Biomedical subjects

Tohru Ishitani

Publications and source records attributed to Tohru Ishitani.

At least 19 recordsLinked to original sources

PubMed2006

Influence of a combination of random noise and pattern-edge width (in pixels) on contrast-to-gradient image resolution in scanning electron microscopy.

The contrast-to-gradient (CG) method has been proposed previously for the evaluation of image resolution in scanning electron microscopy (SEM). In the CG algorithm, the image to be evaluated is gradually reduced to form the 1/r-reduced image (r = 1, 2, 3, ...) until the representative features of local patterns are accurately recognizable after the corresponding r-th noise reduction. We have studied the influence of a combination of random noise and pattern-edge width (in pixels) on the CG resolution R using both PC-made test images and typical SEM images. As the random noise is larger, the value of R becomes larger (or poorer). The increase in R due to the noise influence is more significant for the narrower width of the pattern edge. This satisfactorily agrees with the common idea that the image resolution is to reflect the sharpness of the pattern edge recognized in the image noise. By revising the form of DeltaR from the weighted standard fluctuation of R to the differential of R, a significantly weak N(dot)-dependency on R has been favorably confirmed, with keeping firmly the noise-dependency on R. Here, N(dot) is the number of pattern dots in the image.

Journal Article↗

PubMed2005

Influence of random noise on the contrast-to-gradient image resolution in scanning electron microscopy.

The contrast-to-gradient (CG) method has been proposed previously for the evaluation of image resolution in scanning electron microscopy (SEM). The CG resolution is based on the local resolution, which is defined as the distance to recognize the object's local surface at each pixel position. Then, the CG resolution R is inherently influenced by random noise contained in the image. The present study demonstrates the influence of random noise on the R-values using both personal computer-made dot patterns in various sizes and densities and SEM test micrographs. It is found that the R-values increase with increasing random noise intensity N and their increases are smaller for higher-density patterns, but the R-values are independent of their image sizes under constant pattern density. In addition, the standard CG algorithm is modified especially for straightly fabricated patterns along the x- (or y-) axis, resulting in an improvement in the measurement accuracy of y- (or x-) directional resolutions. The straightness as known information is utilized fully to recognize the local pattern in the random noise.

Journal Article↗

PubMed2005

STAT3 regulates Nemo-like kinase by mediating its interaction with IL-6-stimulated TGFbeta-activated kinase 1 for STAT3 Ser-727 phosphorylation.

Signal transducer and activator of transcription 3 (STAT3) is activated by the IL-6 family of cytokines and growth factors. STAT3 requires phosphorylation on Ser-727, in addition to tyrosine phosphorylation on Tyr-705, to be transcriptionally active. In IL-6 signaling, the two major pathways that derive from the YXXQ and the YSTV motifs of gp130 cause Ser-727 phosphorylation. Here, we show that TGF-beta-activated kinase 1 (TAK1) interacts with STAT3, that the TAK1-Nemo-like kinase (NLK) pathway is efficiently activated by IL-6 through the YXXQ motif, and that this is the YXXQ-mediated H7-sensitive pathway that leads to STAT3 Ser-727 phosphorylation. Because NLK was recently shown to interact with STAT3, we explored the role of STAT3 in activating this pathway. Depletion of STAT3 diminished the IL-6-induced NLK activation by >80% without inhibiting IL-6-induced TAK1 activation or its nuclear entry. We found that STAT3 functioned as a scaffold for TAK1 and NLK in vivo through a region in its carboxyl terminus. Furthermore, the expression of the STAT3(534-770) region in the nuclei of STAT3-knockdown cells enhanced the IL-6-induced NLK activation in a dose-dependent manner but not the TGFbeta-induced NLK activation. TGFbeta did not cause STAT3 Ser-727 phosphorylation, even when the carboxyl region of STAT3 was expressed in the nuclei. Together, these results indicate that STAT3 enhances the efficiency of its own Ser-727 phosphorylation by acting as a scaffold for the TAK1-NLK kinases, specifically in the YXXQ motif-derived pathway.

Amino Acid Motifs↗

PubMed2005

Nrarp functions to modulate neural-crest-cell differentiation by regulating LEF1 protein stability.

