The histoautoradiography and fine structure of the adrenal medulla in Spontaneously Hypertensive Rat (SHR, Okamoto and Aoki) was examined in the prenatal stage. The labeling index of the fetal adrenal medulla in SHR was 10±1.38 on the 20th day of gestation and became lower on the 22nd day of gestation. This tendency was consistent with the decrease of mitotic index. The autoradiographical grain count per labeled cell on the 20th day of gestation didn't differ from that on the 22nd day of gestation. These results indicate that the generation time of the adrenal medullary cell in SHR became more longer on successive day of gestation. However, there was no significant difference of the histology and histoautoradiography between SHR and control. Ultrastructurally, most of medullary tissue on the 20th day of gestation were composed of the intermediate cells containing many intermediate granules. These granules increased in number during the fetal life. In most of the adrenal medullary cells, the fine structure was similar to that observed in the control group. These findings suggest that the difference of cell proliferation and cell differentiation in the adrenal medullary cells between SHR and control appears after birth, because the significant difference in the medullary cells of both groups has previously reported in the adult rats.
The localization of NAD-linked polyol dehydrogenase (sorbitol dehydrogenase) was studied in mouse liver and kidney, using new histochemical technique in light and electron microscopy. The enzyme activity was mostly found in the parenchymal cells in the liver. The distribution pattern of the enzyme in each liver lobule differed from each other. In the kidney, the reaction product was concentrated in the brush border of the proximal convoluted tubules. In addition, the biochemical data showed that the enzyme activity was mainly found in the soluble fraction. Since the reaction product was not recognized in the control section, the result obtained by this method was found to represent the specific reaction site of NAD-linked polyol dehydrogenase (sorbitol dehydrogenase) in the tissue.
Polyglucose particles artificially synthesized under histochemical conditions from glucose 1-phosphate by phosphorylase and branching glycosyltransferase in the nuclei of skeletal muscle fibers, satellite cells and endothelial cells of the blood capillary of the normal rat were studied at the electronmicroscopic level. Newly formed polyglucose particles appeared in the karyolymph and interchromatin area of nuclei as a large macromolecular structure of spheroidal branching bodies. The size of intranuclear polyglucose particles ranging from 400 Å to 1600 Å in diameter was slightly larger than the cytoplasmic particles. There was no evidence that newly formed particles in the nuclei had a relation to the chromatin or the nuclear membrane.
Histochemical technique using autoradiographic procedure was presented which can be used to demonstrate non-labeled mercury in paraffin-embedded tissues of the central nervous system and other organs. Compared to other histochemical methods, this technique allows the use of formalin-embedded, stored, serial paraffin sections and appears more sensitive for the demonstration of intracellular mercury in Minamata disease (methylmercury poisoning). The technique forms reaction products of high quality which can easily be seen in cell bodies and does not disturb the structure of tissue cells during preparation.
Histochemical detection for aminopeptidase in tissues and organs was made to employ aromatic substrates with conventional azo coupling method. Several amino acids combined with β-naphthylamide and aromatic amines with tosyl, carbasole and benzoyl groups were employed to histochemical use in the rat and guinea pig. Amino acids β-naphthylamide splitting enzymes were similarly localized histochemically despite of the use with several kinds of amino acids, though except for a few cases which were dipeptide β-naphthylamide. Enzymatic activities were intensely localized at the proximal convoluted tubules, intermediate in distal tubules and tracable in glomerulus in the kidney, and they were restricted at villi of small intestine, as previously described (18) There were a few differences in distribution between the rat and guinea pig, i. e., in the rat kidney the outer zone of cortex was more reactive than the inner zone, however, in the guinea pig the inner zone was more marked to outer zone. Histochemical stainability of tissue sections were stronger in the guinea pig than those of the rat. The substrate specificity of aminopeptidase in the rat kidney was obtained to plot the two type Lineweaver-Burk curves. An optimal pH of the enzyme was detected ranging from 6.0 to 7.0 in the rat kidney. Zymogram from the rat kidney and small intestine using thin layer polyacrylamide gel electrophoresis showed two migrating bands with L-leucine β-naphthylamide. Zymogram for serum revealed a sharp band at anodal site and unclear two bands at the γ-globulin site.
