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A variety of methods have been reported using polymerase chain reaction (PCR)-based nucleic acid testing (NAT) because of its potential to be used in highly sensitive inspection systems. Among these NATs, fusion-PCR (also called as overlap-extension-PCR) has been focused on this study and adopted to generate the fused amplicon composed of plural marker gene fragments for detection. Generally, conventional agarose gel electrophoresis followed by gel staining is employed to check the PCR results. However, these are time-consuming processes that use specific equipment. To overcome these disadvantages, the immunochromatographic test (ICT) for the detection of PCR amplicons with hapten-labels that were generated by PCR using hapten-labeled primers was also adopted in this study. Based on these concepts, we constructed the systems of hapten-labeled fusion-PCR (HL-FuPCR) followed by ICT (HL-FuPCR-ICT) for the two and three marker genes derived from pathogenic microbe. As a result, we successfully developed a two marker genes system for the pathogenic influenza A virus and a three marker genes system for the penicillin-resistant Streptococcus pneumoniae. These detection systems of HL-FuPCR-ICT are characterized by simple handling and rapid detection within few minutes, and also showed the results as clear lines. Thus, the HL-FuPCR-ICT system introduced in this study has potential for use as a user-friendly inspection tool with the advantages especially in the detection of specific strains or groups expressing the characteristic phenotype(s) such as antibiotic resistance and/or high pathogenicity even in the same species.

Small-angle X-ray scattering (SAXS) coupled with computed tomography (CT), denoted SAXS-CT, enables the spatial distribution of the characteristic parameters of nanoscale structures inside samples to be visualized. In this work, a new scheme with Tikhonov regularization was developed to remove the effects of artifacts caused by streak scattering originating from contour regions of the sample., Small-angle X-ray scattering (SAXS) coupled with computed tomography (CT), denoted SAXS-CT, has enabled the spatial distribution of the characteristic parameters (e.g. size, shape, surface, length) of nanoscale structures inside samples to be visualized. In this work, a new scheme with Tikhonov regularization was developed to remove the effects of artifacts caused by streak scattering originating from the reflection of the incident beam in the contour regions of the sample. The noise due to streak scattering was successfully removed from the sinogram image and hence the CT image could be reconstructed free from artifacts in the contour regions.

We attempted to produce three dimensional (3D) aero-sponges using liquid silk (LS) extracted from the silk glands of fully grown larvae of Bombyx mori silkworms by combining an ice-crystal-sublimat...

We attempted to produce three dimensional (3D) aero-sponges using liquid silk (LS) extracted from the silk glands of fully grown larvae of Bombyx mori silkworms by combining an ice-crystal-sublimation method and a water-annealing treatment. The pore structure of the 3D LS-aero-sponge was constructed by the branched fibrous substrates and the films with small holes. It was assumed that the branched fibrous structure was mainly comprised of fibroin whereas the films were comprised of the fibroin interacting with the sericin fractions. Most of the WAXD peaks from the as-molded 3D LS-aerosponge could be assigned to the silk1 crystal diffraction peaks of fibroin. The silk1 crystal modification of fibroin formed in the course of the production of the 3D LS-aero-sponge could bring about a good thermal shrinkproof property. Furthermore, a gamma ray irradiation treatment applied prior to the cell proliferation tests of the 3D LS-aero-sponges was found to be effective to form cross-linking in the sericin fractions. As a result, the yield stress in the compression tests of the 3D LS-aero-sponges was increased. The 3D LS-aerosponge exhibited a good living cell proliferation properly in the culture tests using NH3T3 cells (Swiss albino mice embryonic fibroblasts) although that of the 2D LS-cast films was very poor. We suggest the good cell proliferation property of the 3D LS-aerosponge was due to the continuous branched fibrous structure comprised of fibroin.

Many insects use tactics to protect their eggs from predation, infection, and water loss, among other risks. Some assassin bugs only in the New World tribe Apiomerini are known to use plant substances, such as resin, instead of secretions to protect their eggs. Here, we report a novel storage mechanism and the utilization of plant resin for egg protection in Asian assassin bugs, Velinus nodipes and Agriosphodrus dohrni (tribe: Harpactorini). Adult females of both species were observed to take in plant resin through their genitalia. We reported on the handling of resin by these species and confirmed that A. dohrni adult females stored resin in their subrectal glands and that the substance covering their egg masses was derived from plant resin. This is the first report on the storage of plant resin inside the bodies of assassin bugs and on resin-protected eggs in Harpactorini. Such internal resin storage was not accompanied by external morphological features, as observed in some Apiomerini assassin bugs, thereby suggesting that this tactic has been overlooked and may have evolved in broader tribes of assassin bugs than previously thought.