Nrarp (Notch-regulated ankyrin repeat protein) is a small protein that has two ankyrin repeats. Although Nrarp is known to be an inhibitory component of the Notch signalling pathway that operates in different developmental processes, the in vivo roles of Nrarp have not been fully characterized. Here, we show that Nrarp is a positive regulator in the Wnt signalling pathway. In zebrafish, knockdown of Nrarp-a expression by an antisense morpholino oligonucleotide (MO) results in altered Wnt-signalling-dependent neural-crest-cell development. Nrarp stabilizes LEF1 protein, a pivotal transcription factor in the Wnt signalling cascade, by blocking LEF1 ubiquitination. In accordance with this, the knockdown phenotype of lef1 is similar to that of nrarp-a, at least in part, in its effect on the development of multiple tissues in zebrafish. Furthermore, activation of LEF1 does not affect Notch activity or vice versa. These findings reveal that Nrarp independently regulates canonical Wnt and Notch signalling by modulating LEF1 and Notch protein turnover, respectively.

Animals↗

PubMed2004

Wnt-1 signal induces phosphorylation and degradation of c-Myb protein via TAK1, HIPK2, and NLK.

The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. Here we report that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps.

Animals↗

PubMed2004

Monte Carlo simulation of topographic contrast in scanning ion microscope.

Topographic contrast of secondary-electron (SE) images in a scanning ion microscope (SIM) using a focused gallium (Ga) ion beam is investigated by Monte Carlo simulation. The SE yield of heavy materials, in particular, due to the impact of 30 keV Ga ions increases much faster than for the impact of electrons at < or =10 keV as a function of the angle of incidence of the primary beam. This indicates the topographic contrast for heavy materials is clearer in a SIM image than in a scanning electron microscope (SEM) image; for light materials both contrasts are similar to each other. Semicircular rods with different radii and steps with large heights and a small wall angle, made of Si and Au, are modeled for comparison with SE images in SEM. Line profiles of the SE intensity and pseudo-images constructed from the profiles reveal some differences of the topographic contrast between SIM and SEM. We discuss not only the incident-angle effect on the contrast, but also the effects of re-entrances of primary particles and SEs to the neighboring surface, the effect of a sharp edge on the sample surface, and the effects of pattern size and beam size.

Gold↗

PubMed2004

Contrast-to-gradient method for the evaluation of image resolution taking account of random noise in scanning electron microscopy.

We have taken random image noise into consideration in the contrast-to-gradient (CG) method for the evaluation of image resolution R in scanning electron microscopy (SEM). When looking at the local fine pattern in the SEM micrograph containing much random noise, viewers gradually expand the region of interest (ROI) to improve the signal-to-noise ratio (SNR) and to recognize the pattern. We employed this approach in the CG algorithm to evaluate potential resolution Rpot, which is defined as the minimal/most accurate CG resolution calculated in the ROI expanding process. The image noise or SNR also is evaluated by the parameter deltaR / R, where deltaR is a standard deviation of R. The Rpot values depending on SNR are useful for comparison among images containing different random noise.

Algorithms↗

PubMed2004

Evaluation of TEM samples of an Mg-Al alloy prepared using FIB milling at the operating voltages of 10 kV and 40 kV.

Transmission electron microscopy (TEM) samples of an Mg-Al alloy has been prepared using a Ga-focused ion beam (FIB) milling at two different operating voltages of 10 kV and 40 kV to investigate the influence of the FIB energy on the sample quality. The fine structures of the samples have been studied using a high resolution TEM, and the concentration of the implanted Ga was analysed using an energy dispersive X-ray (EDX) analysis. The result of the TEM observation revealed that point defects were introduced to the sample finally milled at 40 kV but not at 10 kV. However, crystal lattice images and electron diffraction patterns were clearly observed on both the samples. The typical influence of the FIB energy was indicated in the elemental analysis. The relative Ga concentration in the thin sample finally milled at 10 kV was 1.0-2.0 at% that is less than half of 4.0-6.0 at% of the Ga concentration in the sample finally milled at 40 kV. A comparison between the experimental results of the Ga concentration measurement with simulation was also discussed.

Alloys↗

PubMed2004

Application of a FIB-STEM system for 3D observation of a resin-embedded yeast cell.

