Biomedical subjects

V Mizuhira

Publications and source records attributed to V Mizuhira.

At least 19 recordsLinked to original sources

PubMed2000

Fixation and imaging of biological elements: heavy metals, diffusible substances, ions, peptides, and lipids.

We tested various fixation and analysis methods to demonstrate by electron microscopy elemental imaging in tissues and cells, i.e., soluble substances such as many kinds of ionic elements, water soluble low molecular peptides, and even organic solvent soluble substances such as lipids. For the ionic elements, we tested frozen dried or freeze-substituted methods and organic or inorganic special chemical precipitation methods combined with microwaved fixation methods. The data were analyzed with electron beam X-ray microanalysis, electron energy filtered imaging analysis, and electron microscope autoradiography. The data were demonstrated as elemental distribution images and were calculated quantitatively. For the soluble low molecular peptides, we developed a tannic acid and aldehyde method combined with microwaved fixation. We discuss the theoretical background of the tannic acid fixation and microwaved fixation methods. For the organic solvent soluble substances, i.e., lipids including steroids, we successfully tested the use of a mixed fixative of aldehyde and osmium, digitonization, and osmification with the use of p-phenylendiamine or imidazole. We also proposed some new ideal biotracers for electron beam X-ray microanalysis and electron energy filtered imaging analysis.

Animals↗

PubMed1997

Microwave fixation and localization of calcium in synaptic terminals using x-ray microanalysis and electron energy loss spectroscopy imaging.

The distribution of calcium ions is demonstrated in synaptic terminals by means of a two-step chemical precipitation of calcium ions in the rat brain. K-oxalate/K-antimonate chemical replacement with simultaneous computerized microwave irradiation was used. This precipitate in nerve cell structures was investigated by computerized electron probe x-ray microanalysis (EDX) and electron energy loss spectroscopic (EELS) imaging. The values obtained by EDX agreed with those of the standard sample and theoretical values of Ca-antimonate. Typical EELS spectra of Ca:L, O:K, and Sb:M were obtained from nerve terminals in the same tissue block as that used for EDX analysis. Excellent net Ca:L and Sb:M EELS digital images were obtained after their background images were subtracted. Calcium ions were distributed in the nerve terminals, synaptic vesicles, mitochondria, and synaptic membranes.

Animals↗

PubMed1996

High resolution localization of endothelin receptors in rat renal medulla.

The cellular localization of endothelin receptors in the inner medulla of the rat kidney was investigated by using high resolution light and electron microscopic autoradiography, with the microwave irradiation fixation methods. Kidney slices were incubated with 125I-endothelin-1 alone or with selective ligands for the endothelin ETB and/or ETA receptors for light microscopic autoradiography. At the microscopic level, 125I-endothelin-1 was found to bind specifically to the glomeruli, arterioles and peritubular spaces in the cortex and vasa recta and surrounding tissues in the inner medulla. These bindings were also observed when the tissue slices were incubated in the presence of IRL1620 (ETB receptor agonist) or 97-139 (ETA receptor antagonist). Electron microscopic autoradiography using 125I-endothelin-1 in the inner medulla revealed silver grains over endothelial cells of the vasa recta and interstitial and collecting duct cells. No grains were detected over inner lining cells of the thin limbs of Henle's loop. These interstitial cells contained abundant microorganelles and lipid droplets, and had extensive cytoplasmic processes that closely related to the basement membranes of the vasa recta and loop of Henle. These findings demonstrate that type 1 interstitial cells are also primary sites for endothelin receptors as well as endothelial cells of the vasa recta and collecting duct cells in the inner medulla.

Animals↗

PubMed1996

Microwave fixation method for cytochemistry. For conventional electron microscopy, enzymo-immunocytochemistry, autoradiography elemental distribution studies and staining methods.

More than a decade ago, we introduced a microprocessed household microwave oven for microwave-stimulated fixation. In this paper we present an overview of our experience. We have introduced the use of tannic acid (TA, 0.1%), which is very effective for fixing soluble peptides or proteins, except in the case of enzymo-cytochemistry. In the latter case, a combination of TA with aldehyde in the microwave-fixation step brings perfect fixation, but inactivation of the enzymes and their enzymatic activities.