Grazing-incidence small-angle X-ray scattering coupled with computed tomography (CT) has enabled the visualization of the spatial distribution of nanostructures in thin films. In this study, to optimize the CT image quality, total variation regularization is introduced to minimize sinogram image noise and artifacts., Grazing-incidence small-angle X-ray scattering (GISAXS) coupled with computed tomography (CT) has enabled the visualization of the spatial distribution of nanostructures in thin films. 2D GISAXS images are obtained by scanning along the direction perpendicular to the X-ray beam at each rotation angle. Because the intensities at the q positions contain nanostructural information, the reconstructed CT images individually represent the spatial distributions of this information (e.g. size, shape, surface, characteristic length). These images are reconstructed from the intensities acquired at angular intervals over 180°, but the total measurement time is prolonged. This increase in the radiation dosage can cause damage to the sample. One way to reduce the overall measurement time is to perform a scanning GISAXS measurement along the direction perpendicular to the X-ray beam with a limited interval angle. Using filtered back-projection (FBP), CT images are reconstructed from sinograms with limited interval angles from 3 to 48° (FBP-CT images). However, these images are blurred and have a low image quality. In this study, to optimize the CT image quality, total variation (TV) regularization is introduced to minimize sinogram image noise and artifacts. It is proposed that the TV method can be applied to downsampling of sinograms in order to improve the CT images in comparison with the FBP-CT images.

An attempt has been made to combine small-angle scattering of X-rays or neutrons with scanning electron microscopy in reciprocal space, in order to establish a structural analysis method covering a wide range of sizes from micro- to macro-scales. A system with a binary contrast, in which scattering objects with a homogeneous density are dispersed in vacuum (or air), is considered. A topological surface image, detected by secondary electron emission, is converted by means of a Fourier transform into a two-dimensional scattering amplitude in reciprocal space. The method was first tested by studying a dilute system of monodisperse SiO2 particles, with respect to calibrations for brightness inversion, noise reduction and two-dimensional Fourier transform, to obtain a scattering amplitude that agrees well with the analytical amplitude for a spherical particle. Secondly, the microstructure of a carbon-supported Pt catalyst for polymer electrolyte fuel cell applications was examined with the combined method, covering length scales from 10 µm down to nanometres. After two-dimensional Fourier transformation, the secondary electron emission images with low magnification are able to overcome the limitation of the minimum wavenumber (q min) detectable by ultra-small-angle scattering.

Injection-molding was performed using ethylene–propylene copolymer as the matrix by varying the physicochemical treatment of the reinforcement, i.e., core portion of kenaf stem that was dry-distilled and coated with or without β-sheet protein derivatives. Although dry distillation reduced the moisture content, the Charpy impact strength of the molded composites was low. When molding was performed using the fillers coated with β-sheet protein derivatives, especially coated with liquid silk (LS), a satisfactory Charpy impact strength of the molded composites was markedly gained. Thus, the β-sheet protein derivatives had potential to work as a compatibilizer. Coating the fillers with β-sheet proteins was also effective in suppressing the generation of silver streaks on the surface of composites and in preventing the occurrence of defective injection molding.

Three-dimensional (3D) cell culture systems have been used to obtain multicellular spheroidal cell aggregates, or spheroids, from cancer cells. However, it is difficult to efficiently prepare large tumor-derived spheroids from cancer cells. To circumvent this problem, we here used a tool equipped with removal membrane, called Spheroid Catch, for the selection and enrichment of large-sized and/or size-matched spheroids from human squamous cell carcinoma (SAS cells) without loss of recovery. After a five-round process of selection and enrichment, we successfully isolated a subpopulation of SAS cells with augmented spheroid-forming capability, named eSAS: the efficiency of spheroid formation is 28.5% (eSAS) vs 16.8% (parental SAS). Notably, we found that some of eSAS cells survived after exposure of high doses of cisplatin in 3D culture. Moreover, orthotopic implantation by injecting eSAS cells into the tongues of nude mice showed reduced survival rate and increased tumor growth compared with those of nude mice injected with SAS cells. These results suggest that spheroids exhibiting properties of higher spheroid forming capacity can be efficiently collected by using Spheroid Catch. Indeed, genome-wide cDNA microarray and western blot analyses demonstrated higher mRNA and protein levels of hedgehog acyltransferase (HHAT), which is associated with stem maintenance in cell carcinoma by catalysing the N-palmitoylation of Hedgehog proteins, in eSAS cells than in SAS cells. We propose that Spheroid Catch could be useful for the study of spheroids, and potentially organoids, in the basic and clinical sciences, as an alternative method to other type of cell strainers.