The combination of a focused ion beam (FIB) system and a scanning transmission electron microscope (STEM) has been applied to the three-dimensional (3D) observation of a resin-embedded yeast cell. Using a FIB microsampling technique, a sample with a thickness of tens of micrometres was extracted from a resin-embedded block sample. The extracted sample was transferred to a FIB-STEM-compatible specimen rotation holder and trimmed by FIB milling for 3D STEM observation. Although the FIB milling was carried out at an operating voltage of 40 KV, the sample was cross sectioned without forming a harmful damage layer on its surface. Cell structures, such as cell wall, cell membrane, mitochondria, peroxisomes, endoplasmic reticulum and vacuoles, were observed clearly in a pillar-shaped sample of 20 microm long, 4 microm wide and 3 microm deep.

Animals↗

PubMed2004

Improvements in performance of focused ion beam cross-sectioning: aspects of ion-sample interaction.

A gallium (Ga) focused ion beam (FIB) has been applied increasingly to 'site-specific' preparation of cross-sectional samples for transmission electron microscopy (TEM), scanning TEM, scanning electron microscopy and scanning ion microscopy. It is absolutely required for FIB cross-sectioning to prepare higher-quality samples in a shorter time without sacrificing the site specificity. The present paper clarifies the parameters that impose limitation on the following performances of the FIB cross-sectioning: milling rate, cross-sectioning at a right angle with respect to the sample surface, curtain structures formed on the cross sections, ion implantation and ion damage. All of these are discussed from the viewpoint of ion-sample interaction. Improvements for these performances achieved by diminishing their limiting origins or by correcting the resultants are described. Especially, the FIB scanning speed is significantly utilizable to improve the milling rate. A microsampling method, which allows the FIB incidence in a sidewards or upwards direction as well as downwards with respect to the microsample surface, is very effective to minimize the curtain structures.

Gallium↗

PubMed2004

A method for multidirectional TEM observation of a specific site at atomic resolution.

A new technique has been developed for the three-dimensional structure characterisation of a specific site at atomic resolution. In this technique, a focused ion beam (FIB) system is used to extract a specimen from a desired site as well as to fabricate the electron transparent specimen. A specimen holder with a specimen stage rotation mechanism has also been developed for use with both an FIB system and a high-resolution transmission electron microscope (TEM). The specimen holder allows both the FIB milling of a specimen and its observation in TEM without remounting the specimen from the specimen holder. A specimen for the three-dimensional TEM observation is extracted using the FIB micro-sampling technique and shaped into a pillar to mount on a tip of a needle stub enabling a multidirectional observation. The technique was applied to the multidirectional observation of the crystal structure of an Si single crystal at atomic resolution. The crystal lattice fringes of the two Si(111) planes with distances of 0.31 nm as well as the lattice fringes of the Si(200) with distances of 0.19 nm were clearly observed.

Imaging, Three-Dimensional↗

PubMed2003

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling.

The cytokines IL-1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF-kappaB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1- and TNF-induced activation of the NF-kappaB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-kappaB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-kappaB is not impaired in TAB2-deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-kappaB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. Further more, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1- and TNF-induced activation of TAK1 and NF-kappaB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Comparative study of depth and lateral distributions of electron excitation between scanning ion and scanning electron microscopes.

In order to study the contrast difference between scanning ion microscopes (SIM) and scanning electron microscopes (SEM), the depth and lateral distributions of secondary electrons escaped from surfaces of 17 metals with atomic numbers, Z2, of 4-79 were calculated for bombardment with 30 keV Ga ions and for 10 keV electrons. For both projectiles, the excitation depth generally decreased with increasing Z2, while showing the same periodic change as the secondary-electron yield. However, an opposite trend in Z2 dependence between the Ga ion and electron bombardments was calculated with the lateral distribution of secondary electrons escaped from the surface. Except for low Z2 metals, the lateral distribution, which is much narrower for 30 keV Ga ions than for 10 keV electrons, indicates that the spatial resolution of the secondary-electron images is better for SIM than for SEM, if zero-sized probe beams are assumed. Furthermore, the present calculation reveals important effects of electron excitation by recoiled material atoms and reflected electrons on the lateral distribution, as well as the secondary-electron yield, for the Ga ion and electron bombardments, respectively.

Journal Article↗

PubMed2003

The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling.

