The acid phosphatase activities in the transitional epithelium of the dog urinary bladder were examined ultracytochemically using a modified technique of Gomori with β-glycerophosphate as the substrate. The reaction products were localized on the plasma membrane in addition to the lysosomes and Golgi complexes. This enzyme activity was sensitive for tartrate, sodium fluoride, and heat treatment and insensitive for alloxan, p-chloromercuribenzoic acid and magnesium chloride. No prominent difference between lysosomal and extra-lysosomal acid phosphatase activities was observed. Since the possible participation of alkaline phosphatase was ruled out by use of the appropriate inhibitors, the enzyme reactions on the plasma membrane may be interpreted as reflecting the lysosomal acid phosphatase.
Prolactin synthesis was investigated in trophoblastic cells in the metastatic tumors of choriocarcinoma of the liver by means of the fluorescent antibody technique and electron microscopy. Of interest is the morphological function of the trophoblast in choriocarcinoma which is presumably free from decidual interaction whereas the morphology of the tumor is very similar to that of the cell column in the normal placenta. In the tumor tissue, trophoblasts were observed, though randomly arranged, in gradations with their cell differentiation from cytotrophoblast to syncytium. The intermediate cell of the trophoblasts was demonstrated to be capable of synthesizing prolactin by fine granular distribution in cytoplasm while syncytium in the metastatic tumors was characterized by fluorescence and consisted of fine granules mixed with network formation.
When unfixed cardiac muscle from the rat or hamster was incubated in substrate-free media containing cerous chloride buffered to pH 7.5 with Tris-maleate-sucrose, electron-dense reaction bodies were formed in the sarcoplasm and, to a greater extent, inside and budding from mitochondria. Fixation in glutaraldehyde prevented the reaction product appearing within mitochondria, but had no effect on either the bodies associated with outer mitochondrial membranes or those apparently free in the sarcoplasm or outside the cells. No reaction product was observed in unfixed liver, kidney or skeletal muscle. The electron-dense reaction product within cardiocytes was absent in fixed tissues treated beforehand with acetone and in fresh tissues preincubated with the D-aminoacid oxidase inhibitor kojic acid. It was substantially reduced by preincubation in Tiron (a superoxide scavenger), diethyldithiocarbamic acid (an inhibitor of copper-containing enzymes such as diamine oxidase and superoxide dismutase) or EDTA, and totally if any of these compounds was included in the incubation medium. However, p-chloromercuribenzenesulphonate, a non-penetrating and non-specific inhibitor of NAD (P) H oxidoreductases, and atebrine limited the amount of reaction product formed in all sites by much less. Chlorpromazine inhibited the formation of reaction product in the sarcoplasm but gave rise to a coagulated product within mitochondria. With Clorgyline, in contrast, no product was deposited in mitochondria and large amorphous masses of reaction product appeared in the sarcoplasm. None of the following, when included in the incubation medium, had any significant effect: 3-amino-1, 2, 4-triazole, catalase, dicoumarol, glutathione peroxidase, mannitol, methanol, Pargyline, o-phenanthroline, potassium cyanide, rotenone, sodium azide, sodium benzoate, sodium pyruvate, superoxide dismutase and thiourea. It is concluded that the electron-dense product arises from a reaction of cerium ions with either a lipid peroxide or endogenous hydrogen peroxide generated by a metal-containing thiol enzyme, possibly an oxidised form of monoamine oxidase A with the properties of a diamine oxidase. The significance of the cerium reaction in the ageing of cells is discussed briefly.
In order to correlate light and electron microscopic observations of an enzyme-histochemical study, JB-4 sections were examined. The desired areas, which were confirmed by light microscopy, could be easily recognized by electron microscopy. In the JB-4 embedded tissues, the enzymatic activities of alkaline phosphatase, acid phosphatase and non-specific esterase were well preserved.
Catalase activity in the erythrocytes of beagle dogs was biochemically assayed and compared with human erythrocytes. The mean value of catalase activity in dog erythrocytes was about one-thirtieth of that of human erythrocytes. Wide variations in catalase values were found in the dog erythrocytes. Among 17 dogs tested, one female dog was found to be acatalasemic and two male and one female dogs hypocatalasemic. These four catalase-deficient animals came from a single brood together with one normal female dog. Although the catalase activity of the acatalasemic dog erythrocytes was difficult to detect, the liver and kidney showed half the catalase activity of normal dogs.
Postnatal changes in aminergic terminals were studied electron microscopically in the periventricular region of the rat hypothalamus by means of a potassium permanganate fixation technique. By this method, a NA terminal could be recognized by the presence of small cored vesicles within the terminals. In neonatal rats, NA terminals were already present, although no special contact with other neuronal elements was found. In three day old rats, the NA terminals seemed to gradually make special contact with dendritic processes. Such contacts were similar to the synaptic specialization of Gray's type I, and their membrane specializations existed in all part of apposing membranes of synapses. Hence, they were termed an “immature synapse”. Subsequently, such immature synapses appeared to increase markedly in number with the development until two weeks after the birth. In 21 day old rats, the NA terminals possessing typical synapses of Gray's type I decreased in population of total NA terminals. A large number of synapses in NA terminals displayed partial membrane specializations in this stage, but differed from those of “mature type” in electon-opacities of both intracellular materials in synaptic clefts and cytoplasmic materials associated with postsynaptic membrane, so that they were termed a “transient type”. In 35 day old rats, NA terminals possessed synapses of a “mature type” which were characterized by both few electron-dense intercellular materials in synaptic clefts and poor development of postsynaptic thickening. As several features were found in synapses revealing intermingled characters of all three types, it is suggested that the synapse of the immature type gradually converted to the mature type synapse. These results were confirmed by another approach employing the conventional aldehyde-osmium fixation method in rats pretreated with 5-OH dopamine.
The cellular differentiation and histogenesis of the histochemical architecture of the medial pterygoid muscle of rats were studied by histochemical staining for SDH. From day 18 of gestation to day 18 after birth, the development of two kinds of muscle fibers could be distinguished: fibers with fine formazan crystals diffusely distributed in the sarcoplasm (SS) and fibers with large formazan particles diffusely distributed in the sarcoplasm (SL). After day 18 some muscle fibers showed marked progress of cellular differentiation and became similar to adult muscle fiber types. After day 23 most muscle fibers could be classified into three types; Type A, Type B, and Type C according to Stein and Padykula (47). Until day 2 after birth, regions with high and low percentage distribution of SL fibers (Psl) were distinguishable. By day 3 after birth the organization of the primordial muscle structure observed in cross section had become similar to that of adult muscle and significant differences of Psl values were found in different regions in cross sections. Thereafter the Psl values of each muscle region increased, especially from day 6 to 18. On day 23 after birth the distribution patterns of percentage of Type B plus Type C fibers (Pbc) and the percentage of Type C fibers (Pc) in cross sections became similar to that in young adult muscle. Moreover, the range of Psl, Pbc and Pc values of primary fasciculi in each region showed significant changes during postnatal development.