Injection molding was performed using poly(l-lactide) (PLLA) as a matrix and by varying the reinforcements, i.e., dry-distilled kenaf core (D-core) or untreated kenaf bast fibers, and the physical properties for these composites were subsequently compared. The dry-distillation was able to reduce the moisture content of D-core by a maximum of 4.2% as compared with untreated control core. As a result, the hydrophobicity was increased, which contributed to favorable Charpy impact strength and tensile properties of the D-core/PLLA composite relative to the kenaf bast fiber/PLLA composite. Moreover, the puffing phenomenon, which arises when heating with a microwave oven due to the vaporization of water dispersed within the interfacial regions and associated softening of the PLLA matrix by the heated vapor, was completely suppressed for the D-core/PLLA composite, whereas a large puffing ratio was observed for the kenaf bast fiber/PLLA composite. Reducing the moisture content also effectively suppressed ...

Cholesteric liquid crystal elastomer (CLCE) films, whose helical axis is parallel to a direction in the film plane, exhibit a periodic surface undulation with a period corresponding to a half-pitch (about 100 μm) of the helical director configuration. Temperature variation drives not only a finite macroscopic uniaxial deformation along the helical axis but also a marked variation in the surface undulation. AFM results demonstrate that the surface undulation varies thermoreversibly like a stationary wave with an amplitude of about 600 nm. The top and bottom positions at 40 °C become the bottom and top ones, respectively, at 130 °C, while the undulation disappears near 100 °C. Microspectroscopy reveals the periodic spatial variation in optical birefringence, which reflects the quasi-helical superstructure of local directors in the CLCE films. The combined analysis of the results of AFM and microspectroscopy indicates that the thermally induced variation in the surface undulation becomes largest at positions...

Nonlinear stress-strain behavior of the elastomer foams of high porosity is investigated in several types of biaxial tension, and uniaxial tension and compression. The values of Poisson's ratio for the open- and closed-cell foams are estimated to be about 0.24 almost independently of the imposed tensile strain, indicating that the foams undergo finite volume increase under tension. Unequal biaxial tensile experiments reveal no explicit cross-effect of strains between different axes in the elastomer foams: The deduced strain energy density function ( W ) has no appreciable contribution from the term of I 2 which is the second invariant of deformation gradient tensor representing the explicit cross-effect of strains, while in general W for conventional non-cellular elastomers involves finite contributions from I 2 -term. No explicit cross-effect of strains result in a unique feature in biaxial tensile behavior that the effect of the strain in one direction on the stress in the other direction is considerably small. These features in tensile behavior are commonly observed for the open- and closed-cell foams. In contrast, the cell type has a pronounced effect on the compression behavior accompanying the buckling and collapsing processes of the cells, as observed in earlier studies. The stress-strain relations in compression accompanying the cell buckling are markedly different from the expectation of W obtained from the data of biaxial and uniaxial tension where the cell bucking is not possible.

Images of the spatial distribution of nanostructures in thin films were successfully reconstructed by grazing-incidence small-angle X-ray scattering (GISAXS) coupled with computed tomography (CT) measurements. As a model sample of inhomogeneous thin films, a thin film was patterned with four characters (F, B, S and L) consisting of nanoparticles of gold (Au), platinum (Pt), Au/Pt and Pt/Au, respectively, on a silicon substrate. The characters each produced respective two-dimensional GISAXS images which reflect the nanoparticle structures and their correlations in the thin film. The application of the GISAXS-CT technique to the characteristic scattering GISAXS intensity of each component enables one to reconstruct the images of each character independently. Moreover, it was found that the patterned images could be reconstructed even from very weak scattered intensities at higherqpositions and the diffuse intensities. These results indicate that the GISAXS-CT method is a powerful tool to obtain distinct reconstruction images detailing the particle size, shape and surface roughness.

