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Tatsuo Ushiki

Publications and source records attributed to Tatsuo Ushiki.

At least 19 recordsLinked to original sources
PubMed2006

Evidence of antibody production in the rat cervical lymph nodes after antigen administration into the cerebrospinal fluid.

We previously showed histologically that, in the rat, the cerebrospinal fluid drains from the subarachnoid space along the olfactory nerves to the nasal lymphatics and empties into the superficial and deep cervical lymph nodes. The present study was performed to investigate whether these lymph nodes play a role in the immune response of the central nervous system. For this purpose, keyhole limpet hemocyanin conjugated with fluorescein isothiocyanate (KLH-FITC) was administered into the subarachnoid space of the rat brain, and the time-kinetics and location of FITC and anti-FITC antibody forming cells in the cervical lymph nodes were studied histologically and immunohistochemically. FITC fluorescence was detected in superficial and deep cervical lymph nodes as well as the subarachnoid space and the nasal mucosa 2 h after FITC-KLH injection into the subarachnoid space. The specific antibody-forming cells first appeared in both the superficial and deep cervical lymph nodes on the 4th day after antigen administration although the reaction was more intense in the deep than in the superficial cervical lymph nodes. These cells were located in the medullary cords of the cervical lymph nodes. The number of antibody forming cells increased thereafter, reached a peak around the day 6, and then declined on day 10. These findings indicate that antigens introduced in the cerebrospinal fluid are drained into the cervical lymph nodes through the nasal lymphatics and initiate the antigen-specific immune response there. Thus, the cervical lymph nodes probably act as a monitoring site for cerebrospinal fluid and play a major role in the central nervous system immune response.

Animals↗
PubMed2006

Atomic force microscopy of native human metaphase chromosomes in a liquid.

The present study introduces a method for obtaining three-dimensional images of native (i.e., unfixed) chromosomes by atomic force microscopy (AFM) in a liquid. Human metaphase chromosomes were isolated from a human lymphoblast-like cell line, K562, by the hexylene glycol procedure according to Wray and Stubble- field (1970), adsorbed on a silane-coated glass slide, and observed in a dynamic force mode (i.e., intermittent contact mode) of AFM in a hexylene buffer solution. In adequate operating conditions, the shape of chromosomes with paired chromatids was clearly and three-dimensionally observed by AFM. At high magnification, globular or fibrous structures about 50 nm thick could be found on the surface of each chromaid, implying that chromatin fibers were strongly wound or twisted in the chromatid. Thus, AFM imaging enabled the direct visualization of native chromosomes in a liquid at high resolution--which is comparable with that of scanning electron microscopy--and can serve to analyze the mechanism of chromosome condensation and separation in relation to the structure of chromosomes.

Cell Line, Tumor↗
PubMed2005

CHRONIS: an animal chromosome image database.

We have constructed a database system named CHRONIS (CHROmosome and Nano-Information System) to collect images of animal chromosomes and related nanotechnological information. CHRONIS enables rapid sharing of information on chromosome research among cell biologists and researchers in other fields via the Internet. CHRONIS is also intended to serve as a liaison tool for researchers who work in different centers. The image database contains more than 3,000 color microscopic images, including karyotypic images obtained from more than 1,000 species of animals. Researchers can browse the contents of the database using a usual World Wide Web interface in the following URL: http://chromosome.med.niigata-u.ac.jp/chronis/servlet/chronisservlet. The system enables users to input new images into the database, to locate images of interest by keyword searches, and to display the images with detailed information. CHRONIS has a wide range of applications, such as searching for appropriate probes for fluorescent in situ hybridization, comparing various kinds of microscopic images of a single species, and finding researchers working in the same field of interest.

Animals↗
PubMed2005

Cathodoluminescence investigation of organic materials.

Cathodoluminescence (CL) properties of various kinds of organic material were investigated for the purpose of staining biological specimens and obtaining CL images. Several kinds of organic light emitting device (OLED) material exhibited CL. The europium complex, Eu(dbm)3(phen), showed the strongest CL signal and was chemically modified for biological staining. However, the CL intensity from the stained biological specimen was too weak to build CL images. We discussed the CL properties of organic materials considering their chemical structure and charge distribution in the molecules.

Biotin↗
PubMed2005

Myosin-Va regulates exocytosis through the submicromolar Ca2+-dependent binding of syntaxin-1A.

Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 microM Ca2+. Interference with formation of the syntaxin-1A-myosin-Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca2+ depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca2+.

Amino Acid Sequence↗
PubMed2005

The structure of C-banded human metaphase chromosomes as observed by atomic force microscopy.

