Indirect immunofluorescence and immunoperoxidase techniques were successful in demonstration of progesterone in the cultured mouse adreno-cortical adenoma cells (Y-l). The immunoreactive progesterone was localized in the cytoplasm, but not in the nuclei, and showed increased staining in the cells stimulated by either adrenocorticotropic hormone (ACTH) or dibutyryl adenosine 3′, 5′-cyclic monophosphate (dbc AMP). “Round up”cells, which are considered to be the most active form of steroidogenesis, revealed intensive staining. The mode of localization in the cytoplasm of the immunoreactive steroid will be discussed.
Histochemical studies have been conducted on the interpeduncular nucleus (IPN) of the rat. The neurons of such show a high level of hexokinase activity and are able to receive their energy supply directly from glucose in the circulating blood. These neurons are equally supplied with the enzymes of the Embden-Meyerhof pathway and with those of the hexose monophosphate shunt, indicating a high level of activity in the tricarboxylic acid cycle. Therefore, the rat IPN may belong to the category of“usual nuclei” (15, 16). Neurons were classified into five categories based on variousmorpho-logical patterns in the Golgi apparatus (GA). Type I and II neurons were dominant in the Corpus interpedunculare (Ci), and types III, IV and V neurons were dominant in the magnocellular nucleus (Mi) of the IPN. In general, the activities of the glycolytic enzymes such as hexokinase, aldolase, glycero-aldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and succinate dehydrogenase were higher in the Ci than in the Mi. These results indicate that the rat IPN may be classified histochemically into two subnuclei. The high succinate dehydrogenase activity sporadically localized in the neuropil suggests that it may correspond to the structures formed by the horizontal axonplexus and by the richly ramified dendrites of the neurons in the IPN.
Non-specific esterases of the normal mouse epididymis have been studied by the a-naphthyl acetate (α-NA) and 5-bromoindoxyl acetate (5-BIA) methods and by these techniques in combination with inhibitors. These studies demonstrate that several esterase isozymes are present in the epididymis. The a-NA method produces a strong reaction to at least one isozyme more than is demonstrable by 5-BIA. The latter technique thus distinguishes between different cells which show identical reaction to α-NA. The use of inhibitors confirms these differences. The techniques used enable localization of the cells of origin of various isozymes in a complex organ. The results provide a basis for studies on the genetical control of cell function.
Adenyl cyclase activity was constantly present in the plasma membrane, and granular precipitates suggesting activity in the mitochondria were noted in some fibers after reaction stimulation with activators. In the muscle degenerated by plasmocid, the localization of adenyl cyclase activity did not change, but in general, the intensity of the enzyme activity decreased slightly in comparison to that of the normal muscle. NaF or epinephrine stimulation of injured muscle resulted in a less marked increase of reaction than that of the normal muscle. This was considered to be a possible indication of changes in the plasma and mitochondria membranes in the acute phase of plasmocid injury of skeletal muscles.
Chromium was histochemically stained with Chrome azurol S. The low reactivity of chromium was surpassed by the accelerated reaction of methanol added to the staining medium. Interferences were masked by acetylacetone. Chromium-Chrome azurol S chelate complex colored blue in the sections of the livers and the kidneys of chromium injected rats. The blue coloration of the stained sections was identified as chromium with an electron probe microanalyser.
Young male Wistarking rats (ca. 100-120g.) were kept on a vitamin E-deficient diet for 4, 6, 9 and 13 weeks. Changes in acid phosphatase (ACPase) and thiamine pyrophosphatase (TPPase) activities were investigated at the ultrastructural level in the neurons of the cerebrum and spinal cord of these animals. A parallel observation on the controls was also made under identical conditions and results compared. After 4 to 13 weeks of deficiency, ACPase activity in the Golgi apparatus increased, but the normal distribution of TPPase activity as seen in the controls and after 4 weeks of deficiency decreased at 6, 9 and 13 weeks. During 4 to 9 weeks periods of deficiency, there was no marked change in lysosomal morphology and their ACPase. At 13 weeks, however, lysosomes increased in size and number, with a significant decrease in ACPase activity. An increase in ACPase activity in the Golgi apparatus may be related to its increased secretory activity, while a decrease in TPPase may indicate its decreased carbohydrate metabolism. The decrease of ACPase activity in enlarged lysosomes of 13 weeks deficiency may be due to the following reasons; i) enzyme has been consumed in the process of changes in lipid components and/or ii) the release of enzyme in the cytoplasm because of damaged lysosomal membranes. The increase in lysosomal size may be due to polymerization of oxidized lipids.