Using grazing-incidence small-angle scattering (GISAXS) with computed tomography (CT), we have individually reconstructed the spatial distribution of a thin gold (Au) layer buried under a thin poly(styrene-b-2-vinylpyridine) (PS-b-P2VP) layer. Owing to the difference between total reflection angles of Au and PS-b-P2VP, the scattering profiles for Au nanoparticles and self-assembled nanostructures of PS-b-P2VP could be independently obtained by changing the X-ray angle of incidence. Reconstruction of scattering profiles allows one to separately characterize spatial distributions in Au and PS-b-P2VP nanostructures.

Three-dimensional structural analyses of huge spherulites of poly(oxyethylene) (PEG) using X-ray computerized tomography (CT) in blends of PEG and amorphous poly(d,l-lactic acid) are reported for the first time. Slit-shaped cracks that were straight or curved were clearly observed using X-ray CT. Furthermore, they appeared to form a set of spokes, presenting a signature of a huge spherulite. Several aspects that are representative of an axialite structure are presented, and they are in good agreement with the intuitively proposed structural model for axialite long ago, although the size scale is much larger in our study.

Tomographic images constructed from small-angle X-ray scattering (SAXS) intensities utilizing microbeams have been examined for a model multilayered aluminum alloy sample heat-treated to form spatially modulated composition and microstructures. Spatial distributions of composition and precipitation microstructure have been examined by one-dimensional scanning SAXS measurements and two-dimensional tomographic images of attenuation parameters and integrated intensity of SAXS intensity. Reconstructed images using the integrated intensity reflected the distribution of precipitates in terms of their volume fraction over the sample inside the sample, and agreed with more detailed analysis made in the one-dimensional case.

Two novel phase-separation morphology are reported in the ternary polymer blends of poly(propylene)/nylon-12/poly(lactic acid) by utilizing a high-contrast X-ray computerized tomography. One is a “mosaic-tiling” structure, where each of three components forms equally-sized droplets in a three-dimensional tessellation manner. The other morphology is a “worm-like” structure. Two components of the three form successive droplets like stripe socks. These results strongly suggest the wide variety of the phase-separation morphologies in ternary polymer blends.

We have visualized redox and structural changes in the mitochondria of yeast Saccharomyces cerevisiae as a eukaryotic cell model using a genetically encoded yellow fluorescent protein (Y1-Yellow) and conventional fluorescence microscopy. Y1-Yellow originating from a yellow emitting luminous bacterium Aliivibrio sifiae Y1 was fused with a mitochondria-targeted sequence (mt-sequence). Y1-Yellow fluorescence arising only from the mitochondrial site and the color of yellow fluorescence could be easily differentiated from cellular autofluorescence and from that of conventional probes. Y1-Yellow expressing S. cerevisiae made the yellow fluorescence conspicuous at the mitochondrial site in response to reactive oxygen species (ROS) transiently derived in the wake of pretreatment with hydrogen peroxide. Based on our observation with Y1-Yellow fluorescence, we also showed that mitochondria rearrange to form a cluster structure surrounding chromosomal DNA via respiratory inhibition by cyanide, followed by the generation of ROS. In contrast, uptake of an uncoupler of oxidative phosphorylation is not responsible for mitochondrial rearrangement. These results indicate the utility of Y1-Yellow for visualization of mitochondrial vitality and morphology in living cells.

Since polymers mainly consist of the light atomic elements, the transmission of polymers against X-rays is usually too high to be visualized in X-ray microscopy, and hence it has been considered that polymers are not suitable for X-ray computerized tomography (XCT). We calculate the X-ray absorption coefficients of various polymers and find reasonably good conditions for the XCT observations of polymers: the use of 15 KeV X-rays on average can resolve the polystyrene (PS) and poly (methymethacrylate) (PMMA) in 3 µm spatial resolutions. According to this calculation, we build an XCT and experimentally confirm the visualization of the phase-separation structures of PS/PMMA blends.

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B’γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan–Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer.

