Distribution of injected [U-14C] galactose in adult, pregnant, newborn and infant mice (ICR strain) and 14C incorporation into the acid-soluble and -insoluble substances of organs were examined by whole-body autoradiography. Autoradiography of whole-body cryosections was performed before and after 6% perchloric acid treatment (untreated and treated autoradiographs). In the adult mice, [U-14C] galactose in the peritoneal cavity was completely absorbed into tissues and organs within one hr. Radioactivity peak of blood was attained 5 min after injection. Radioactivities at this time were high in liver and kidney, and relatively high in large and small intestine, myocardium and retina. In the first hr, marked changes of organ radioactivities were seen; Marked decreases in kidney, liver, myocardium, blood, large and small intestine, while increases in salivary glands, Harderian gland and pancreas. However, radioactivity peak in brain was seen at 30min. Incorporation rate of radioactivity into acid-insoluble substances of organs increased at this time: Rates of increase were high for mucous salivary glands, Harderian gland, myocardium and kidney, but relatively low for liver and serous salivary glands and a nearly constant incorporation rate was observed for the brain. Liver and spleen had a heterogeneous distribution of radioactivity: High radioactivities in liver were around central veins at 5min, and such spots were diffusely distributed in the lobules at 15min, while high radioactivities in spleen were in the red pulps. In pregnant mice, fetus showed a different distribution pattern of radio-activity from the maternal body with high radioactivities being noted in the myocardium and cartilage. In the placenta, the incorporation rate of radio-activity into its acid-insoluble substances was higher in the fetal portion. During postnatal growth, distribution of radioactivities in kidney, small and large intestine exhibited higher rates, and activities of brain showed an acute increase from 3 weeks.
In order to confirm the relationship between mitochondrial GSH-PO and steroidogenesis in the rat adrenal cortex, we have studied the immunocyto-chemical localization of GSH-PO in the rat adrenocortical cells of Metopiron administration, which blocks the conversion of deoxycorticosterone to corticosterone. In the control rat adrenocortical cells, mitochondrial GSH-PO was mainly localized in lipid-depleted compact cells, of inner fasciculata cells, but not zona glomerulosa and zona reticularis cells. By Metopiron administration, the adrenal cortex was markedly hypertrophic. Immunohistochemically, GSH-PO was observed in outer fasciculata cells and intracellular localization of GSH-PO was mainly observed in cytosol (cytosol GSH-PO). However, mitochondrial GSH-PO was less than that in the control rat adrenocortical cells. Based on our findings, it is strongly suggested that mitochondrial GSH-PO may be a very important enzyme for steridogenesis, especially, corticosterone biosynthesis.
The effects of hypoxic hypoxia on local cerebral blood flow was studied by using 14C-iodoantipyrine autoradiography. Hypoxia (8%O2 and 6%O2 for 30min) was induced in mice without control of the temperature and blood pressure. Percent decreases in cerebral oxygen tension (P cortex O2) during 8%O2 and 6%O2 hypoxia were 80% and 92%, respectively. Under normal conditions (21%O2), P cortex O2 was 17.4±4.0mmHg. Optical density (OD), main factors responsible for the measurement of local cerebral blood flow were observed by the scanning micro-densitometer. The pyramidal cell layer of the hippocampus, choroid plexus, and venous (sinus) blood did not decrease in ODs during hypoxic hypoxia, while ODs in all other cerebral regions examined significantly decreased as compared with that of the control (P<0.01). In others, amygdala showed a marked decrease (ca. 30%) in ODs during hypoxic hypoxia.
Histochemical investigation of lectin-binding sites was carried out on basal cell epithelioma (BCE) and carcinoma (BCC) cells obtained from human skin lesions. The HRP-conjugated lectin technique was employed (Con A, for detecting glucose and mannose; RCA-1, for Gal; PNA and SBA, for Gal and GaiNAc; DBA, for GalNAc; UEA-1, for fucose; and WGA, for GlcNAc and NANA). Irrespective of histologic variation in BCE and BCC, the tumor cells exhibited rather weak binding to lectin conjugates, and the tumor cells adjacent to keratinizing epithelia in keratotic BCE displayed stronger lectin staining as compared to those in non-keratinized tumors. Some of the desquamated tumor cells found in cystic spaces of cystic BCE had the strongest reaction for the PNA conjugate. The cytochemical distribution of lectin conjugates in BCE and BCC was usually confined to intercellular materials and surface of the tumor cells, but the lectin staining was not strong. No major difference was found between tumor cells of BCE and BCC with respect to lectin-binding.
The ultrastructural localization of lysozyme was studied using the protein A-gold technique in the human nasal gland. There was much in the secretory granules of the serous cells, but not in the mucous cells. Some granules of the serous cells had a central core, and these had more lysozyme. Much lysozyme was also found in the secretory material within the glandular lumen in forms associated with a central core or in amorphous secretion, suggesting its protective role here against bacterial infection.