The ultrastructure of C-banded human metaphase chromosomes was studied by the combined use of light microscopy and atomic force microscopy (AFM). Light microscopy of the C-banded chromosomes showed that the centromeric regions of all chromosomes except the Y chromosome were positively stained. AFM further revealed that the C-positive region was higher than the C-negative region. The area of the C-positive region was specific depending on each chromosome; it ranged from the centromere to the proximal end of the long arm in chromosome 1, while it was restricted to the centromere in chromosomes 2 and 3. At higher magnification, chromatin fibers about 50 nm thick were clearly shown in the entire length of the chromosomes. In the C-positive region, these chromatin fibers were densely packed, while chromatin fibers were loosely packed with gentle twisting in the C-negative region. These AFM findings suggest that certain factors related to the chromatin fiber compaction remain in the C-positive region even after successive C-banging treatment.

Centromere↗
PubMed2004

Neutrophil extravasation in rat mesenteric venules induced by the chemotactic peptide N-formyl-methionyl-luecylphenylalanine (fMLP), with special attention to a barrier function of the vascular basal lamina for neutrophil migration.

The present study was performed to investigate morphologically the process of neutrophil extravasation induced by the synthetic bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in venules of the rat mesentery by the combined use of intravital microscopy and transmission electron microscopy (TEM). Special attention was given to the interaction of the neutrophils with the endothelial cells and endothelial basal lamina. By intravital microscopy, the rolling and adhesion of leukocytes were observed within 3 min in preparations activated by fMLP. Neutrophils remained in the vascular wall for more than 30 min even after penetration of the endothelium. In this period, neutrophils migrating between endothelial cells and their basal lamina were often observed by TEM. After 40 min application of fMLP, some of the migrating neutrophils parted from the vessel wall into the surrounding connective tissues. There were neutrophils which passed through the small pore of the basal lamina with a cytoplasmic constriction. These findings indicate that the endothelial basal lamina acts as a physical barrier against neutrophil extravasation, thus resulting in the transient retainment of neutrophils beneath the endothelial cells of the venular wall.

Animals↗
PubMed2004

Cytoarchitecture of the normal rat olfactory epithelium: light and scanning electron microscopic studies.

The three-dimensional cytoarchitecture of the normal rat olfactory epithelium was examined by scanning electron microscopy (SEM) of KOH digested tissues as well as by light and transmission electron microscopy of plastic sections. Observations specimens from the lateral side of the olfactory epithelium allowed identification of four cell types by their surface structure: olfactory neurons, supporting cells, basal cells, and duct cells of the Bowman's gland. The olfactory neurons were characterized by the presence of a thick apical process (i.e., dendrite) and a thin basal process (i.e., axon). These olfactory neurons tended to be aligned along the vertical axis of the epithelium. Immature olfactory neurons were present at the basal part of the epithelium and had a pear-shaped cell body with a thin and long axon and a short dendrite which failed to reach the epithelial surface. Supporting cells were roughly columnar in shape and occupied the full length of the epithelium. They became thinner in the basal two thirds of their length but had branched foot processes spreading on the basal surface of the epithelium. Basal cells located in the basal epithelial region were oval, round or cuboidal and present among the foot processes of the supporting cells. The ducts of the Bowman's gland entered the epithelium from the lamina propria and took straight, perpendicular courses within the epithelium. These intraepithelial ducts were composed of several slender cells. The acinar cells are sometimes present in the epithelium and appeared as a globular bulge of the duct at the basal part of the epithelium. SEM observation of the basal surface of the olfactory epithelium also clearly showed that axon bundles were surrounded by the sheet-like processes of Schwann cells, the investment being found at the base of the epithelium just before axon bundles leave the epithelium.

Animals↗
PubMed2004

Atomic force microscopy of human metaphase chromosomes after differential staining of sister chromatids.

Human metaphase chromosomes, in which 5-bromo-deoxyuridine (BrdU) had been incorporated into the DNA, were treated with the fluorescent plus Giemsa (FPG) method. Use of this method distinctly stained one of the paired sister chromatids with the Giemsa solution due to the difference in content of BrdU in the two chromatids. These chromosomes with their differential staining of sister chromatids were observed by atomic force microscopy (AFM). In the air-dried specimens, one of the paired chromatids that was stained strongly with Giemsa solution was about two times higher than the counterpart that was stained faintly with Giemsa solution. In the critical point dried chromosomes, the height of the Giemsa positive chromatid roughly matched that of the Giemsa negative counterpart. These findings imply that the arrangement of the Giemsa negative chromatid after FPG staining is fragile and easily collapses due to the surface tension of water during air-drying. At higher magnifications, the surface structure differed between Giemsa positive and negative chromatids; the Giemsa positive chromatid (i.e., unifilarly BrdU-incorporated chromatid) was composed of fibrous structures while the Giemsa negative chromatid (i.e., bifilarly BrdU-incorporated chromatid) exhibited a fine granular appearance. These structural changes in the sister chromatids are thought to arise from the ultraviolet irradiation and heating of the chromosomes during FPG staining.

