In both electromotoneurons (EMNs) and the electric organ of two teleost fishes Eigenmannia virescens and Gnathonemus petersii Calbindin-D28K (CaBP 28K) was localized immunocytochemically. Its distribution in the nerve endings on the electrocytes was compared with that of endogenous calcium. Finally, the concentration of CaBP 28K was estimated by radioimmunoassay in both the electric organ and cerebellum of G. petersii. The concentration of CaBP D28K was low in the somata of EMNs, whereas a high level was found in the axons and nerve endings on the electrocytes of E. virescens and on the stalks of G. petersii. In the cytoplasm nerve endings of both species CaBP 28K was located on membranes of presynaptic sites, synaptic vesicles and mitochondria. Only specific zones of the postsynaptic membrane of E. virescens electrocytes were immunolabelled. The radioimmunoassay showed that CaBP 28K was more concentrated in the electric organ than in the cerebellum of G. petersii. Endogenous calcium was found inside of synaptic vesicles as well as in the endoplasmic reticulum cisternae of the nervous fibers and of the electrocytes of both fishes. Thus CaBP 28K is concentrated in EMNs axon terminals where its distribution differs from that of endogenous calcium.
The localization of two stress proteins, small heat shock protein (HSP27) and αB crystallin, in the developing rat retina were studied immunohistochemically and immunochemically, using the antibodies for HSP27 and αB crystallin. At embryonic day 15, the immunoreactivity to αB crystallin was observed in some cells in the neural retina and in the pigment epithelium (PE). At postnatal day (P) 5, immunoreactivity to HSP27 was first observed in the inner limiting membrane (ILM), nerve fiber layer (NFL) and PE. Immunoreactivity to αB crystallin was present in almost the same portions of the retina. At P10, the differentiating outer segments of the photoreceptor cells (ROS) and PE were intensely immunoreactive to αB crystallin, as compared with other layers, while the ILM, NFL and some portions of PE were immunoreactive to HSP27. At time progressed, immunoreactivity to HSP27 in the IPL and αB crystallin in the ROS increased. Our enzyme immunoassay of the retina revealed that the concentration of HSP27 gradually increased over time. In contrast, the concentration of αB crystallin increased after birth and reached almost to adult level at P30. The concentration of HSP27 was much lower as compared to that of αB crystallin. The differences in locations and changes in the levels of concentration between HSP27 and αB crystallin suggested that these two stress proteins may have different functions in the developing retina.
To visualize proteoglycans histochemically, tissue blocks from rat tracheal cartilage were fixed with a paraformaldehyde solution containing cetylpyridinium chloride, embedded in LR-White, stained with a postembedding method of ruthenium red-tungstate (RR-PTA) sequence and examined in a transmission electron microscope. In the intercellular matrices of the cartilage tissues, a number of RR-PTA reactive, electron dense deposits were scattered in irregularly shaped clumps of different sizes. In the matrices adjacent to chondrocytes, RR-PTA reactive structures were regularly arranged in filamentous shapes. If digested with testicular hyaluronidase, both the RRPTA reactive structures in the extracellular matrices were either markedly decreased or abolished. Thus, these structures could be regarded as molecules of chondroitin sulfate (A and/or C) proteoglycans. In view of the dimensions of the RR-PTA reactive structures adjacent to chondrocytes, they could be taken to represent large molecules of proteoglycan aggregates in the cartilage tissues.
α-actinin is an actin-binding protein, closely associated with actin-containing stress fibers and, in particular, with their membrane-bound terminals. Using the immunohistochemical pre-embedding PAP method, we have demonstrated the presence of actin-binding protein in the cerebellar cortex of adult rats at both light and electron microscopic levels. α-actinin immunoreactivity has been demonstrated within Bergmann glial cells, Golgi and stellate cells, astrocytes and cerebellar capillaries. These results suggest the essential involvement of this actin-binding protein in the cerebellar cortex function.
The distribution pattern of tartrate-resistant acid phosphatase (TRAP) in matrices of developing medullary bone was histochemically examined and compared with the calcifi cation pattern of the matrix employing estrogen-induced medullary bone of male Japanesequail. Medullary bone trabeculae extended inward toward the marrow cavity and the calcification of bone matrix had begun by 4 and 7 days treatment of estrogen. The bone matrix located near the cortical bone was calcified and thin osteoid was observed. On medullary bone trabeculae more distant from cortical bone, the calcification was seen in deeper areas of the matrix with wide osteoid, and frequently the extending tips consisted solely of osteoid. Medullary bone trabeculae were more extended and the calcified areas were also increased in quails treated with estrogen for 7 days. TRAP activity was mostly positive in the matrix near the cortical bone and extending trabeculae, but surface areas and peripheries of the trabeculae were negative for the activity. The TRAP-positive area of medullary bone matrix had increased by 7 days treatment compared to that of 4 days treatment. The similarity between the distribution patterns of TRAP-positive areas in the matrix and the distribution patterns of calcified matrix suggests that the TRAP accumulated in the matrix during medullary bone formation became an active form with calcification of matrix.