Contrasts of the polymer materials under a high contrast X-ray computerized tomography (XCT) are comprehensively investigated. We developed a high contrast XCT, and demonstrated its capabilities to polymer systems, such as polymer blends. Then we got a hypothesis that the pixel values of the cross-sectional image obtained by XCT agree with the X-ray absorption coefficient at 15 keV. This hypothesis is intensively examined by using various polymers. Consequently, we propose an empirical criterion that 0.1 cm −1 difference in the X-ray absorption coefficients at 15 keV is necessary to distinguish the polymers under XCT. This criterion is also confirmed in the polymer blend systems.

Three-dimensional real-space images of the carbon fiber (CF)/polymer composites were obtained by a high-contrast X-ray computerized tomography (CT). Since both CF and polymers do not include heavy atoms, CF has not been supposed to be suitable to X-ray CT observation. In this study, we used the X-ray CT apparatus which is designed to enhance the contrast of the materials consisting only of light-weight atoms. Besides the usage of the appropriate apparatus, the experimental conditions were found to be important : cutting the sample into a thin rod, and obtaining sufficient number of projections. In our case, we used 1mm × 1mm × 4mm sample, and 720 projections with 0.25 degree intervals in order to obtain the 3μm voxel resolution in the reconstructed three-dimensional images. Eventually, each CF in polystyrene was clearly visualized in three dimensions.

We develop the exact WKB analysis of the PII hierarchy introduced by Gordoa et al. [5] in this paper; our particular interest is in the relation between the Stokes geometry of a higher order Painleve equation in the hierarchy and that of its underlying Lax pair. An important observation is that Stokes curves of the Painleve equation may cross, reflecting the higher order character of the equation, and in a neighborhood of the crossing point an unexpected degeneracy of the Stokes geometry of the Lax pair may occur along a curved ray emanating from the crossing point.

Three-dimensional (3D) observation of co-continuous macropore structure was performed using laser scanning confocal microscopy (LSCM). The obtained LSCM images were reconstructed into 3D images, and further subjected to image analysis. The LSCM observation and the following image analysis provided geometrical parameters of the macropore surfaces, which cannot be obtained by classical techniques for macropore characterization, such as scanning electron microscopy (SEM) observation or mercury intrusion method. Based on the obtained structural parameters, influences of viscoelastic properties on the liquid-phase structural formation mechanism were examined.

The excess shear stress after application of large step strains in polymer blend is calculated from observed shapes of deformed droplets in immiscible matrix, based on the Doi–Ohta expression for the interface contribution to the stress. The calculation is made for droplet shapes of flat ellipsoid, rod with end caps, dumbbell and ellipsoid of revolution. The predicted excess relaxation modulus agrees very well with experimental data normalized per one droplet with the volume-averaged radius for a poly(isobutylene)/poly(dimethyl siloxane) blend with narrow distribution of droplet size. Especially, slow stress relaxation in the intermediate stage and faster relaxation thereafter predicted from the rod like and dumbbell shapes are consistent with the experimental data. For a blend of hydroxypropylcellulose solution/poly(dimethyl siloxane) with broad distribution of droplet size, the predicted excess relaxation modulus agrees reasonably well with experimental data by taking account of the size distribution.

We unequivocally verified a success of creating “voided double-gyroid-channel” morphology and identified for the first time its cleavage planes inherent in the morphology on the basis of computer analysis of field-emission scanning electron microscope (FE-SEM) images. This morphology was created by transforming the minor network phase of the double-gyroid cubic structure of block copolymers with Ia3d space group symmetry into empty space. The fabrication process leading to the morphology was proven to conserve the original double-gyroid structure. The FE-SEM images observed on the freeze-fractured surface of the morphology exhibited almost only two kinds of patterns: “periodic double-wave pattern” and “hexagonal doughnut pattern”. Thus, the images observed on the freeze-fractured surfaces are “deterministic”, compared with those observed under transmission electron microscopy on the ultrathin-sectioned specimens which essentially vary randomly, depending on the sectioning directions relative to the cubi...

Nerve growth factor (NGF) is a well-known neurotrophic factor and the NGF signaling through the receptor, TrkA, plays important roles in regulating neuronal differentiation and survival. A recent study has demonstrated that the TrkAs expressed in undifferentiated PC12 cells were associated with caveolae, which were invaginated small pits on the plasma membrane. Caveolae are frequently seen in many cell types such as endothelial cells, fibroblasts and hepatocytes, but few in neurons. In the present study, we performed immunocytochemistry of TrkA in differentiated PC12 cells and analyzed the ultrastructural localization of TrkA by conventional electron microscopy and high-voltage electron microscopic (HVEM) tomography. The TrkA immunoreactivities were mainly associated with the cytoplasmic vesicles (10-30 nm in diameter) and a part of the plasma membrane. The HVEM tomography showed that the TrkA immunoreactivities were often assembled into ring-like structures (400-800 nm in diameter) near the plasma membrane, unlike typical flask-shaped invaginations of caveolae (50-100 nm in diameter). These results suggest that TrkA are not localized in the caveolae, at least in differentiated PC12 cells, but other invaginations are involved in a novel process of internalization of ligand-bound TrkA.

