The authors observed substance P-, somatostatin- and β-endorphin-like immunoreactivities ultrastructurally in the perikarya of the primary sensory ganglion cells by means of Nakane's enzyme-labeled antibody technique. As the results, the immunoreactivities of the neuropeptides were observed as irregularly shaped masses always encapsulated with membrane-bound contours of which diameters were 200nm to 2μm. Such neuropeptide-containing immunoreactive masses encapsulated by the membrane were usually seen in the vicinity of the Golgi apparatus. These immunoreactivities of the neuropeptides were more often observed in smaller nerve cells, though they were sometimes observed in the larger ones, too. The authors also observed substance P- and somatostatin-like immunoreactivities in the Rexed's lamina II in the posterior horn of the spinal cord as nerve terminals. Substance P- and somatostatin-containing nerve terminals were often stained diffusely by positive immunoreactivity and within them there were denser immunoreactive vesicles uniform in size. These electron dense vesicles with positive immunoreactivity were noted along the nerve axon to the terminal.
Motor innervation of cat diaphragm was studied by retrograde tracer methods using HRP and fluorescent substances of primuline (Pr) and propidium iodide (Pi). In HRP study 20% HRP was injected into the left hemidiaphragm of kittens. After 48hr animals were perfused with glutaraldehyde and paraformaldehyde. Spinal cord from C1 to Th13 were resected, cut into 40μm serial frozen sections, and provided for TMB reaction. Under the darkfield microscope a cluster of HRP positive neurons was observed in the ventromedial portion of the left anterior horn of the lower cervical (C5 to C6) and lower thoracic (Th9 to Th12) spinal cord, ipsilaterally to the injected side. For the survey of the interrelation of motor innervation between the diaphragm and intercostal muscles, fluorescent dual labeling method was applied. 10% Pr was injected into the left hemidiaphragm, while 3% Pi was injected into the left 8th to 13th intercostal muscles in kittens simultaniously. After 36hr animals were perfused with 10% formalin. Resected spinal cords were cut into 25μm serial frozen sections and observed under the fluorescence microscope. At 360nm excitation wavelength a small cluster of neurons was illuminated in the specific yellow fluorescence for Pr, while at 550nm another cluster of neurons was visualized in the specific orange fluorescence for Pi. These two clusters formed a cell group in the ventromedial portion of the anterior horn from 9th to 12th thoracic cords. Based on these experiments, motor innervation of cat diaphragm can be ascribed not only to the lower cervical but also to the lower thoracic motoneurons, probably via intercostal nerves. The present results might reappraise and consequently support the old hypothesis of “extra-phrenic motor innervation of diaphragm”.
A modification of Goldfischer's method for demonstration of activity for arylsulfatase has been employed. At pH 6.5 two forms and localizations of reaction product can be distinguished in the kidney: A supranuclear granular reaction and a diffuse cytoplasmic staining. In the liver only discrete granules are present. The nature of the reaction product depends considerably on fixation and pH. At pH 8.0 non-specific precipitation takes place. At pH lower than 5.5 no reaction product is formed.
Simultaneous demonstration of two different antigens in a single cell was performed on the electron microscopic level by combined staining with both HRP-labeled antibody and ferritin-labeled antibody. For HRP-labeled antibody method, 4-chloro-1-naphthol was employed as the substrate in place of diaminobenzidine to visualize the reactive sites, since the final product of diaminobenzidine was not dense enough to show the localization of antigens in high magnification in electron microscopy. Two groups of enzymes, α-amylase and chymotrypsinogen A or α-amylase and trypsinogen, were found to be present concurrently in a single zymogen granule. This pattern of the distribution of enzymes was recognized in all the zymogen granules found in a section. These results support the view of Scheele and Palade (1975) and that of Tartakoff et al. (1975) within the limits of the final step of the secretory process, who maintain the opinion of parallel processing of the proteins in the pancreas.
Prolactin, a peptide hormone in pituitary glands, shows a remarkable increase in amount in both plasma and amniotic fluid during pregnancy. In the plasma of the pregnant female, there is a rectilineal relationship between the increase of a measurable amount of prolactin and the progress of gestation. The highest level of amniotic prolactin is observed between the 15th and the 17th week of pregnancy. The production site as well as the function of prolactin during pregnancy have been disputed. In the present paper, the authors describe the production and localization of prolactin materializing tissues of human placenta and decidua in early pregnancy (from the 7th to the 9th week after last menstruation) obtained by either hysterectomy or curette. Immunohistological investigations revealed that syncytial trophoblast is a production site of prolactin. Prolactin was recognized in decidua tissue which does not produce but only deposits the prolactin. By a method of double staining, the production and localization of either beta-subunit of human chorionic gonadotropin (hCG-β) or human placental lactogen (hPL) were manifestatively differentiated from that of prolactin in syncytial trophoblast.
Two patients with hCG-producing tumors were investigated. One was a lung tumor in a postmenopausal patient and the other a primary choriocarcinoma of the uterus. Tissue of the liver in the former and the circulating blood of both patients were also investigated. The present paper describes the distribution of hCG among those tissues in order to make a histological characterization of trophoblast-like cells found in the lung at autopsy as to whether they compose primary or metastatic choriocarcinoma including trophoblastic differentiation of carcinoma cells. The authors performed an immunohistological detection of tissue hCG using beta-subunit of hCG (hCG-β) obtained from normal pregnant women. There was no dissimilarity of intracytoplasmic distribution between trophoblast-like cells and syncytial trophoblasts in uterine choriocarcinoma, which implies that hCG-β from normal pregnant women seems not to be helpful to cytological differentiation for hCG-producing tumors because of the suspected heterogeneity of hCG-β due to its origin. Further examination is required to submit an acceptable significance of the findings that hCG-β is demonstrated in not only cells enclosed in pulmonary alveoli or hepatic cells but also leucocytes in circulating blood.