Wnt signaling controls a variety of developmental processes. The canonical Wnt/beta-catenin pathway functions to stabilize beta-catenin, and the noncanonical Wnt/Ca(2+) pathway activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca(2+) pathway activated by Wnt-5a antagonizes the Wnt/beta-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/beta-catenin signaling pathway. Here we show that activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor (beta(2)AR-Rfz-2) containing the ligand-binding and transmembrane segments from the beta(2)-adrenergic receptor (beta(2)AR) and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the beta-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits beta-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca(2+) pathway and antagonizes canonical Wnt/beta-catenin signaling.

Adrenergic beta-Agonists↗

PubMed2003

Regulation of lymphoid enhancer factor 1/T-cell factor by mitogen-activated protein kinase-related Nemo-like kinase-dependent phosphorylation in Wnt/beta-catenin signaling.

The Wnt/beta-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic beta-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. Previously, we have shown that a specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. In this study, we investigated the relationships among NLK, beta-catenin, and LEF-1/TCF. We found that NLK interacts directly with LEF-1/TCF and indirectly with beta-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the beta-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity.

Amino Acid Sequence↗

PubMed2002

Origins of material contrast in scanning ion microscope images.

A Monte Carlo simulation of ion-induced kinetic electron emission (KE) was carried out to study the material contrast in scanning ion microscope (SIM) images, i.e. secondary electron (SE) yields decreasing with atomic number Z2 of the target, which is opposite to that for scanning electron microscope (SEM) images. The simulations show that SE yields decrease with increasing Z2 for the targets of Al (Z2 = 13), Cu (Z2 = 29) and Au (Z2 = 79) bombarded by 10 approximately 40 keV gallium (Ga) ions. Details of the SE yield according to the collision partners (i.e. Ga ion, recoiled target-atom and excited electron) clarify the origins of material (or Z2) contrast in the Ga-SIM images. Cause and effect on the material contrast are as follows: the heavier (or slower) collision partner transfers less energy to the excited electrons and leads to a poorer multiplication of other excited electrons in the cascade process. The simulation also predicts that the Ga-SIM images are more sensitive to the outermost target surface than the SEM images. material contrast, atomic number contrast, secondary electrons, secondary electron yield, scanning ion microscope, scanning electron microscope

Journal Article↗

PubMed2002

Contrast-to-gradient method for the evaluation of image resolution in scanning electron microscopy.

In a previous study, we proposed the contrast-to-gradient (CG) method for evaluating image resolution. Here, the CG resolution is defined as a weighted harmonic mean of the local resolution, which is proportional to the quotient of the threshold contrast divided by the local gradient. The local gradient is calculated from the quadratic function that best fits the local pixel intensities over the region of interest (ROI) of 3 x 3 or 5 x 5 pixels in size. To refine the CG method, some modifications are carried out in the present study. Directional resolutions are employed to evaluate images, including astigmatism or strongly directional patterns as well as isotropic patterns. Here, CG resolution is redefined so as to keep the same value even for the image reversed in black-and-white contrast, because of no difference in the image information during contrast reversing. Besides, CG resolution is characterized to be independent of the brightness/contrast change unless these changes do not bring about both cut-off and saturation in the pixel intensities. Dependencies of the denoising effect and the resolution accuracy on ROI size are demonstrated as a function of image-noise.

Journal Article↗

PubMed2001

Method for Cross-sectional Thin Specimen Preparation from a Specific Site Using a Combination of a Focused Ion Beam System and Intermediate Voltage Electron Microscope and Its Application to the Characterization of a Precipitate in a Steel.

In this study, we discuss a method for cross-sectional thin specimen preparation from a specific site using a combination of a focused ion beam (FIB) system and an intermediate voltage transmission electron microscope (TEM). A FIB-TEM compatible specimen holder was newly developed for the method. The thinning of the specimen using the FIB system and the observation of inside structure of the ion milled area in a TEM to localize a specific site were alternately carried out. The TEM fitted with both scanning transmitted electron detector and secondary electron detector enabled us to localize the specific site in a halfway milled specimen with the positional accuracy of better than 0.1 &mgr;m. The method was applied to the characterization of a precipitate in a steel. A submicron large precipitate was thinned exactly at its center for the characterization by a high-resolution electron microscopy and an elemental mapping.

Journal Article↗