Alkaline Phosphatase↗

PubMed1996

Angiotensin II-induced structural and functional alterations in spontaneously hypertensive rat kidney.

The purpose of this study was to compare functional and structural changes in the kidneys of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats during prolonged administration of angiotensin II. Rats were pretreated with captopril, and the effects of exogenous angiotensin II (200 ng.kg-1.min-1 for 7-14 days sc) on renal hemodynamics and renal vascular structure were examined. Angiotensin II induced significant reductions of renal blood flow and glomerular filtration and increases of renal vascular resistance in SHR but not WKY. Furthermore, angiotensin II induced an increase of the media/lumen ratio in the interlobular arteries (0.33 +/- 0.02 to 0.56 +/- 0.02) and arcuate arteries (0.27 +/- 0.02 to 0.53 +/- 0.07) of SHR without significantly altering the media/lumen ratio in the interlobular arteries (0.34 +/- 0.03 to 0.34 +/- 0.02) and arcuate arteries (0.30 +/- 0.02 to 0.36 +/- 0.05) of WKY (two-factor analysis of variance, strain x treatment; P = 0.0002 for interlobular arteries and P = 0.0319 for arcuate arteries). Results from this study indicate that the SHR kidney is more responsive than the WKY kidney to the functional and structural effects of prolonged angiotensin II infusion.

Angiotensin II↗

PubMed1995

Temperature-induced changes in the number of vesicles in the free nerve endings of temperature neurons of the snake.

By observing ultrastructural changes under the electron microscope, we illustrated exocytosis and recycling of vesicles in the infrared receptor, a kind of free nerve ending in the pit organ of the crotaline snake, Trimeresurus flavoviridis. While maintaining the snake pit organs at stable temperatures of 15 degrees C, 25 degrees C, and 30 degrees C, we fixed them by perfusion and then processed them for transmission electron microscopy. The largest number of clear and coated vesicles appeared in the terminals at the lowest temperature. The perimeter and area of a terminal were enlarged at 30 degrees C, and "opening waves" on the plasma were prominently found at the highest temperature. We also observed coated vesicles that budded from the plasma membrane in the terminals. The configuration of mitochondria in the terminals was quantitatively different between lower and higher temperatures. The data suggest that exocytosis and endocytosis in these terminals operate in a manner similar to that observed in other cell types.

Animals↗

PubMed1994

Microwave fixation and localization of calcium in synaptic vesicles.

The distribution of Ca2+ ions is demonstrated in the synaptic terminals by means of a 2-step chemical precipitation of Ca2+ ions in nervous tissue. K-oxalate/K-antimonate chemical replacement with simultaneous computerized microwave irradiation is used. This precipitate in cell structures was investigated by computerized electron probe X-ray micro-analysis. The calculated values (from the theoretical, standards and sections), elemental binding ratios and elemental molecular weight ratios were compared. Each calculated value coincided with the theoretical value. This method can reliably detect Ca2+ ions at the micromolar level. Ca2+ ions were distributed in the synaptic vesicles and surrounding membranes. Further progress is expected in freeze-substitution and in the application and propagation of the EELS-Imaging system in calcium determinations.

Animals↗

PubMed1992

Ultrastructural localization of calcium in mechanoreceptors of the oral mucosa.

Cytochemical localization of Ca2+ in Meissner corpuscles and Merkel cell-neurite complexes in the palatine mucosa of the Mongolian gerbil was studied by a combined oxalate antimonate-microwave irradiation procedure. The reaction products obtained were identified as calcium antimonate by EGTA solubility and X-ray microanalysis. Meissner corpuscles in the normal palatine rugae could be roughly classified into three types by amount and localization of Ca2+. Type I corpuscles were characterized by a high Ca2+ content in both the terminal axoplasm and caveolae of the lamellar plates, type II, by a low Ca2+ content in the terminal axoplasm and a high Ca2+ content in the lamellar cytoplasm. Type III corpuscles showed intermediate characteristics. Palatine rugae stimulated mechanically during fixation contained an increased number of type I corpuscles. On the other hand, two patterns were distinguished in the distribution of Ca2+ in Merkel cells in palatine rugae fixed under normal conditions. One showed abundant Ca2+ dispersed throughout the cell, while in the other, Ca2+ was specifically localized in the Golgi apparatus and mitochondria. Similar distribution patterns also were observed in palatine rugae that had received mechanical stimulus during fixation. Axon terminals of most Merkel cell-neurite complexes in normal palatine rugae were poor in axoplasmic Ca2+, whereas those in most Merkel cell-neurite complexes in mechanically stimulated palatine rugae contained abundant Ca2+ in their axoplasm.

