Biomedical subjects

T Ushiki

Publications and source records attributed to T Ushiki.

At least 37 records · Page 2Linked to original sources

PubMed2000

Time-lapse viscoelastic imaging of living fibroblasts using force modulation mode in AFM.

Using the force modulation mode in atomic force microscopy, we have succeeded in capturing time-lapse viscoelastic images of living mouse fibroblasts (NIH3T3) for several hours in a physiological condition without damaging the fibroblasts. Elongation of the lamellipodia and swelling of blebs were observed in time-lapse topographic images, which were taken every 10 min. The corresponding viscoelastic responses at a frequency of 600 Hz were visualized as consecutive images. The stiffer part of the cell body was fairly stable and did not show morphological changes for over 1 h. This is probably due to excess condensation of the actin network, hardening the cell cortex, and lowering the cytoskeletal activity. The nuclear portion of the cell body seems to be slightly less viscous than the peripheral region.

3T3 Cells↗

PubMed2000

Structure of rice starch granules in nanometre scale as revealed by atomic force microscopy.

Atomic force microscopy (AFM) was performed for the analysis of the fine structure of rice starch granules in the nanometre scale which were prepared by a physical destruction method. The present study directly demonstrated that fine particles of approximately 30 nm in diameter were present inside each granule and occasionally formed straight chain arrangements. We considered that these fine particles correspond to the individual single cluster in the cluster model which has been proposed in previous studies on the starch granule structure.

Microscopy, Atomic Force↗

PubMed2000

Scanning electron microscopic studies of the vascular smooth muscle cells and pericytes in the rat heart.

The cytoarchitecture of smooth muscle cells and pericytes in the rat cardiac vessels was studied by scanning electron microscopy after the removal of connective tissue matrices using a modified KOH-collagenase digestion method. The initial stem of the coronary arteries had groups of smooth muscle cells which ran in various directions on the outermost layer of the media. Although smooth muscle cells in coronary arteries of more than 100 microm in the outer diameter were arranged in a rough circle around the vessel axis, oblique and/or longitudinal muscle bundles were often present in the medio-adventitial border of the vessels. The presence of irregularly oriented muscular bundles is probably connected with resistance against the stretching force induced by the beating of the heart. As the vessel size decreased toward the periphery, almost all of the smooth muscle cells became spindle-shaped with several tiny processes and ran circularly or helicaly to the vessel axis. In the precapillary arterioles (6-12 microm), smooth muscle cells acquired various cytoplasmic processes which helicaly surrounded endothelial cells. Unmyelinated nerves were often associated with arterioles. Blood capillaries were morphologically divided into three segments: arterial capillaries which had pericytes with wide and circularly oriented processes, true capillaries whose pericytes extended long and thin primary processes bilaterally along the vessel axis, and venous capillaries surrounded irregularly and loosely by wide pericytic processes. The stellate pericytes in the postcapillary venules (10-30 microm) gradually changed into flat tape-like smooth muscle cells, which ran circularly in the collecting venules and veins (30-200 microm). The large collecting veins were finally overwhelmed by superficial thin layer of the myocardium, their own smooth muscle cells being very sparse. This suggests that large veins have poor ability to contract by themselves but are influenced by the surrounding myocardial cells.

Animals↗

PubMed2000

The subfibrillar arrangement of corneal and scleral collagen fibrils as revealed by scanning electron and atomic force microscopy.

The present study was designed to analyze the subfibrillar structure of corneal and scleral collagen fibrils by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Isolated collagen fibrils of the bovine cornea and sclera were fixed with 1% OsO4, stained with phosphotungstic acid and uranyl acetate, dehydrated in ethanol, critical point-dried, metal-coated, and observed in an in-lens type field emission SEM. Some isolated collagen fibrils were fixed with 1% OsO4, dehydrated, critical point-dried and observed without metal-coating in an AFM. Isolated collagen fibrils treated with acetic acid were also examined by SEM and AFM. SEM and AFM images revealed that corneal and scleral collagen fibrils had periodic transverse grooves and ridges on their surface; the periodicity (i.e., D-periodicity) was about 63 nm in the cornea and about 67 nm in the sclera. Both corneal and scleral collagen fibrils contained subfibrils running helicoidally in a rightward direction to the longitudinal axis of the fibril; the inclination angle was about 15 degrees in the corneal fibrils and 5 degrees in the scleral fibrils. These findings indicate that the different D-periodicity between corneal and scleral fibrils depends on the different inclinations of the subfibrils in each fibril. The present study thus showed that corneal collagen fibrils differ from scleral collagen fibrils not only in diameter but also in substructure.