Enterochromaffin-like (ECL) cells, which are numerous in the oxyntic mucosa of the rat stomach, proliferate in the presence of hypergastrinemia. We investigated the distribution of cells expressing gastrin receptor (GR) in Sprague-Dawley rats in which hypergastrinemia was induced by partial gastric corpectomy. The ECL cell density was determined by immunohistochemical staining for histamine. The expression of GR mRNA was investigated using digoxigenin-labeled complementary RNA probes. The labeling index of the generative cell zone was determined by 3H-thymidine autoradiography. The ECL cell density and the labeling index in the generative cell zone increased in association with increased plasma levels of gastrin after 24 weeks (p<0.05). The expression of GR mRNA was seen not only in ECL, generative, parietal and chief cells, but also in stromal cells of hypergastrinemic rats. The expression of GR mRNA became more marked in the oxyntic mucosa as plasma levels of gastrin increased, and it was suggested that GR mRNA expression acts as one indication in the development of ECL cell hyperplasia.
The expression of transforming growth factor-beta-1 (TGF-beta-1) was investigated in five normal human pituitary glands and 48 human pituitary adenomas using immunoreactivity to polyclonal antibodies against human TGF-beta-1. The relationship between hormone production and the expression of TGF-beta-1 was also investigated. TGF-beta-1 was detected in both normal cells of pituitary gland and in tumor cells of pituitary adenoma. Fifteen of 48 pituitary adenomas showed positive immunoreactivity for TGF-beta-1. Immunohistochemical staining of serial sections revealed coexpression of TGF-beta-1 and pituitary hormones in pituitary adenomas (PRL, ACTH, GH) and in normal pituitary glands (PRL, ACTH, LH). Intense and widespread positive immunoreactivity was observed in lactotroph cell adenomas. These results suggest TGF-beta-1 may be important in the differentiation and proliferation of the cells of normal adenohypophysis and adenoma.
We have investigated the localization of HCV in the liver and the expression of Fas antigen, involved in hepatocellular death through apoptosis, to examine the relation between HCV infection and Fas antigen cellular expression. The localization of HCV was investigated by in situ hybridization (ISH), and the expression of Fas antigen was determined by immunohistochemistry using an anti-Fas monoclonal antibody, in 20 surgically-resected liver specimens from anti-HCV positive patients. The Methylgreen/Pyronin Y technique clearly showed well-preserved tissue RNA in 8 of 20 livers. ISH determined the co-localization of signals for plus and minus strands of HCV-RNA in the cytoplasm of hepatocytes. Fas antigen was more strongly expressed on the plasma membrane of the periportal hepatocytes than on the plasma membrane of the centrilobular hepatocytes, even when HCV-RNA was found to be widely distributed in the hepatocytes throughout the liver lobule. Furthermore, Fas antigen positive hepatocytes were frequently observed close to the area of piecemeal necrosis associated with a high incidence of apoptosis. The results suggested that the expression of Fas antigen does not occur by HCV infection alone, but probably requires several other factors.
Septal cells in the alveolar interstitium of the lung have been assumed to be contractile and involved in the regulation of ventilation/perfusion ratio. We studied two cytoskeletal components of the septal cell in the rat lung. One contains thick microfilament bundles: by decoration with heavy meromyosin, the bundle was shown to be composed of actin filaments of opposite polarity. The other contains intermediate filaments: by pre-and post-embedding immunogold electron microscopy, they were shown to contain both desmin and vimentin. Double labeling immunoelectron microscopy further revealed that some intermediate filaments are co-polymers of desmin and vimentin. The features revealed in the present study support the hypothesis that septal cells can contract and change the architecture of the air-blood barrier.
To determine whether N-acetylgalactosamine (GaINAc)-or galactose (Gal)-containing components are present on the plasma membrane of hepatocytes, and, if so, to examine the distribution of the components on the plasma membrane, the binding of Dorichos biflorus agglutinin or soybean agglutinin to the plasma membrane of mouse hepatocytes was demonstrated histochemically. In addition, to determine the nature of the components containing GaINAc or Gal, the lectin binding sites were extracted from the plasma membrane and subjected to electrophoresis followed by lectin binding assay. Plasma membrane fractions from mouse hepatocytes included GaINAc-or Gal-containing components, and the components were glycolipid. The terminal sugar in the sugar chain of the glycolipid molecules was GaINAc. The glycolipid molecules containing GaINAc were predominantly localized along the basolateral (sinusoidal plus lateral) domain of the plasma membrane. Colchicine inhibited the transport of the glycolipid molecules to the plasma membrane. Therefore, glycolipid molecules containing GaINAc are selectively transported to the basolateral domain of the plasma membrane of mouse hepatocytes via vesicular transport.
