Vinci Mizuhira

Separation and purification of (Cd, Cu, Zn)-metallothionein in carp hepato-pancreas

1. Cd-binding protein was isolated from the hepato-pancreas of carp administered CdCl2 (2 mg/kg). 2. This protein had a high absorption at 254 nm derived from Cd-mercaptide binding, and a low absorption at 280 nm derived from the absence of aromatic amino acids; the authors recognized the presence of two types. 3. The amino acid composition of the proteins was determined. The contents of cysteine residues were 34.24% and 31.90% in MT-I and -II respectively. Tyrosine, phenylalanine, tryptophan, histidine, leucine and arginine residues were absent in both types.

AN ANALYSIS OF THE HISTOCHEMICAL PROCEDURE FOR SODIUM ION DETECTION

Some aspects of the histochemical technique for the detection of sodium ions with potassium antimonate have been analyzed. When potassium antimonate is used for the detection of sodium ions the pH should not decrease below 7.2 during fixation. Potassium antimonate was also precipitated by ethanol used for dehydration, resulting in dilution of the water used as solvent. This should not be confused with the histochemical product. Precipitate may be formed with calcium ions and magnesium ions. Potassium phosphate buffer used to control the pH seems to inhibit the precipitation reaction of potassium antimonate with sodium ions; the nonbuffered fixative produces a good result at the electron microscopic level.

Characteristics of Capillary Permeability in Nasal Mucosa

The transendothelial passage of horseradish peroxidase (HRP) and colloidal carbon and diffusion of HRP in pericapillary space, injected intravenously into rats, was studied at the light microscopic and ultrastructural level in the nasal mucosa. In the lamina propria of the mucosa in the middle third of the nasal septum of rats, the capillaries directly beneath the epithelium were mostly with fenestrae, while the capillaries around the nasal gland were without the fenestrae. The permeability of the capillaries in the lamina propria of the nasal mucosa was very high and some of the endothelial cells were wide open, like liver sinusoid, allowing free passage of carbon particles. Marked transendothelial passage was also noted at ten seconds after HRP infusion through the capillaries without fenestrae located around the nasal gland. On the other hand, no extracapillary leakage of HRP was noted even at 150 seconds after its infusion in the capillaries without fenestrae located in the muscles. The permeability of small venules located in the lamina propria of nasal mucosa also was extremely high. The reasons for the greater permeation of HRP through capillaries of nasal mucosa are as follows. First, the endothelial cell junction was loose in the nasal mucosa and in some capillaries endothelium had holes like liver sinusoid. Second, interstitium surrounding the capillary was loose in nasal mucosa.

AN ELECTRON STAIN FOR ELASTIC FIBERS USING ORCEIN

Orcein was found to be useful as an electron-opaque stain for elastic fibers in epoxy-sections. Ultra-thin sections of aorta were treated with elastica stain containing 0.1-0.3% orcein and counterstained in uranyl acetate and lead citrate. Elastic fibers were densely and specifically demonstrated in the stroma and near smooth muscle cells. The result of orcein staining has a comparable appearance under both light and electron microscopes.

Dysmyelination in the sciatic nerves of dystrophic mice

Ultrastructural alterations were observed in the sciatic nerve of dystrophic mice. Myelin sheaths were abnormal in shape, abruptly ceased beyond a node of Ranvier, leaving the axon naked. These changes were seen in both afferent and efferent nerve fibres. Apparent embryonal Schwann cells and Schwann cells which were associated with increased lysosomes in the cytoplasm were observed in the proximal portion. There is a relative decrease in Schwann cells in the cross-section of the radicular parts, and a relative increase in the distal parts. The mean number of neurotubules per unit area was smaller while the mean number of the neurofilaments was larger in U-axons, in the dystrophic mice than in the controls. In M-fibres, neurotubules and neurofilaments showed no significant difference between systrophic and control mice.

Calcium transport mechanism in crayfish gastrolith epithelium correlated with the molting cycle

SummaryPeriodical 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. These results provide the strong evidence that Ca2+-ATPase may act strongly in the calcium transport system of crayfish molting.