Animals↗

PubMed2000

The presence of specialized epithelial cells on the bronchus-associated lymphoid tissue (BALT) in the mouse.

The aggregation of lymphoid cells in the bronchial mucosa has been named the bronchus-associated lymphoid tissue (BALT) and investigated in comparison with the gut-associated lymphoid tissue (GALT). To elucidate precisely the structure and function of the BALT, the present study examined the age-related change in the mouse BALT by light microscopy. We also observed the characteristics of the overlying epithelium, especially the lectin-binding properties of the epithelial cells, by the combined use of light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). By LM, lymphoid aggregates were not recognizable in the bronchial mucosa of young (8-10 week-old) mice, while they were commonly found at the second to fourth branching portions of the bronchial tree in older (32-40 week-old) mice. The epithelium overlying the lymphoid aggregates of the mature mice often contained a large number of mononuclear cells. Lectin cytochemistry revealed that UEA1 (Ulex europaeus agglutinin 1) positive cells were not only restricted to the overlying epithelium of the BALT in the older mice but also found in a cell group in the mucous epithelium at the branching portions in the young mice. Comparison between the LM and SEM images of the UEA1-stained whole mount specimens clarified the surface morphology of the lectin-stained epithelial cells, showing them to be non-ciliated cells with a large number of short microvillous projections on the apical surface. TEM studies further demonstrated that the UEA1 reaction products appeared on the plasma membrane of the non-ciliated cells which often enfolded lymphocytes in the old mice. Latex microbeads, which were administrated intratracheally, were selectively taken up by the UEA1-positive cells of the BALT. These results indicate that the mouse BALT has specialized epithelial cells similar to the UEA1 positive M cells in the GALT and probably functions as a part of the mucosal immune system. This study also showed the possibility that the UEA1 positive cells appear in the mucous epithelium before the formation of the BALT.

Age Factors↗

PubMed1999

Immunohistochemical demonstration of bovine viral diarrhoea virus antigen in the pancreatic islet cells of cattle with insulin-dependent diabetes mellitus.

The pancreatic islets were studied in seven cattle with insulin-dependent diabetes mellitus (IDDM) associated with persistent bovine viral diarrhoea virus (BVDV) infection. BVDV antigen was detected immunohistochemically in the pancreatic islet cells. There was a decrease in the size and number of islets, vacuolar degeneration of residual islet cells, and lymphocytic insulitis. The atrophied islets were composed of small uniform cells with limited amounts of cytoplasm, containing a small number of insulin- and chromogranin-positive granules. Enlarged islets consisting of islet cells with vacuolated cytoplasm were also frequently observed. Many of the vacuolated islet cells differed from the cells of normal islets in containing fewer cytoplasmic insulin- and chromogranin-positive granules. Mild lymphocytic insulitis was observed frequently in enlarged islets but rarely in atrophied islets. Immunoreactivity with BVDV antibody was found in the acinar cells of the pars exocrina in all seven cattle and in the residual cells of the islets of Langerhans of four cattle. BVDV antigen-positive cells were seen more frequently in the enlarged islets than in the atrophied islets. Some islets with lymphocytic infiltrates showed a small number of antigen-positive cells. These findings suggest that autoimmune IDDM was induced by persistent BVDV infection, resulting in gradual destruction of the islet beta cells.

Animals↗

PubMed1999

Scanning electron microscopic observation of apical sites of open-type paraneurons in the stomach, intestine and urethra.

The apical region of open-type paraneurons in tubular organs functions as a receptor site for chemical information in the lumen. Electron microscopic studies have demonstrated a tuft of microvilli on the luminal surface of cells, but failed to visualize it three-dimensionally. The present scanning electron microscope (SEM) observation succeeded in viewing, from the luminal side, open-type paraneurons distributed in epithelia of the stomach, intestine, and urethra. The pyloric antrum of avian species and the duodenum of human fetuses, the latter forming an endocrine cell colony at every villus tip, were chosen for SEM observation in order to eliminate visual obstruction by adjacent epithelial cells with developed microvilli. The luminal surface of gut endocrine cells was consistently covered with a tuft of 80-200 microvilli. Pyloric paraneurons possessed thick and stiff microvilli as compared with those of exocrine cells. The microvilli on intestinal paraneurons were more irregular in length and more loosely grouped than those composing the striated border of enterocytes. Urethral paraneurons containing serotonin were surrounded by three or four polygonal epithelial cells. Their narrow apical surface was provided with 30-100 microvilli which varied in length from cell to cell, and which were conspicuously projected above the luminal surface of the urethra. The microvillous crown of the gut and urethral paraneurons was so prominent and constant a structure on the apical surface as to allow easy identification of open-type paraneurons under the SEM.

