Fusayoshi Murata

Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy

SummaryCultured HeLa cells or mouse liver and pancreas tissues were labeled with 3H-thymidine, -uridine or -glycine for varying periods in vitro, frozen in liquid nitrogen and cut on an LKB ultrotome equipped with LKB Cryokit. Dry ultrathin sections were mounted on grid meshes and were either air-dried, freeze-substituted or freeze-dried, and were covered with dry films of radioautographic emulsions, exposed, developed, stained and were observed in electron microscopes.After a number of trials, it was possible to obtain fairly good preservation of both cell structure and radioisotopes by means of freeze-dried and drymounted ultrathin frozen sections. However, the results are not completely satisfactory at the present time.

Histochemical Studies of Mucosubstances in Metaplastic Epithelium of the Stomach, with Special Reference to the Development of Intestinal Metaplasia

SummaryProcesses in the development of intestinal metaplasia of the stomach were investigated from the morphological and histochemical approaches using light and electron microscopic techniques. The specimens taken from 38 gastric carcinomas and 15 gastric and/or duodenal ulcers were subjected to this study. Morphological appearances of the intestinal metaplasia observed in routine examination with hematoxylin and eosin staining was able to be divided into complete and incomplete metaplasia by the light and electron microscopic histochemical stainings of the mucosubstances. The columnar cells at the area of the incomplete metaplasia had both the properties of the intestinal epithelia and the gastric foveolar epithelia. The incomplete as well as the complete metaplasia arose from the generative cells at the isthmus of the gland. The generative cells, however, sometimes gradually transformed to produce the complete metaplastic cells. The two processes of the development of the intestinal metaplasia were proposed and discussed.

Nucleic acid synthesis in proliferating peroxisomes of rat liver as revealed by electron microscopical radioautography

SummaryThirty albino rats were fed with a diet containing 1, 2 or 4% di-(2-ethylhexyl)-phthalate (DEHP), a peroxisome-proliferating agent. Others were fed with normal diet as controls. Both groups were sacrificed at varying intervals from 3 days to 4 weeks. The livers were either removed and fixed in glutaraldehyde and osmium tetroxide or fixed in glutaraldehyde, incubated in a diaminobenzidine (DAB) medium, postfixed, embedded in Epon, and sectioned. Other tissues were incubated in Eaglés MEM containing either [3H]thymidine or [3H]uridine, fixed, embedded in Epon, sectioned, and radioautographed. Specimens were observed in a Hitachi H-700 electron microscope.The number of peroxisomes showing DAB reactivity increased in DEHP-fed animals as compared with normal controls In radioautograms of normal rats labelled with [3H]thymidine, no silver grains were, observed, whereas grains were observed over some nuclei, mitochondria and peroxisomes of DEHP-fed animals. In contrast, radioautograms of tissue labelled with [3H]uridine revealed a few grains in nuclei and mitochondria or endoplasmic reticulum of normal rats, although grains appeared in nuclei, mitochondria, endoplasmic reticulum and peroxisomes of DEHP-fed animals more frequently.From these results, it is concluded that [3H]thymidine and [3H]uridine were incorporated in the proliferating peroxisomes, suggesting that nucleic acid synthesis had taken place.

Demonstration of intracytoplasmic glycogen of megakaryocytes and blood platelets by means of the periodic acid-thiocarbohydrazide-silver proteinate method.

SummaryThe glycogen of megakaryocytes and blood platelets has been investigated in glutaraldehyde and osmium tetroxide fixed tissues by the periodic acid-thiocarbohydrazide-silver proteinate method (PA-TCH-SP). The PA-TCH-SP method involves the staining of intracytoplasmic glycogens more densely than the routine lead citrate method. Glycogen having a mean particle diameter of 21.1 nm has been shown localizing in the matrix of mature megakaryocytes, while that of glycogen in the platelets was 26.2 nm. The staining pattern of the glycogen in blood platelets was classified into three groups according to staining intensity.It is found that the PA-TCH-SP method is a very suitable one for the demonstration of intracytoplasmic glycogen from the viewpoints of reaction specificity, reproducibility, fineness of reaction products, sufficiency of electron density, and experimental cost. This method is also a very useful one for differentiating intracytoplasmic glycogens and ribosomes.

Electron microscopic demonstration of lipase in the pancreatic acinar cells of mice

SummaryA method for electron microscopic demonstration of lipase has heen developed by means of lead precipitation technique, modifying the original technique for light microscopy by Gomori. Care was taken to reduce the concentration of lead nitrate solution for substitution in order to prevent both the non-specific lead deposition and the diffusion.Applying the method to the pancreatic acinar cells of mice, specific activity was observed in rough surfaced endoplasmic reticulum, Golgi apparatus, zymogen granules and secretory canaliculi.

Fine structural localization of arylsulfatase B activity in the rabbit blood platelets

SummaryFine structural localization of arylsulfatase in the rabbit blood platelets has been investigated in this study. Among many cell organellae, reaction products were exclusively observed in the alpha granules of the platelets. Whithin the alpha granules, arylsulfatase activity appeared to localize in variable patterns, i.e. reaction products confined mainly at the peripheral region in many granules, while they deposited heavily throughout the granule matrices in some others. In a blood platelets, each alpha granule showed the different staining pattern which indicated more variable functional heterogeneity in the granules.

Supplemental studies on the method for electron microscopic demonstration of lipase in the pancreatic acinar cells of mice and rats.

SummaryThe method for electron microscopic demonstration of lipase which was previously reported by the present authors was supplementally studied on the prefixation and substrate in normal animals as well as in animals with experimental pancreatitis.As for the fundamental techniques, no significant difference was observed between the concentrations of the glutaraldehyde for prefixation, neither the kinds of Tweens used, while a significant difference was observed between the duration of incubation. A longer incubation (16–18 hours) brought about larger end products, a shorter incubation (1–3 hours) smaller products.In the animals with experimental pancreatitis, the lipase activity observed in endoplasmic reticulum and Golgi apparatus was not changed as compared with the normal controls, while the activity in secretory granules and glandular lumen decreased. Furthermore, the lipase activity appeared in focal cytoplasmic degradation and around lipid droplets which were not found in the normal controls.

A modified wire-loop method for quantitative electron microscopic radioautography.

SummaryA simplified method of emulsion coating and development for quantitative electron microscopic radioautography without any special instrument is introduced.Seven grids carrying ultrathin sections are attached to a glass block with double-coated Scotch tape. They are coated with a wire-loop, 2.5 cm in diameter. Ten glass blocks are then stuck on a slide, exposed, developed, fixed and stained simultaneously.Employing this method, the emulsion coating and the following procedures are easily accomplished under an identical condition.

Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow.

SummaryThe glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.

Fine structural and ultracytochemical studies on the lymphocytes in three types of genetic mucopolysaccharidoses.

SummaryLymphocytes from 6 patients with 3 types of genetic mucopolysaccharidoses (Hurler’s syndrome, Hunter’s syndrome and Morquio’s syndrome) contained numberous vacuoles in their cytoplasm.The size of the vacuoles ranged from approximately 300 nm to 750 nm. The percentage of the lymphocytes with vacuoles varied from 10% to 38%. The vacuoles showed acid phosphatase activity, which indicated their lysosommal nature. Staining with dialyzed iron solution usually localized acid mucosubstance in the peripheral region of these vacuoles after glutaraldehyde fixation. Ferritin and horseradish peroxidase were observed in the vacuoles after incubation of the patient’s lymphocytes with these tracers. This finding indicates the participation of endocytosis in the formation of these vacuoles.