-1) were affected only slightly by the second stimulus, showing that attachment of a small number of myosin heads to actin can make a cooperative structural change. In overstretched muscle, the intensity increase of the troponin reflection in response to the second stimulus was smaller than that to the first stimulus. These results show that the summation is not due to an increased Ca binding to troponin and further suggest a highly cooperative nature of the structural changes in the thin filament that are related to the regulation of contraction.

Monitoring the structural behavior of troponin and myoplasmic free Ca2+ concentration during twitch of frog skeletal muscle.

The interaction of troponin molecules on the thin filament with Ca(2+) plays a key role in regulating muscle contraction. To characterize the structural changes of troponin caused by Ca(2+) and crossbridge formation, we recorded the small-angle x-ray intensity and the myoplasmic free Ca(2+) concentration using fluo-3 AM in the same frog skeletal muscle during twitch elicited by a single electrical pulse at 16 degrees C. In an overstretched muscle, the intensity of the meridional reflection from troponin at 1/38.5 nm(-1) began to change at 4 ms after the stimulus, reached a peak at 10 ms, and returned to the resting level with a halftime of 25 ms. The concentration of troponin-bound Ca(2+) began to increase at 1-2 ms after the stimulus, reached a peak at 5 ms, and returned to the resting level with a halftime of 40 ms, indicating that troponin begins to change conformation only after a sizable amount of Ca(2+) has bound to it, and returns to the resting structure even when there is still some bound Ca(2+). In a muscle with a filament overlap, crossbridge formation appears to slow down Ca(2+) release from troponin and have a large effect on its conformation.

Flight muscle myofibrillogenesis in the pupal stage of Drosophila as examined by X-ray microdiffraction and conventional diffraction

In the asynchronous flight muscles of higher insects, the lattice planes of contractile filaments are strictly preserved along the length of each myofibril, making the myofibril a millimetre-long giant single multiprotein crystal. To examine how such highly ordered structures are formed, we recorded X-ray diffraction patterns of the developing flight muscles of Drosophila pupae at various developmental stages. To evaluate the extent of long-range myofilament lattice order, end-on myofibrillar microdiffraction patterns were recorded from isolated quick-frozen dorsal longitudinal flight muscle fibres. In addition, conventional whole-thorax diffraction patterns were recorded from live pupae to assess the extent of development of flight musculature. Weak hexagonal fluctuations of scattering intensity were observed in the end-on patterns as early as approximately 15 h after myoblast fusion, and in the following 30 h, clear hexagonally arranged reflection spots became a common feature. The result suggests that the framework of the giant single-crystal structure is established in an early phase of myofibrillogenesis. Combined with published electron microscopy results, a myofibril in fused asynchronous flight muscle fibres is likely to start as a framework with fixed lattice plane orientations and fixed sarcomere numbers, to which constituent proteins are added afterwards without altering this basic configuration.

Optimization of calcium concentration of saliva with phosphoryl oligosaccharides of calcium (POs-Ca) for enamel remineralization in vitro

OBJECTIVE Phosphoryl oligosaccharides of calcium (POs-Ca) are highly soluble calcium source made from potato starch. The aim of this study was to investigate the optimal concentrations of POs-Ca for the remineralization of subsurface enamel lesions in vitro. DESIGN Demineralized bovine enamel slabs (n=5) were remineralized in vitro for 24h at 37°C with artificial saliva (AS) containing 0-0.74% POs-Ca to adjust the Ca/P ratio to 0.4-3.0, then sectioned and analysed by transversal microradiography (TMR). The data were analysed by Scheffes post hoc test. The Ca/P ratio with most remineralization was used to investigate the effect of calcium on enamel remineralization (n=11). The demineralized slabs were treated with AS with calcium-chloride- (CaCl2-) or POs-Ca with an identical calcium content, and sectioned for TMR and wide-angle X-ray diffraction (WAXRD) analyses to evaluate the local changes in hydroxyapatite (HAp) crystal content. The data were analysed using the Mann-Whitney U-test. RESULTS The highest mineral recovery rate resulted from addition of POs-Ca to adjust the Ca/P to 1.67. At this ratio, the mineral recovery rate for AS containing POs-Ca (24.2±7.4%) was significantly higher than that for AS containing CaCl2 (12.5±11.3%) (mean±SD, p<0.05). The recovery rate of HAp crystallites for AS containing POs-Ca (35.7±10.9%) was also significantly higher than that for AS containing CaCl2 (23.1±13.5%) (p<0.05). The restored crystallites were oriented in the same directions as in sound enamel. CONCLUSIONS POs-Ca effectively enhances enamel remineralization with ordered HAp at a Ca/P ratio of 1.67.

Structural Alterations of Thin Actin Filaments in Muscle Contraction by Synchrotron X-ray Fiber Diffraction

Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray fiber diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.

Measurement of persistence in YAG:Ce3+ scintillator with pulsed synchrotron X-rays

The decay time of YAG:Ce3+ scintillator was estimated to be 60 ns by using a fast CMOS camera and synchrotron bunches.

X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.