Chronological changes of adenylate cyclase in blood cells and the uptake of L-dopa at each-clock time were investigated. Supplementally, the uptake of an exogenous foreign substance (neutral red) was also examined. From the experiment, it was confirmed that the change of adenylate cyclase activity of leucocytes at each clock-time was closely associated with the spontaneous daily changes of biogenic amines in leucocytes and with uptake ability for administered L-dopa. In regard to granulocytes, the reaction products were distributed mainly along the cytoplasmic membranes at 0200 hr. The reaction products increased markedly at 1400 hr and were seen not only on the cytoplasmic membranes, but in intracellular structures. However, at 2000 hr, only a small number of deposits were observed. Furthermore, in order to enhance the uptake ability of leucocytes for biogenic amines, a combination of L-dopa and prednisolone was administered. Here, the activity of adenylate cyclase in granulocytes increased significantly, although adenylate cyclase in the other blood cells showed no considerable differences. Subsequently, it seems to be possible that the activity of adenylate cyclase in leucocytes was facilitated by L-dopa, and furthermore, that granulocytes are possibly provided with a receptor for L-dopa.
The immunofluorescent method (Coons' indirect method) with the use of a specific antisera for melatonin and N-acetylserotonin has been used to show the localization of melatonin in the enterochromaffine cells of the gastrointestinal tract. Despite the fact that in a number of cases antisera will apparently give cross-reactions to melatonin+N-acetylserotonin, it is possible that the active synthesis of melatonin does take place in the enterochromaffine cells. Herein, it is suggested that melatonin-producing cells (epiphysis pinealocytes, corresponding cells in the cerebellum, enterochromaffine cells of the gastro-intestinal tract) be united into a single group of cells, respresenting the main part of the“APUD”-system which plays an important role in homeostasis of the body.
Non-specific esterase in the liver, kidney and small intestine of nine different mammalian species (monkey, dog, cat, mink, rabbit, hamster, guinea pig, rat and mouse) was studied by light and electron microscopy, with thiolacetic acid as a substrate. In the liver and kidney, strong activity was observed in all of the species except in the mink liver. Both smooth and rough endoplasmic reticulum, including the nuclear envelope, were intensely reactive. A marked difference of species was shown in the small intestine, for an intense reaction occurred in the apical region of epithelial cells in all species except the dog, cat and mink. These results were in close agreement with those obtained from a biochemical assay.
DivisiFerritin-conjugated lectins were utilized for L cells as cytochemical stains for sugar residues in the complex carbohydrates of the cell membranes. Concanavalin A, Ricinus communis toxin, and Bauhinia purpurea hemagglutinin were coupled to ferritin with glutaraldehyde and purified by affinity chromatography. On fixed L cell plasma membranes, all of these ferritinconjugated lectins were uniformly distributed. The number of binding sites of these lectins on L cells was determined as 1.4×106 for concanavalin A, 7.7×106 for Ricinus communis toxin, and 0.2×106 for Bauhinia purpurea hemagglutinin, from binding experiments with 131I-labeled lectins. On unfixed L cell plasma membranes, ferritin-conjugated concanavalin A became unevenly clustered at a low temperature (4°C). A small proportion of ferritinconjugated Ricinus communis toxin clustered, but ferritin-conjugated Bauhinia purpurea hemagglutinin did not cluster under the same conditions. Incubation at 37°C for 30 minutes, after the binding of ferritin-conjugated concanavalin A and Ricinus communis toxin, induced invagination of the plasma membranes, accompanied by internalization of the bound ferritin-conjugates.