The transport of dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH) in guinea pig sciatic nerves was studied by measuring the accumulation of these enzyme activities to a ligature. Axonal flow of DBH protein in rat sciatic nerves was also studied by immunofluorescence, and the anatomical results were compared with the biochemical ones. The DBH immunofluorescence was increased in the proximal 3-mm segment of a ligature up to 48 hr after ligation. The DBH activity was also highly accumulated in the proximal 3-mm segment until 48 hr after ligation. Retrograde axonal flow of DBH or TH was observed indouble ligation experiments at 5 hr after ligation, but at 24 hr the retrograde axonal flow apparently changed to the orthograde. The mean, or absolute axonal flow rate (mm/24 hr) of DBH in guinea pig sciatic nerves was 100, or 180, while that of TH was 20, or 32, respectively. The results suggest that DBH and TH may be localized in different intracellular compartments; DBH may be transported at a fast flow rate as a particulate enzyme, while TH at an intermediate flow rate mostly as a cytoplasmic enzyme.
Glycolytic aspect in dark and light adapted retinas of the chicks was studied by the use of histochemical techniques for phosphorylase activity. In the dark adapted retina, the paraboloid of the accessory cone contained synthesized polyglucose particles which were approximately 300 Å in diameter. These particles were less stainable with lead citrate and located in the cytoplasmic matrices. In the retina incubated in the medium for phosphorylase activity under exposure to light, synthesized polyglucose particles in the paraboloid varied from 95 to 380 Å in diameter and were less stainable with lead citrate. Synthesized polyglucose particles in the accessory cone were so many that the tubular structure was flattened and the cytoplasm was expanded. Therefore, phosphorylase activity in the accessory cone of the chick retina is higher in light adaptation than in dark adaptation.
Chemical and histochemical profiles of central nervous system tissues, skeletal and cardiac muscles and visceral organs of experimental calciummagnesium deficient rats and control animals were studied. Six to eight weeks after a feeding schedule of a calcium-magnesium deficient diet, the calcium concentration in the kidney increased twenty-five fold over that of the control and there was a moderate increase in the spinal cord and skeletal muscle. Fluctuation of the magnesium concentration was insignificant. A decrease in succinic dehydrogenase activity demonstrated in the cerebral cortex, cerebellum, spinal cord, kidney and skeletal muscle corresponds to tissue morphology, for example, atrophy and a decreased SDH type II muscle fiber was noted in gastrocnemius tissue specimens. Acetylcholinesterase activity in the motor end-plate of the gastrocnemius muscle showed a slight decrease in enzyme activity and a swelling of the motor end-plate. The severe histochemical changes of SDH and acid phosphatase activities in renal tubules and hepatic parenchymal cells suggest a progression of cellular catabolism related to lysosomal enzyme activity.
In vitro transcription was made on chromatin fibers isolated from rat liver and several types of tumor cells using E. coli RNA polymerase and 3H-uridine triphosphate-containing substrates. Electron microscopy of the chromatin fibers after the transcription reaction revealed a characteristic structure with RNA-like fibers attached to the chromatin fibers. Electron microscopic autoradiography of these specimens demonstrated silver grains along the RNA-like fibers. This finding provided direct evidence that these fibers were real RNA molecules newly transcribed in vitro. Analysis of the ultrastructure of these transcription complexes revealed that the transcriptionally active regions of the chromatin had extended fibers bound with proteins. Long stretches of free DNA were not found in the active regions.
Carbamoyl phosphate, an intermediate metabolite of ammonia, was found to be markedly hydrolyzed in the rat brain tissues. This activity was demonstrated with light and electron microscopy certainly being indicated as enzymatic nature, by capturing released phosphate through the activity with lead ions present in the incubating medium. The reaction products were localized mostly in the endoplasmic reticulum and nuclear envelope of the nerve and glial cells. Inner cisternae of the Golgi apparatus were also occasionally stained. The nature of the activity was (1) Briefly fixed tissue with glutaraldehyde showed the best results. Formaldehyde and acetone, on the other hand, markedly destroyed the activity. (2) Sodium fluoride present in the incubating medium completely inhibited the activity, but phloridzin did not. (3) The pH optimum was between 5.0 and 7.0. These results were compared with those of hepatocytes in relation to glucose 6-phosphatase, acylphosphatase and other phosphatases.
The spinal cord of the rat in the different developing stages was light and electron microscopically investigated by a modified ZIO impregnation technique. As the animal matured, both the myelin sheath and synaptic vesicles in nerve endings increased their affinity to ZIO impregnation. However, ZIO reactivity of the glial cells, on the contrary, was reduced with the maturation. This method appears to be useful and convenient for the demonstration of myelinated axons without further staining and for the detection of some kind of the glial cells in the early developing stage.