Female Wistar-Imamichi rats, were given 7, 12-dimethylbenz(a)anthracene (DMBA), and then three different high fat (HF) diets containing olive, safflower, and corn oil, respectively, for 120 days. Mammary tumors developed in O of 10 rats fed on a corn oil diet (C), 2 of 10 rats fed on an olive oil diet (O), and 2 of 10 rats fed on a safflower oil diet (S). In the tumor-bearing rats fed on O and S diets with DMBA treatment, the levels of the plasma progesterone were higher than those of control rats on the same diets without DMBA treatment. The levels of plasma progesterone of the tumor bearing rats fed on C diet were no difference compared with that of control. The plasma 17β-estradiol levels in tumor-bearing rats on different diets were similar. Changes of 3β-hydroxysteroid dehydrogenase (3β-HSD; EC 126.96.36.199) activity in the ovaries of mammary tumor bearing rats fed on HF diets were examined by the microspectrophotometric method. In mammary tumor-bearing rats of HF diets, the activity was higher than that of control rats in the corpus luteum and interstitial gland in the ovaries, but not in the thecal membrane. The activity tended to increase with diet in the order C, S, and O. In particularly, in the ovaries of the tumor-bearing rats fed on O, the secondary corpus luteum (CL 2) and the primary corpus luteum (CL 1) showed high activity. These showed that high content of oleic acid contained in olive oil may induce an increase of progesterone production in their ovaries. In control rats, the activity in CL 2 in the ovaries was lower than that in CL 1. These results suggest that the increases in plasma progesterone in tumor-bearing rats on O or S may have been caused by prolonged corporus luteum function and the active formation of interstitial glands in the ovaries. This resulted in a low incidence of mammary tumors because continuous stimulation of progesterone caused lobulo-alveolar proliferation, thereby protecting the mammary epithelium from carcinogens.
To study the non-immunogenic defense mechanism on organisms against foreign substances, reactions of cells to anionic colloidal particles were observed on macrophages from rat peritoneal cavity and liver tissues. As the non-antigenic anionic particles, iron colloids obtained by coating ferric hydroxide colloidal particles with potassium polyvinyl sulfate or chondroitin sulfate were used. Observations revealed that peritoneal macrophages and Kupffer cells develop surface receptors having specific affinity to anionic particles by which the cells adsorb them on the surface and internalize them into the cytoplasm for decomposition or detoxication. The other general somatic cells did not have any such receptors and were unable to take up the foreign particles having a negative surface charge.
The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid method were used for localization of EGF in the intercalated duct secretory granules of the submandibular glands of adult A/J “female” mice. Immunoreactive EGF was detected in the secretory granules of the intercalated duct cells and granular convoluted tubule cells. Electron microscopically, EGF was localized only in the secretory granules with the gold particles, indicative of EGF not being distributed homogeneously over the granules. A characteristic finding was an EGF-positive “peripheral part” and an EGF-negative “central part”. However, the density of labeling by gold particles over the peripheral part was lower than that within the secretory granules of the granular convoluted tubule cells. The findings suggest that the intercalated duct secretory granules are only a minor source of EGF in the submandibular glands of adult female mice.
The experimental teeth (upper right first molars of adult Wistar rats) were moved in the buccal direction for 4 days by a wire spring and subsequently moved in the lingual direction for 24 and 72hr. The ultrastructual changes and localization of acid phosphatase (ACPase) activity of the osteoblasts were examined in the lingual periodontium (pressure side) of the teeth moved in the experimental periods of 24 and 72hr. The following results were obtained: 1) Osteoblasts in the lingual areas of the periodontium of the 24hr experiment contained autophagic vacuoles in the cytoplasm, while rough-surfaced endoplasmic reticulum and mitochondria were reduced. 2) ACPase activity in the osteoblasts adjacent to the bone surface was noted in lysosomes, Golgi apparatus and autophagic vacuoles. The enzyme activity was also present in the lysosomes showing lysosomal wrapping mechanism and in the lysosomes fusing with the vacuoles. 3) The lingual areas of the periodontium in the 72hr experiment were characterized by necrosed tissues and none of the reaction products of the enzyme activity could be found in these areas.
Twelve cases of benign and malignant cartilaginous tumors were subjected to cytological and cytochemical studies using human fetal cartilage as control. They included 4 cases of chondrosarcoma, 2 cases of benign chondroblastoma, and 3 cases each of enchondroma and osteochondroma (exostosis). An electron microscope was used. The ultrastructural features and cytochemical localization of the reaction product of polysaccharide and alkaline phosphatase activity (ALPase) of the tumor cells of well-differentiated chondrosarcoma, enchondroma and osteochondroma were similar to those of the distal zones of the epiphyseal plate of the controls. These results suggested tumor cell functions may be similar to those of the controls as well as normal chondrocytes. Undifferentiated chondrosarcoma cells, however, had poorly developed organellae and a few vacuoles with deposits of periodic-acid methenamine-silver staining (PAM) in their cytoplasm. An abundance of ALPase reaction product was observed in their cytoplasmic membranes but scarcely any in their cytoplasm. These findings suggest decreased matrix synthesis (especially proteoglycans) in the cells of undifferentiated chondrosarcoma. Benign chondroblastoma cells demonstrated well-developed rough-endoplasmic reticula (r-ER) in contrast to the Golgi apparatuses, and PAM-reaction products were not seen in their cytoplasm. On the basis of the ultrastructural localization of ALPase activity, two different cell types were seen in benign chondroblastoma: fetal cartilagelike cells (type A), and peculiar cells which had abundant deposits of ALPase in the enlarged r-ER but not on the cytoplasmic membrane (type B). These findings suggested an interference in the matrix synthesis process of r-ER in type B benign chondroblastoma cells. It was concluded that the ultrastructural localization of both ALPase and polysaccharide reaction products could be useful in defining cell function and the level of cell differentiation in cartilaginous tumors.
Adenylate cyclase in rat cerebellum was investigated on the light microscopic level with adenylyl imidodiphosphate (AMP-PNP) as substrate. The results show that the reaction is overlapped by other enzymes with high activity and inhibited by NaF and dithiothreitole. It does not seem possible to demonstrate adenylate cyclase light microscopically in rat cerebellum because adenylate cyclase activators inhibit the conversion of AMP-PNP. The biochemical results of Johnson and Welden (7) could be confirmed histochemically.