Animals↗

PubMed1999

Scanning electron microscopic studies on the route of neutrophil extravasation in the mouse after exposure to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP).

The present study was performed to demonstrate three-dimensionally the process of neutrophil extravasation induced by the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in mice. Thirty to 40 min after the injection of fMLP to the mouse lip, the tissues were fixed with glutaraldehyde and examined by scanning electron microscopy (SEM) as well as by transmission electron microscopy (TEM). Observation of fMLP-injected tissues showed many neutrophils adhering to the inner wall of postcapillary venules. Some of these adherent neutrophils attached to each other to form groups of two to six. There were also many neutrophils migrating through the endothelium. Most of these neutrophils took a transcellular route, in that they penetrated the cytoplasm of the endothelial cells. The junction of two neighboring endothelial cells did not open, and endothelial pores free from migrating neutrophils were scarcely observed. There were bulging portions on the vascular wall which were probably produced by the presence of underlayed neutrophils. Thus, the present study gave direct evidence of neutrophil migration via a transcellular route in response to fMLP. Our findings also indicate that the pores of the endothelium close quickly after neutrophil extravasation.

Animals↗

PubMed1999

Growth of collagen fibrils produced by human osteosarcoma cells: high-resolution scanning electron microscopy.

To demonstrate three-dimensionally the process of the collagen fibril growth, the bottom of culture dishes with human osteosarcoma cells (NOS-1) and their extracts were examined by conventional scanning electron microscopy (SEM). Backscattered electron (BSE) imaging of SEM was also applied to the specimens, which were stained with phosphotungustic acid and uranyl acetate. Conventional SEM images showed several stages of collagen fibril assembly. Short collagen fibrils with tapered ends were distributed at the bottom of the dish just beneath and/or around the cultured cells; they were 1 microm long and 20-30 nm in diameter at the thickest middle portion. These fibrils were often twisted and united in a right helical direction, and consequently increased in length (5-10 microm) and diameter (more than 100 nm). In BSE images, the periodical bands stained with phosphotungstic acid and uranyl acetate were visualized throughout the individual fibrils. The banding pattern indicated that the polarity of the collagen molecules was unidirectional; namely, that all molecules were pointed in the same direction throughout the length of the fibrils.

Biopolymers↗

PubMed1999

Imaging of living cultured cells of an epithelial nature by atomic force microscopy.

The present paper describes the applicability of atomic force microscopy (AFM) to the observation of living cultured cells of an epithelial nature (human esophageal squamous cell carcinoma cells, or C7 subclone of KESC2 cells) in a culture medium. For this purpose, we made a fluid chamber system which allows a constant-speed perfusion of fluid at a regulated temperature in the chamber. Using this system, AFM images of living cells were successfully obtained for over one hour at time intervals of 2-4 min during continuous perfusion of the fresh culture medium. A series of these AFM images proved useful for examining the movements of cellular processes in relation to subcellular cytoskeletal elements. Time-lapse movie records produced by sequential AFM images further verify the reality of the cellular dynamics.

Carcinoma, Squamous Cell↗

PubMed1999

Microhardness evaluations of resin-dentin bonding areas by nano-indentation.