A technique for cytofluorometric measurement of contents of Hb and nuclear DNA on a single erythroid cell is described. Smears of mouse bone marrow were fixed with methanol and treated with 0.2 M mercaptoethylamine hydrochloride (MEM) dissolved in 1.5 M perchloric acid, irradiated by UV light under a fluorescence microscope. This is a modification of method of Granick et al. (1965) to convert hemoglobin into fluorescent porphyrin. The smears were then stained with Feulgen nuclear reactions. The non-specific fluorescence in the background was eliminated by pre-, or post-irradiation method with the wavelength 543 nm (Fujita and Fukuda, 1974). The amounts of Feulgen nuclear DNA and intracellular porphyrin converted from heme or Hb were determined by cytofluorometric measurement on a single erythroid cell. With the two quantitative parameters. erythroid cells in bone marrow were classified into seven classes of different maturation stages. “Proerythroblasts” which were identified on the bases of morphological criteria had in general aneuploid amounts of nuclear DNA with disproportional contents of Hb showing rather aberrations from normal steps of cell maturation. The DNA amounts of “orthochromatic erythroblasts” showed continuous decrease from diploid range to almost zero suggesting that the removal of nuclear DNA is not exclusively due to mechanical expulsion of a whole intact nucleus.
In the hope of gaining some insight into the staining mechanism of the Feulgen nucleal reaction in situ, the staining reaction of three kinds of hydrolyzed nucleoprotein-containing films with Schiff's reagent was studied from both the physicochemical and the chemical points of view. The establishment of an approximate equilibrium state in staining was assumed, and the mode of the adsorption isotherm in this state was examined. The Feulgen nucleal reaction in the heterogeneous phase conforms to Langmuir's adsorption isotherm and thus exhibits a saturation value in staining. Based on this fact, the stoichiometry of the reaction was determined. The saturation value of the stoichiometry was proved to be four.
Little is known about the spectral characteristics of leucocytes stained by routine methods and their values in blood cytodiagnosis. In this work a new method was developed for measuring color differences per cell between different cells, and the applicability of this method in characterization of leucocyte types was examined. In this new method the relative color spectrum was measured over the range of 370 nm to 700 nm as the ratio of the absorption at each wavelength to the maximum absorption (A/Amax, %). The relative color spectrum was obtained from the absorption curve by a wavelength-scanning method using a double-beam microspectrophotometer with a projection beam area larger than the cell size. The error due to staining was least when blood cells are stained by a modification of the method of Wright. Several types of human and animal leucocytes were distinguished on the basis of their relative color spectra, and these spectral differences seemed suitable for blood cytodiagnostic purposes, because a fixed spectral pattern was obtained for most leucocyte types, even including lobated-nuclear neutrophils. However, it has not yet been possible to distinguish eosinophils from pseudoeosinophils in fowl blood or monocytes from some lymphocytes.
The histochemical localization of nonspecific alkaline phosphatase activity (ALPase) on the limiting membrane of seminiferous tubules was investigated in the testes from the rats aged 1, 7, 14, 21, and 42 days. On thick (ca 40μ) sections incubated in Burstone's medium for ALPase, a cribriform distribution (about 36-45μ in width) of the strong activity began to be detected on 14 days of age and it was established by 21 days postnatal. Closing about the basement membrane of seminiferous tubules, an inner cellular layer of the flattened cells showing many of the characteristics of smooth muscles began to be demonstrated ultrastructurally on 14 days of age due to the differentiation of the outer cellular layer. At that time, moderate activity of ALPase began to be seen throughout a myoid cell layer by Ogawa et al. 's ultracytochemical method. Abundant reaction products for ALPase could be proved more than 21 days of age. Many pinocytotic vesicles within the cytoplasm of the myoid cells were filled with final products, and the strong activity was also observed in the space surrounding the myoid cells. Finally, the peritubular zone appeared virtually adult in ultrastructure and in both localization and activity of ALPase by 21 days postnatal. By electrophoresis on starch gel, the isozyme pattern of ALPase demonstrated by azo dye method was studied on the testicular tissues at the same stages of postnatal development as design of the histochemical investigation. Type II of the isozyme increased moderately more than 14 days after birth. A cribriform distribution of ALPase on the wall of seminiferous tubules might be related to the activity surrounding the myoid cell, which would be Type II of ALPase.
The colloidal iron method here introduced is an improved form of the Chèvremont-Fréderic method for the SH+SS groups. The principal point of improvement is the use of Müller's colloidal iron hydroxide instead of ferric sulphate as the ferric iron reacting with the ferrocyanide ion. This modification significantly reduced staining contamination, significantly increasing staining contrast; therefore, it made to be possible the electron micro scopical demonstration of the protein bound SH+SS groups. However, strict care must be taken against non-specific reduction caused by the tissue components and their adsorption reactivity. In the present experiment, this method was applied to the electron microscopic demonstration of the SH+SS groups in rat pancreatic acini, in which the internal portion and the limiting membrane of the secretory granules yielded clearly positive results.