Antimetabolites↗
PubMed2004

Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy.

Cathodoluminescence (CL) is the light that is emitted from a material irradiated by an electron beam. The present study was undertaken to show the applicability to biological studies of a scanning electron microscope (SEM) equipped with a high-sensitive cathodoluminescence detection system. For this purpose, we injected inorganic fluorescent powders (P43) suspended in phosphate buffered saline into rat blood circulation, fixed the animals with glutaraldehyde within a day, and observed the hepatic tissues with a SEM. Our instrument enabled the simultaneous collection of both secondary electron (SE) and CL images of these tissues. Backscattered electron (BSE) images of the same portion were also able to be obtained with this microscope. SE and BSE images clearly showed the three-dimensional structure of the hepatic tissues including hepatocytes, Kupffer cells, Ito cells, and sinusoidal epithelial cells, while CL images visualized cathodoluminescence signals emitted from P43 as bright spots. We observed non-coated tissues under a low-vacuum condition and metal-coated tissues under a high-vacuum condition, and found that the high-vacuum observation of metal-coated tissues provided high quality CL images of P43 in the Kupffer cells. The superimposition of the CL images onto the corresponding SE or BSE images revealed that bright spots in the CL images were produced by the fluorescent powders uptaken by Kupffer cells. These findings indicate that the detection of CL as well as SE or BSE signals by SEM all provide us with useful information on the distribution of fluorescent tracers in tissues and cells in three-dimensional images.

Animals↗
PubMed2003

Embryonic development of the myenteric nerve plexus in the rat small intestine as revealed by light and scanning electron microscopy.

This study was performed to clarify developmental changes in the structure of the myenteric nerve plexus in the rat small intestine. The small intestines of fetal and neonatal rats were examined by light microscopic immunohistochemistry for protein gene product 9.5 (PGP-9.5) and alpha-smooth muscle actin (alpha-SMA). Direct observation of the three-dimensional structure of the neuronal and muscular elements was accomplished by scanning electron microscopy of tissues after KOH treatment. At the embryonic day 14 (E14), PGP-9.5 immunopositive (neuronal) cells first appeared in the mesenchymal tissue outside an alpha-SMA positive (muscle) cell layer. These cells increased in number to form a thin and continuous sheet, which was sandwiched between the inner and outer muscular layers at E17. This neuronal layer consisted of immature ganglion cells associated with supportive (enteroglial) cells, and was gradually segregated into groups by incomplete separation through slit-like interruptions at E18. With the widening of these slits at E19, the neuronal sheet was observed as a ladder composed of transversely elongated ganglia and their connecting nerve strands, resulting in the typical structure of the myenteric nerve plexus. Fibroblastic cells of a peculiar shape appeared on the outer surface of the myenteric plexus at E19. These cells (presumably interstitial cells) interconnected their long branching projections to form a cellular network on the myenteric plexus until birth.

Animals↗
PubMed2002

Molecular visualization of immunoglobulin switch region RNA/DNA complex by atomic force microscope.

Immunoglobulin heavy-chain (IgH) class switch recombination (CSR) is initiated by DNA breakage in the switch (S) region featuring tandem repetitive nucleotide sequences. Various studies have demonstrated that S-region transcription and splicing proceed to genomic recombination and are indispensable for CSR in vivo, although the precise molecular mechanism is largely unknown. Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an atomic force microscope (AFM). The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate that may stall replication and increase the recombinogenicity of the S-region DNA.

Immunoglobulin Class Switching↗
PubMed2002

Simultaneous collection of topographic and fluorescent images of barley chromosomes by scanning near-field optical/atomic force microscopy.

The present study investigated the applicability of scanning near-field optical/atomic force microscopy (SNOM/AFM) to observations of YOYO-1 stained barley chromosomes. Using this technique, topographic and fluorescent images in the same portion were obtained simultaneously, thus enabling the precise analysis of fluorescent structures in relation to the morphology of chromosomes. In YOYO-1 stained chromosomes, the fluorescent intensity roughly reflected the local amount and/or density of DNA in chromosomes. R-banding of chromosomes stained with YOYO-1 and methyl green was also observed clearly as bright spots of various sizes by this microscope. Thus, the SNOM/AFM is expected to become a useful tool for analysing the structure and function of chromosomes more precisely than before.

Benzoxazoles↗
PubMed2002

Chromosome binding site of latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus is essential for persistent episome maintenance and is functionally replaced by histone H1.

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (DeltaN-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of DeltaN-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.

Antigens, Viral↗
PubMed2002

Collagen fibers, reticular fibers and elastic fibers. A comprehensive understanding from a morphological viewpoint.