The purpose of this experiment was to determine the hardness values of the hybrid layer and its surroundings through the continuous use of a microhardness measuring device. Black's Class V cavities were prepared in nine dog teeth. The cavities were divided into four groups according to the dentin adhesive system applied. The adhesive systems were: "Bond One System", "Liner Bond II sigma System", "One Step System", and "Single Bond System". The treated teeth were observed at seven days post-application. Specimens were cross-sectioned perpendicularly or horizontally to the resin-dentin interface and embedded in epoxy resin. Their surfaces were polished. The microhardness of the resin-dentin bonding area was measured with a nano-indentation tester. The hardness values at a point of 10 microns distant from the interface in the direction of the dentin differed between systems. It appeared that this was influenced by the presence of the decalcified dentin not impregnated by resin, differences in the chemistry forming the hybrid layer, and the composition of the bonding resin. The hardness of the dentin-bonding interface and its surroundings was determined, and these areas were observed using SEM. Three layers were confirmed the healthy dentin layer, the composite resin layer, and the hybrid layer, (in which decalcified dentin impregnated by resin and that not impregnated by resin are considered to be mix). In the hybrid layer, no impression was found by SEM although the hardness in the bonding interface was significantly different. These layers appear to be more elastic and softer than the healthy dentin.

Animals↗

PubMed1999

[Observation of human corneal and scleral collagen fibrils by atomic force microscopy].

PURPOSE: We attempted to analyze the three-dimensional ultrastructure of human corneal and scleral collagen fibrils with an atomic force microscope (AFM). METHODS: A normal eye removed from a 66-year-old male was used in the study. Suspended corneal and scleral collagen fibrils were individually attached to glass slides by centrifugation. These collagen fibrils were air-dried and observed with a non-contact mode AFM in air. RESULTS: AFM imaging provided information on the surface topography of both corneal and scleral collagen fibrils. The corneal collagen fibrils had a height of 11.9 +/- 1.0 (mean +/- standard deviation) nm and the scleral fibrils of 82.5 +/- 35.6 nm. A periodic banding pattern of grooves and ridges was clearly found in both types of fibrils; the D-periodicity and the groove depth were 65.7 +/- 0.8 nm and 1.46 +/- 0.50 nm in the corneal fibrils, and 67.3 +/- 1.1 nm and 6.16 +/- 1.23 nm in the scleral fibrils. CONCLUSIONS: Surface topographic images of human corneal and scleral collagen fibrils were clearly obtained with the AFM. This technique provides quantitative information on the surface morphology of the collagen fibrils at high resolution.

Aged↗

PubMed1998

A novel type of myosin encoded by the mouse deafness gene shaker-2.

The mouse recessive deafness mutation, shaker-2(sh-2), represents a plausible model for an autosomal recessive form of human non-syndromic genetic deafness, DFNB3. Here we report the use of a positional cloning approach to show that the gene mutated in sh-2 mice encodes a novel type of unconventional myosin. A G-to-A transition changing cysteine to tyrosine in the conserved actin binding domain is detected in sh-2 but absent in laboratory strains and wild mice belonging to different mouse subspecies and species. This suggests that the novel myosin gene is a strong candidate for DFNB3.

Actins↗

PubMed1998

Differentiation of forbidden T cell clones and granulocytes in the parenchymal space of the liver in mice treated with estrogen.

Estrogen was administered to B6 (NK1.1+ strain), BALB/c (Mls-1b2a, V beta 3+ cells being forbidden clone), or (B6 x BALB/c) F1 mice (1 mg/mouse). On days 3 and 10, the number of cells yielded by the liver doubled, whereas that yielded by the thymus decreased prominently. The numbers of cells in the spleen, bone marrow, and blood were unchanged. c-kit+ stem cells, which give rise to multilineage cells, were present in the liver and bone marrow. The proportion of such c-kit+ cells in the liver increased while that in the bone marrow decreased on day 3. Therefore, the absolute number of c-kit+ stem cells increased severalfold in the liver and clusters of lymphoid cells became visible in the parenchymal space. At that time, the expression of recombination activating gene-1 and -2 mRNAs became prominent. Reflecting these phenomena, the number and proportion of IL-2R beta+ CD3int cells (i.e., primordial T cells) increased in the liver on days 3 and 10. An increase in the number of proportion of such CD3int cells was seen even in the thymus and uterus. In parallel with the increase of CD3int cells, the proportion of granulocytes also increased in various organs on day 3. Forbidden clones were present in either the NK1.1+ or the NK1.1- subset of CD3int cells in (B6 x BALB/c) F1 mice treated with estrogen and liver mononuclear cells in such mice acquired potent cytotoxicity against syngeneic thymocytes. These results reveal that estrogen has the ability to potentiate the generation of self-reactive T cells and granulocytes in the liver and other organs.

Animals↗

PubMed1998

Three-dimensional cytoarchitecture of rat pulmonary venous walls: a light and scanning electron microscopic study.