Ultrastructural localization of calcium around the membrane of the surface connected system in the human platelet

SummaryThe localization of calcium in the membrane system of human platelets was determined by ultrahistochemical methods equipped with an electron probe x-ray microanalyzer. After potassium oxalate-glutaraldehyde treatment large amounts of electron opaque precipitates were observed around the membrane of the surface connected system. Electron probe x-ray microanalysis clearly defined that the precipitates were composed of calcium oxalate. The localization of calcium on the membrane of the surface connected system was also confirmed even after treatment of the platelets with potassium antimonate-OsO4. These results support a model which depicts the surface connected membrane system taking part in the store and the transport of calcium.

The fine structure of the spermatozoa of two species of Rhacophorus (arboreus, schlegelii): I. Phase-contrast microscope, scanning electron microscope, and cytochemical observations of the head piece☆

Green tree frogs, Rhacophorus arboreus and Rhacophorus schlegelii, living on the main island of Japan have spermatozoa in the form of a counterclockwise corkscrew composed of about 20 coils arranged in the shape of a cone. A head piece coiled loosely and tightly 5-6 times each. It was composed of two subcoils, one outside and the other inside. The outside subcoils composed the nuclear head, and the inside coils the acrosome. Following the head piece, a middle piece which had a mitochondrial sheath coiled 1 1/2 times. A tail piece which followed the middle piece coiled 3-10 times irregularly in 5-6 microns width with a 20- to 30-microns straight tail in length. There was a crystalline composed of 500-600 microtubules which surrounded a pair of cilial structures in the tail piece. The tannic acid-aldehyde-osmium tetroxide fixation method produced excellent electron density and good electron conductivity for scanning electron microscope observations.

The surface connected canalicular system of carp (Cyprinus carpio) thrombocytes: Its fine structure and three-dimensional architecture

SummaryThe presence and the three dimensional distribution of the surface connected canalicular system (SCCS) in thrombocytes of a teleost, Cyprinus carpio, were studied using a transmission electron microscope, a high voltage electron microscope and a scanning electron microscope. When the specimens were fixed routinely in glutaraldehyde followed by osmium tetroxide, numerous electron lucent vesicles and canaliculi were distributed throughout the cytoplasm. As ruthenium red-positive reaction product was observed on the inner surface of the vesicles and canaliculi, these are defined as the SCCS of carp thrombocytes. In the stereo-pair of the photographs of thick sectioned specimens and the plastic reconstruction of serially sectioned thrombocytes, we succeeded in finding the whole structure of the SCCS which is composed of numerous anastomosing canaliculi. Scanning electron micrographs revealed many crater-like depressions throughout the cell surface which seem to be the openings of the SCCS.

Calcium transport mechanism in molting crayfish revealed by microanalysis.

Crayfish provide a good model in which to study the transport mechanism of Ca ions. During the molting stage, decalcified Ca ions are transferred into the blood and accumulate in the gastrolith epithelium, after which a gastrolith is formed on the surface of the epithelium. The gastrolith is dissolved in the stomach after molting, and the Ca is reabsorbed and redistributed throughout the newly formed exoskeleton. We studied the mechanism of Ca transport by cytochemical precipitation of Ca ions and by electron microanalysis, including X-ray microanalysis (EDX) and electron energy-loss spectroscopy (EELS), with a computer. In EDX analysis, the fine precipitates of K-antimonate in the gastrolith mitochondria clearly defined Ca with antimony; we also observed a large amount of Ca-oxalate in the mitochondria, and Ca-K X-ray pulses were clearly defined. Ca-K X-rays were also detected from fresh freeze-substituted mitochondria. Finally, we succeeded in taking a Ca-L EELS image from the mitochondria of fresh freeze-substituted thin sections. Only a very small amount of Ca was detected from the cell membrane and other organelles. Ca-adenosine triphosphatase (ATPase) and Mg-ATPase activity was also very clearly demonstrated in the mitochondria. These enzymes may play an important role in Ca metabolism.