The authors studied the distribution of met-enkephalin, α-endorphin, β-endorphin and LHRH within the central nervous system of rats by the indirect immunofluorescent technique, and observed α-endorphin-like immuoreactivity in the median eminence adjoining the portal vein as nerve terminals of dot-like immunofluorescence. This finding deserves special attention since α-endorphin has been considered physiologically inactive. Nerve terminals of α-endorphin-, β-endorphin- and met-enkephalin-like immunoreactivities were immunohistochemically confirmed in the medial hypothalamus. These results positively suggested that this area was playing comprehensive roles in physiological functions of the hypothalamo-pituitary system. To visualize neuropeptide-containing nerve cell bodies, we applied the immunofluorescent technique on various neuropeptides to intraventricularly colchicine injected rats. Some LHRH and met-enkephalin containing nerve cell bodies were observed without colchicine, though. All of substance P-, LHRH- and met-nekephalin-positive nerve cell bodies were visualized in the ventromedial hypothalamic nucleus, substance P- and β-endorphin positive nerve cell bodies in the arcuate nucleus, substance P-positive ones in the posterior hypothalamic nucleus and met-enkephalin-positive ones in the dorsomedial hypothalamic nucleus. Besides, substance P-containing and metenkephalin-containing nerve cell bodies were coexisting in the mesencephalic central gray matter. α-Endorphin-containing nerve cell bodies were also noticed in the caudate nucleus. In addition, calcitonin-like immunoreactive cell bodies were observed in the anterior lobe of the pituitary body.
The immunohistochemical localization of hen egg-white proteins, ovalbumin, lysozyme and ovomucoid, was investigated by means of the fluorescein and enzyme-labeled antibody technique, using the new GMA-Quetol 523-instead of the conventional paraffin embedding method. This method is simpler than paraffin embedding because the embedding matrix need not be removed. Also, it is easy to cut 1-2μm sections with glass knives on a JB-4 microtome. The distribution pattern of each protein could be observed far more precisely than with paraffin sections. The three proteins showed different secretory features, depending on the stage of the ovulation cycle. Ovalbumin was secreted into the oviduct lumen prior to the descent of the egg. While the ovum with a completely calcified shell remained in the uterus, the tubular gland cells were filled with immuno-positive small granules. This was thought to represent the synthesizing stage of ovalbumin in preparation for the next ovulation. Lysozyme was secreted before and after the descent of the egg. When the complete egg was located in the uterus, the immunopositive lysozyme could not be detected in any region of the oviduct. Ovomucoid was also secreted prior to the descent of the egg.
Intestinal metaplasia in human stomach was classified macroscopically into areas with both sucrase and trehalase activities on the mucosal surface (complete type) and areas with sucrase but no trehalase activities on the mucosal surface (incomplete type) by the TES-TAPE method. Five serial frozen sections were taken from tissues of these two types located with TES-TAPE. The sections were examined histochemically for intestinal marker enzymes and mucin, and the metaplastic tubules were also examined histologically. In mucosa from areas of the complete type, the striated border of almost all the metaplastic tubules gave positive reactions for leucine aminopeptidase (LAP) and alkaline phosphatase (ALP) and contained Paneth's cells. No high-iron diamine (HID) positive mucin was detected by staining with high-iron diamine-Alcian blue (HID-AB). In mucosa from areas of the incomplete type, metaplastic tubules giving a positive reaction for LAP, a HID-positive reaction and a negative reaction for ALP were frequently observed. However, other patterns, such as negative reactions for LAP, ALP and HID, and positive reactions for LAP and ALP, but a HID-negative reaction were also observed. Goblet cells were present but no Paneth's cells were found. Thus a variety of phenotypical expressions of intestinal marker enzymes and mucin were observed in metaplastic tubules, especially in areas of the incomplete type of intestinal metaplasia.
This article deals with the behavior of ATPase activity in rat osteoclasts under the effect of calcitonin, or following pretreatment with MgCl2 or ouabain. Pretreatment with MgCl2 seems to cause a slight rise of ATP ase activity in various organelles in osteoclasts, but it does not increase the activity of the lysosomal enzyme. It seems that the Mg++ ion rather has an effect as a inhibitory agent on the lysosomal ATPase. The plasma membrane of the ruffled border does not possess any significant ATPase activity, hence no reactivation of the enzyme seems to occur by pretreatment with MgCl2. It is further proved by the finding that ouabain does not inhibit the ATPase activity in the area of the ruffled border. Short term treatment of calcitonin prevents exocytosis temporarily, thereby causing a significant rise of the enzyme activity in various organelles except in lysosomes. The enzyme activity seems to be reduced by serial administration of calcitonin. An endogenous phosphatase activity, being reactive with ATP-omitted medium, is always observed.
The Regan isoenzyme, a heat-stable alkaline phosphatase, which has been thought to be derived only from cancer or the placenta, has been reported in non-cancer, non-pregnant patients. We performed histochemical studies to determine whether the heat-stable alkaline phosphatase exists in normal cells of the female reproductive organ. For this purpose, the heat-stability test and the L-phenylalanine inhibition test were used. Heat-stable and L-phenylalanine-sensitive alkaline phosphatase was found in normal cells of Muellerian duct origin, e.g., uterine cervical reserve cells, cervical columnar cells and epithelial cells of the endometrial surface. Our findings suggest that heat-stable alkaline phosphatase, such as the Regan enzyme, may be produced by normal cells of Muellerian duct origin.