Animals↗

PubMed1991

Chronic tubulointerstitial changes induced by germanium dioxide in comparison with carboxyethylgermanium sesquioxide.

Chronic nephrotoxicity was investigated in rats orally administered germanium dioxide (GeO2) and carboxyethylgermanium sesquioxide (Ge-132) for 24 weeks. Increased BUN and serum phosphate as well as decreased creatinine clearance, weight loss, anemia and liver dysfunction were apparent at week 24 only in the GeO2 treated group. Vacuolar degeneration and granular depositions were observed by light microscope in the degenerated renal distal tubules in the rats of this group, with the semiquantitative scores of tubular degeneration being 95 +/- 9% in the GeO2 group, 3 +/- 1% in the Ge-132 group and 1 +/- 1% in the control group, respectively. Electron microscopy revealed electron-dense inclusions in the swollen mitochondrial matrix of the distal tubular epithelium in the GeO2 group. Although systemic toxicities were reduced after GeO2 was discontinued at week 24, renal tubulointerstitial fibrosis became prominent even at week 40 (16 weeks after discontinuation). A Ge.K alpha X-ray spectrum was clearly demonstrated in the mitochondrial matrix of the distal tubular epithelium in the GeO2 group with the help of electron probe X-ray microanalysis. On the other hand, neither toxic effects nor renal histological abnormalities were manifested in either the Ge-132 or the control group. The renal tissue content of germanium was high at weeks 24 and 40 in the GeO2 group. From these results, it is concluded that GeO2 causes characteristic nephropathy while Ge-132 does not. In addition, it appears that residual GeO2 remains for a considerably long time even after the cessation of GeO2 intake.

Animals↗

PubMed1991

The permeability of capillaries in the eustachian tube mucosa.

The transendothelial passage of horseradish peroxidase (HRP) and diffusion of HRP into the pericapillary space, injected intravenously into cats, was studied at the light and electron microscopic level in the mucosa of the eustachian tube. The permeability of capillaries in the pharyngeal two-thirds of the tube was higher than in the tympanic one-third though all the subepithelial capillaries were of the continuous type. In the lamina propria of the subepithelium in the pharyngeal two-thirds much HRP was found 10 min after its intravenous injection, but in the tympanic one-third, it was absent in the perivascular spaces. The capillaries of the pharyngeal two-thirds had many projections and marginal folds inside the capillary lumen and large pinocytotic vacuoles containing HRP as well as many micropinocytotic vesicles. On the other hand, the capillaries in the tympanic one-third had many micropinocytotic vesicles too, but only a few large pinocytotic vacuoles.

Animals↗

PubMed1990

[Electron microscopic and immunological studies concerning the effect on the antitumor activity of sizofiran (SPG) combined with radiotherapy for cervical cancer].

It has been well known that Sizofiran (SPG) cultured from the Schizophyllum commune Fries activates the macrophages and induces the cytotoxic lymphocytes in some cancers. In this study, we observed electronmicroscopically the macrophages around the cancer tissue from the patients with uterine cervical cancer after the treatment with SPG. At the same time, their immune responses were also examined by analyzing lymphocyte subsets, ADCC and NK activity in peripheral blood. A considerable number of erratic macrophage with well developed Golgi apparatus, endoplasmic reticulum and mitochondria were found in the uterine cervical cancer tissue from the patients treated with SPG under radiotherapy. Simultaneously, we identified the lysosome granules with a bright filament structure which appeared to be specific for SPG. In the immune responses evaluated by analyses of peripheral blood, the number of CD 16+ cells and NK activity significantly increased in the patients treated with SPG as compared with non-treated group. The present results indicate that SPG-immunotherapy combined with radiotherapy not only induces the cytotoxic activity of macrophage but also augments NK activity in the patients with uterine cervical cancer.

Carcinoma, Squamous Cell↗

PubMed1987

Metabolism of circulating renin by liver and kidney of rats.