The film specimens having the double gyroid network structure with Ia 3 ¯ d space group symmetry were prepared by solution casting of poly(2-vinylpyridine)-block-polyisoprene mixed with a small but varying amounts of polyisoprene with chloroform as a solvent. The network phase composed of poly(2-vinylpyridine) was first crosslinked with 1,4-diiodobutane and then the matrix phase composed of polyisoprene was degraded by ozonolysis to obtain “free-standing” double gyroid network texture in which the two sets of networks are interlaced without contacts with one another in vacant space via their grain boundary connections. The network structures before and after ozonolysis were investigated, respectively, by transmission electron microscopy and field-emission scanning electron microscopy with the aid of numerically calculated three-dimensional computer graphics (3d CG) of the structure. The results clarified a success of the nano-fabrication on the free-standing double gyroid structure. We propose that the 3d CG-SEM, as discussed in the text, on the morphology of the freeze-fractured surface is particularly useful for the identification of the structure.

Three-dimensional (3D) image patterns of phase-separated poly(methyl methacrylate) (PMMA)/poly(styrene-ran-4-bromostyrene) (PS-Br) blends heated at 180°C for 6, 8, and 10 h were observed by 3D NMR microscopy with 3D X-ray microscopy in order to characterize the 3D structure of the polymer blends using a standard 3D reconstruction protocol program for obtaining the 3D digital array of the PS-Br/PMMA morphologies. The phase-separated structure of the polymer blends in several 10-μm scales was reasonably characterized. The phase-separated structure of polymer blends by 3D NMR images was quantitatively consistent with that by 3D X-ray images. It can be said that 3D NMR microscopy is a very useful means for analyzing the 3D structure of phase-separated polymer blends as well as 3D X-ray images. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 470–475, 2007

We examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization. Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 degrees C and that its formation was completed in approximately 3 h. A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to approximately 90 monomers and a bowl-like structure with long and short axes of 400 and 190 A, respectively. Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 A-1 corresponding to the 4.8-A spacing of beta-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine. These results imply that beta-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried beta-pleated sheets in fibril. The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine.

The commercial imaging plate (IP) system, R-AXIS IV++ (RIGAKU Co., Japan) is an IP detector integrated with a read-out system. We evaluated it and made some improvements for R-AXIS IV++ to optimise it for the use of synchrotron X-ray solution scattering measurements. Rigorously controlling self-decay of stored images on the IP, a maximum 5% decrease in sensitivity was observed by changing the end time for IP erasure to the start time of exposure. By replacing standard IP material with another commercial material that gives a slower fading period, the decrease disappeared. It suggested that not only the self-decay of stored intensity, but also the relaxation of the IP was important in a quick measurement cycle (ca. 5 min). After these improvements were made, we compared its suitability for solution scattering with other detectors. We found scattering profiles amenable to quantitative analysis. However, in wide scattering regions with low signal strength, its dynamic range is still very sensitive to measuring conditions.

In recent years, several methods for determining ab initio three-dimensional shape from solution scattering have been reported. In order to clarify the common and unique features of these programs, we verified the stability and convergence of three major programs, 'dammin', 'saxs3d' and 'dalai_ga', for experimental data. Two different types of samples were prepared: troponin-C and denatured apomyoglobin. With all of the three programs, the stability of the models and fitting among different runs increased as the diameters of the packing spheres were reduced in the region larger than 4Å. In the case of troponin-C, the models obtained always bore a striking resemblance irrespective of different runs of three algorithms. The averaged model coincided with the averaged structure of multiple nuclear magnetic reasonance models. In the case of denatured apomyoglobin, the best models from different algorithms also have similar features. These results suggest that if the molecule contains highly mobile portions, the resulting model corresponds to the averaged structure.

Withaferin A (WA), a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.