Cytochemical localization of NAD (P) H oxidase, a hydrogen-peroxide generating enzyme, has been investigated using the cerium method in several mammalian exocrine glands: (a) salivary glands of the rat, Mongolian gerbil, house musk shrew and man, (b) lactating mammary glands of the rat and Mongolian gerbil and (c) exorbital lacrimal gland of the rat. The NAD (P) H oxidase activity could be exclusively localized in association with the myoepithelial cells (MEC) of all the exocrine glands examined. The reaction products in the MEC were found mostly on the plasma membranes facing the neighboring cells (acinar cells, ductal cells and MEC) and in the caveolae. These localizations imply that a membrane-bound NAD (P) H oxidase specific for the MEC could exist in mammalian exocrine glands, and that it may be involved in some intercellular regulatory system between MEC and other parenchymal cells of the glands. With regard to the reactivities on MEC, there were apparent diversities among different glands. Intense and constant activities were found in both the lactating mammary glands of the rat and Mongolian gerbil and the salivary glands of house musk shrew, in which the MEC are extremely developed. The usefulness of NAD (P) H oxidase as a marker enzyme of MEC and the significance of its localization in MEC are discussed.
A new explanation of why calcification develops in arterial vessels is proposed. Some of recently described CD1a+/S-100+vascular dendritic cells have been found to undergo destruction in athero-prone areas of the aorta and in atherosclerotic lesions (Bobryshev and Lord, Arch. Histol. Cytol., 1995). Lysis of these vascular dendritic cells should release their cellular components into the extracellular space, including S-100 which belongs to the family of calcium-binding proteins. In the present study we examined the possible association of vascular dendritic cells with calcification in atherosclerosis and found that vascular dendritic cells were present in early foci of calcification in the arterial intima and in atherosclerotic lesions. This finding suggests the possible involvement of vascular dendritic cells in the process of arterial wall calcification. Vascular dendritic cells and calcifying vascular cells may be related where a subset of vascular dendritic cells represents calcifying vascular cells in the arterial wall. Extracellularly distributed S-100 protein was also found in association with early calcified deposits. The detection of calcium-binding S-100 protein in the extracellular matrix suggests that molecular mechanisms are involved in atherosclerotic calcification, which is quite different from the previously postulated mechanisms involving bone-associated proteins.
Distribution of extracellular free Ca2+ was examined in the rat periodontal ligament and periosteum of alveolar bone during tooth movement, using fluo-3. To stimulate bone formation and osteoclastic bone resorption, upper molars of adult rats were moved lingually for 7 days. After careful removal of the chestal ribs, fluo-3 was injected into the left ventricle of throbbing hearts, immediately after clamping the descending aortae with forceps. 90sec after the injection, the upper jaw was excised out rapidly and immersed in liquid nitrogen. 5μm unfixed undecalcified frozen sections from the frozen tissues were prepared for fluorescent microscopy and also electron microprobe analysis (EPMA). Green fluorescence was emitted from the whole periodontal space and pulp cavity. The pattern of bone formation in the periodontal ligament was quite different from that in the periosteum. A considerable amount of osteoid formed in the buccal periodontal ligament (tension side) and also in the periosteum of lingual alveolar bone (compensative bone apposition). The fluorescence emitted from the osteoid was more intense than that from the periodontal ligament or subperiosteal tissue. EPMA data also showed a higher peak of Ca in the osteoid than in the periodontal ligament or subperiosteal tissues. In addition, a narrow linear green fluorescence was emitted from the interface between the osteoclasts and the bone in the lingual periodontal ligament (pressure side). These results suggest that osteoid contains more free Ca2+ than periodontal ligament and subperiosteal tissue.
To investigate the possible role of manganese superoxide dismutase (Mn-SOD) in steroidogenesis, we studied the expression of Mn-SOD mRNA in the adrenal gland and ovaries of normal and hypophysectomized rats by in situ hybridization. Very strong expression of Mn-SOD mRNA was detected in the zona fasciculata and the zona reticularis of the rat adrenal cortex. The expression was very low in the zona glomerulosa, and was almost negative in the adrenal medulla. The mRNA levels in the adrenal cortex and the ovaries were markedly reduced 4 days after hypophysectomy. Marked adrenocortical atrophy occurred 14 to 24 days after the operation, while levels of Mn-SOD mRNA in the cortex remained low. Mitochondrial structure in the fasciculata cells of normal and hypophysectomized rats were compared by electron microscopy. Mitochondria in the fasciculata cells of normal rats were characterized by dense vesicular Cristae. Four days after hypophysectomy, the cristae became tubulo-vesicular in form and their density decreased significantly. Elongated mitochondria with tubular cristae appeared. Oval mitochondria with a few tubulo-vesicular cristae were present in these cells 14 days after the operation. Our results are consistent with the hypothesis that high Mn-SOD levels might play an important role in the regulation of steroidogenesis. The possible roles of superoxide radicals and hydrogen peroxide in the formation of vesicular cristae in the mitochondria are discussed with respect to steroidogenesis.