The three-dimensional architecture of the rat pulmonary veins was studied by light microscopy (LM) and scanning electron microscopy (SEM). For LM, the left lungs were fixed with formalin, sectioned and immunostained with an anti-alpha-smooth muscle actin (alpha-SMA) antibody in addition to conventional staining. For SEM, the specimens were fixed with glutaraldehyde and immersed in 30% KOH solution for 8 min followed by treatment of collagenase solution for more than 5 h. By LM, the smooth muscle cells stained with anti-alpha-SMA showed discontinuous, periodical thickenings of circular bundles in the wall of the venules, but they became thin and continuous in the larger vessels (or veins) that had a cardiac muscle layer on the outside. Under SEM, the smooth muscle cells formed circular-oriented bundles at constant intervals along the venules less than 100 microm in diameter. These bundles had circumferential constrictions in the lumen. The cardiac muscle cells, which appeared in large pulmonary veins of more than 100 microm, ran in a circular or oblique direction and completely surrounded the vessel wall outside of the thin continuous layer of smooth muscle cells. The muscle arrangements were considered to play a significant role in the return blood flow in rat pulmonary veins.

Actins↗

PubMed1998

Fabrication of a new substrate for atomic force microscopic observation of DNA molecules from an ultrasmooth sapphire plate.

A new stable substrate applicable to the observation of DNA molecules by atomic force microscopy (AFM) was fabricated from a ultrasmooth sapphire (alpha-Al2O3 single crystal) plate. The atomically ultrasmooth sapphire as obtained by high-temperature annealing has hydrophobic surfaces and could not be used for the AFM observation of DNA. However, sapphire treated with Na3PO4 aqueous solution exhibited a hydrophilic character while maintaining a smooth surface structure. The surface of the wet-treated sapphire was found by x-ray photoelectron spectroscopy and AFM to be approximately 0.3 nm. The hydrophilic surface character of the ultrasmooth sapphire plate made it easy for DNA molecules to adhere to the plate. Circular molecules of the plasmid DNA could be imaged by AFM on the hydrophilic ultrasmooth sapphire plate.

Aluminum Oxide↗

PubMed1998

Low-voltage backscattered electron imaging of non-coated biological samples in a low-vacuum environment using a variable-pressure scanning electron microscope with a YAG-detector.

A variable-pressure scanning electron microscope (VP-SEM) equipped with a high-sensitive backscattered electron (BSE) detector of the YAG type was applied to studies of biological tissue samples. The rat kidney and trachea were fixed, dehydrated in ethanol, critical point-dried and examined in the VP-SEM under a specimen chamber pressure of 1 to 150 Pa. The high-resolution surface images of the non-coated specimens were obtained in a low-vacuum (1-20 Pa) environment at an accelerating voltage of 5 kV, while the images at 10-20 kV contained information beneath the surface of the specimens. The observation in the VP-SEM equipped with YAG detector in a low-vacuum (1-20 Pa) environment at low (3-5 kV) accelerating voltages is useful for the three-dimensional analysis of the surface morphology of biological non-coated samples.

Animals↗

PubMed1998

Identification of arterial and venous segments of blood vessels using alkaline phosphatase staining of ink/gelatin injected tissues.

The present study describes a method for discriminating between the arterial and venous segments of blood vessels in mouse tissues and organs using alkaline phosphatase (ALPase) staining of ink/gelatin injected tissues. Anesthetized mice were injected through the left ventricle with blue ink/gelatin, and various organs and tissues were removed from the body and fixed by immersion in 10% formalin. Sections 50-100 microm thick were incubated for ALPase in a medium containing naphthol AS-BI phosphate and fast red TR by the azocoupling method. In such specimens as the brain and skeletal muscles, ALPase activity was found in arterioles and capillaries on the arterial side, whereas it was absent in capillaries on the venous side and in venules. In the liver, only branches of the hepatic artery were positive. ALPase activity was absent in the vessels of the lung except for a positive reaction in branches of the bronchial arteries. These findings indicate that the ALPase activity is confined to the arterioles and arterial segments of the capillaries in the systemic circuit. Thus, ALPase staining of ink/gelatin injected specimens is a useful method for differentiating the arterial and venous segments of the micro-vascular bed in various organs and tissues in mice.

Alkaline Phosphatase↗