Fibrous components of the extracellular matrix are light-microscopically classified into three types of fibers: collagen, reticular and elastic. The present study reviews the ultrastructure of these fibrous components as based on our previous studies by light, electron, and atomic force microscopy. Collagen fibers present a cord- or tape-shape 1-20 microm wide and run a wavy course in tissues. These fibers consist of closely packed thin collagen fibrils (30-100 nm thick in ordinary tissues of mammals), and exhibit splitting and joining in altering the number of the fibrils to form a three-dimensional network as a whole. Individual collagen fibrils (i.e., unit fibrils) in collagen fibers have a characteristic D-banding pattern whose length is ranges from 64 to 67 nm, depending on tissues and organs. During fibrogenesis, collagen fibrils are considered to be produced by fusing short and thin fibrils with tapered ends. Reticular fibers are usually observed as a delicate meshwork of fine fibrils stained black by the silver impregnation method. They usually underlie the epithelium and cover the surface of such cells of muscle cells, adipose cells and Schwann cells. Electronmicroscopically, reticular fibers are observed as individual collagen fibrils or a small bundle of the fibrils, although the diameter of the fibrils is thin (about 30 nm) and uniform. Reticular fibers are continuous with collagen fibers through the exchange of these collagen fibrils. In silver-impregnated specimens, individual fibrils in reticular fibers are densely coated with coarse metal particles, probably due to the high content of glycoproteins around the fibrils. Elastic fibers and laminae are composed of microfibrils and elastin components. Observations of the extracted elastin have revealed that elastin components are comprised of elastin fibrils about 0.1-0.2 microm thick. Elastic fibers and laminae are continuous with networks and/or bundles of microfibrils (or oxytalan fibers), and form an elastic network specific to individual tissues. The fibrous components of the extracellular matrix are thereby morphologically categorized into two systems: the collagen fibrillar system as a supporting framework of tissues and cells, and the microfibrilelastin system for uniformly distributing stress to maintain the resilience adapted to local tissue requirements.

Collagen↗
PubMed2002

Three-dimensional cytoarchitecture of angiogenic blood vessels in a gelatin sheet implanted in the rat skeletal muscular layers.

To demonstrate the structure of angiogenic blood vessels three-dimensionally, a gelatin sponge sheet immersed in a vascular endothelial growth factor (VEGF) solution was implanted in the rat dorsal muscular layer, and examined by light microscopy and scanning electron microscopy (SEM) 5 days to 2 weeks after implantation. Light microscopy of anti-collagen IV antibody immunostained specimens enabled a determination of the basement membrane tube of newly formed blood vessels in the implanted sponge sheet. The tubes were 5-40 microm in diameter, and sometimes tapered to a slender cord within the vascular network. The SEM study of 30% KOH treated tissues revealed two types of tapering ends of newly formed blood vessels. One consisted of endothelial cells with microprojections, and lacked any investment of pericytes over the length of 5-20 microm. The other type was a tapering tip of the endothelial tube covered with pericytic processes. The presence of long processes of pericytes extending beyond the tip of the endothelial tube and connecting to the adjacent vessel wall indicates that this type was produced by endothelial tube regression. Thus, the present study supports the ideas that endothelial tube formation is followed by pericyte coverage at the sprouting tip, and that endothelial tube regression precedes pericyte detachment at the regressing site.

Absorbable Implants↗
PubMed2002

The structure of human metaphase chromosomes: its histological perspective and new horizons by atomic force microscopy.

Studies on the structure of the human chromosome were reviewed from the histological perspective and discussed in connection with our recent findings obtained mainly by atomic force microscopy (AFM). In this paper, we introduce several hitherto known models of the high-order structure of the metaphase chromosome and discuss the actual structure of chromosomes in relation to such structures as spiral chromatids, chromosome bands, and chromosome scaffolds. In chromosomes treated with Ohnuki's hypotonic solution, the chromosome arms were elongated and showed a characteristic spiral pattern of chromatid fibers. On the other hand, alternating transverse ridges and grooves were clearly observed on the surface of chromosomes treated with 0.025% trypsin for G-banding, and these ridges and grooves corresponded to the dark and pale bands of G-banded chromosomes. Similar findings were also found in chromosomes treated with quinacrine mastards for Q-banding. Fibers bridging the gap between the sister chromatids were often observed in G/Q-banded chromosomes; these fibers tended to be restricted within the G/Q-positive portions, suggesting the presence of chromatin fibers bridging these regions. Based on these findings in conjunction with previous studies, we outlined the high-order structure of the human chromosome. Recent advances in nanotechnology have provided new AFM techniques for the imaging and handling of materials at nano-scale resolution. Application of these techniques to chromosome research is expected to provide valuable information on the chromosome structure in relation to its function.

Chromatids↗