Rat renal renin, highly purified and labeled with 125I, was intravenously given to conscious rats, to study the fate of the circulating renin. Rat antirenin anti-serum was used to identify the labeled renin. The disappearance of [125I]-renin from the plasma showed two exponential components and the metabolic clearance rate was 11.4 +/- 1.0 ml/min/kg. Both 70% hepatectomy and bilateral nephrectomy decreased the clearance rate by about 50%. [125I]-renin accumulated mainly in the liver and kidney, and high performance liquid chromatography (HPLC) analysis indicated the degradation of [125I]-renin by these organs. Biliary excretion of [125I]-renin was negligible and urinary excretion accounted for 2% of the injected dose. Light- and electron-microscopic autoradiography indicated that [125I]-renin is taken up mainly by Kupffer cells and proximal convoluted tubular cells in the liver and kidney, respectively, and thereafter distributes to the lysosomes. In conclusion, both the liver and kidney are responsible for the clearance of circulating renin.

Animals↗

PubMed1987

Cellular and subcellular distribution of exogenously administered renal renin in rat liver and kidney.

Using highly purified 125I-labeled rat renal renin, we have demonstrated that both the liver and kidney are responsible for clearance of renal renin. In the present study, light and electron microscope autoradiography of intravenously administered 125I-labeled rat renal renin was performed in rat liver and kidney to observe the cellular and subcellular distribution of the renin. Fifteen and 60 min after the injection, the liver and kidneys were removed and fixed with 0.1 M sodium phosphate buffer, pH 6.8, containing 2.5% glutaraldehyde and 0.5% tannic acid, which allowed for the removal of breakdown products of the labeled renin, and then autoradiography was performed. In the liver, silver grains were mainly localized in Kupffer cells and not in hepatocytes. In the kidney, silver grains were evident in the proximal tubule cells. In both liver and kidney cells, silver grains were mainly located over the lysosomes. Gel permeation high-performance liquid chromatography analysis of the liver and kidney extracts indicated two main fractions, including immunoreactive 125I-renin and the breakdown products (free 125I and 125I-tyrosine). In conclusion, circulating renal renin is mainly taken up by Kupffer cells and proximal tubule cells and is subsequently transported to the lysosomes to be degraded. These results, taken together with our previous finding that nonglycosylated submaxillary renin does not distribute in the liver, suggest that the carbohydrate moieties of renal renin are necessary for the recognition by Kupffer cells.

Animals↗

PubMed1986

Negative-staining autoradiography: a new technique for ultracryotomy utilizing an interposed film.

A new radiocytochemical technique is reported for ultrastructural localization of diffusible substances, using negatively stained ultra-cryostat sections. A sheet of film interposed between the cryostat section and the emulsion layer has rendered negative-staining autoradiography (NSA) practical. The rationale of NSA is that the film completely shields the section from all moisture-producing autoradiographic processes, so that phosphotungstic acid (PTA) can stain the section either before or after autoradiography (ARG), without the possibility of ultrastructural damage by alkaline solutions, interference between PTA and photoprocessing compounds, and superimposed images of a gelatin layer stained with PTA. As a model to demonstrate the newly developed procedure of NSA, rat brains were labeled with [125I]-triiodothyronine, fixed with tannic fixative, immersed in a cryoprotectant, frozen in liquefied propane, and cryostat sectioned. The resulting higher yield of radioactivity (85%) on the section was confirmed by a radiation counter. The retention rate was approximately 20% greater than that of conventional sections. Developed silver grains were found on synaptic vesicles and mitochondria in the polymorphic layer of the dentate gyrus. In this report we will also discuss the problems associated with cryostat sectioning of fresh tissues, the concept of ARG resolution, the distribution pattern of developed silver grains, and the possible applications of NSA.

Animals↗

PubMed1985

Structural and functional properties of adult rat heart myocytes lysed with digitonin.

Low concentrations of digitonin disrupt the sarcolemma of adult rat heart myocytes selectively and completely. When the digitonin lysis is carried out in the presence of 10 mM Mg-ATP, the permeabilized cells retain the rod-cell morphology typical of heart cells in situ and show spontaneous phasic contractions. The rate of contraction is a function of the free Ca2+ concentration from a pCa of 7.2 to 5.2. Higher levels of free Ca2+ result in hypercontracture of the myocytes into round cells with characteristically distorted morphology. The sarcoplasmic reticulum of digitonin-lysed myocytes takes up Ca2+ in an ATP-dependent reaction that is inhibited and reversed by caffeine and strongly enhanced by procaine or ruthenium red. The Ca2+ accumulation has a Km of 0.6 microM Ca2+, depends on Pi (Km of 13 mM), and is strongly inhibited by bicarbonate ion. The hypercontracture of digitonin-lysed myocytes is a function of both the pCa and the Mg-ATP concentration of the suspending medium. Hypercontracture requires ATP. Hypercontracture due to Ca2+ overload occurs at lower Ca2+ concentrations when Mg-ATP is decreased from 10 to 1 mM. However, at low concentrations of Mg-ATP (in the range from 1 to 10 microM), hypercontracture also occurs and is essentially Ca2+-independent. Since hypercontracture of heart myocytes appears analogous to the formation of contraction bands in situ, these observations may be relevant to the phenomena of oxygen paradox and of Ca2+ paradox in intact myocardial tissue.

Adenosine Triphosphate↗

PubMed1985

Renotropic stimulation of DNA synthesis of proximal tubules and endothelial cells in the outer medulla.

We explored the effects of ovine pituitary-derived renotropin on renal DNA synthesis in castrated hypophysectomized mice. Administration of the preparation at a dose of 47 micrograms for 5 days was followed by significant increases in renal DNA (134% of controls), and in kidney dry weight, protein and RNA. A time course study showed that [3H]-thymidine incorporation into renal DNA peaked at 8-10 h after one injection (60 micrograms) 1.9 times higher than in controls. Autoradiographic studies indicated that labeling indices increased significantly in proximal tubules (17 times) and endothelial cells (4 times) in the outer renal medulla of treated mice compared to controls. Nuclear areas in these cells also increased significantly. Our studies demonstrated a time course of new DNA synthesis stimulated by a renotropin and identified renotropin target cells.

Animals↗

PubMed1984

Satellite cells in the tail muscles of the urodelan larvae during development.

The incidence and ultrastructure of satellite cells in the tail muscles of urodelan larvae were examined during development during which the number of satellite cells is gradually reduced. They are found more frequently in red than in the white fibres in all four stages examined (stage 53, 64, 66+ and juvenile). As development proceeds, intercellular space between satellite cell and muscle fibre is in general gradually extended and is mostly filled with basal lamina. Small muscle cells, satellite fibres, which are situated under the basal lamina of the parent fibre, are morphologically similar to satellite cells but contain a small amount of myofibrils. Three types of satellite fibres are distinguishable on the basis of differences in K2-EDTA-treated ATPase activity, width of Z line, and parent fibre type. Neuromuscular junctions are visible in satellite fibres.

Animals↗

PubMed1984

Calcium transport mechanism in crayfish gastrolith epithelium correlated with the molting cycle. II. Cytochemical demonstration of Ca2+-ATPase and Mg2+-ATPase.

Periodical changes in Ca2+-ATPase and Mg2+-ATPase activity were observed cytochemically in the crayfish gastrolith epithelium during the molting cycle in relation to the calcium transport mechanism. The ATPase activity was demonstrated by a new one-step lead citrate method. The reaction products were mainly restricted to the matrix of type II cell mitochondria. The Ca2+-ATPase activity was intensely observed in two calcium moving stages, the small gastrolith period which indicates the beginning of gastrolith formation, and the aftermolt , when the calcified gastrolith has been dissolved in the stomach and then reabsorbed from the stomach epithelium into the newly formed soft exoskeleton through the blood. Although the intensity of reaction products of Mg2+-ATPase varied in each stage, the enzymatic activity was observed throughout all molting stages. Reaction products were observed in all mitochondria, basement membranes, apical cytoplasmic membranes, and in some lysosomes. In conclusion, periodical changes in the two types of ATPase activity were seen in the mitochondria of gastrolith epithelium during the molting cycle, but Ca2+-ATPase activity seemed to be more prominently synchronized to the calcium movement in the gastrolith epithelium than Mg2+-ATPase activity. There results provide the strong evidence that Ca2+-ATPase may act strongly in the calcium transport system of crayfish molting.

Adenosine Triphosphatases↗