Ca++-activated adenosine triphosphatase (Ca++-ATPase) in the epidermal cells of rat skin was demonstrated with the method of Ando et al. (1981), and the presence of enzyme activity on the epidermal gap junctions was obtained. Furthermore, the gap junctions showing enzyme activity were examined morphometrically under a digigramer model-G (Muto Kogyo Co. Ltd., Japan). The electron microscopic observation of epidermis showed reaction products of Ca++-ATPase on the entire cell surface continuously except desmosomes and basal cell membranes facing the basal laminae in the stratum Malpighii and a few reaction products in the stratum granulosum. With keratinocytes differentiating, activities seemed to be diminishing. The reaction products were also found on the cytoplasmic side of the gap junctional membranes. Gap junctions showing the reactions were more often detected in the upper portion of the stratum Malpighii and/or in the lower portion of stratum granulosum of the rat pad epidermis. 1. The findings of Ca++-ATPase in the rat epidermis might reflect changes in the intracellular level of calcium which occur during the proliferation and the differentiation of epidermal cells. 2. The unequal distribution of the epidermal gap junctions appears to be involved in the synchronous differentiation process and in the maintenance of the epidermal thickness.
In order to investigate the ultrastructure and Ca++-activated adenosine triphosphatase (Ca++-ATPase) activity of gap junctions under near-to-native conditions, frozen thin section and isolated gap junctons from rat liver or heart were examined under a 200kV electron microscope equipped with a cryo-transfer system. The connexon arrangement observed from a face-on view in isolated, negatively stained gap junctions revealed quite a different pattern between cryo- and conventional images, although transverse views in frozen thin section showed no notable findings on gap junction structure. That is to say, whereas a well-ordered hexagonal connexon arrangement could be recognized in conventional liver gapjunction images, connexons in cryo-images did not exhibit any regularity in their arrangements. Furthermore, the average diameter of connexons in cryo-images was a little bigger than that of the conventional images. The localization of Ca++-ATPase activity on isolated heart gap junctions also displayed a different appearance between cryo- and conventional images. These results suggest a structural change of isolated gap junctions during dispensable treatments, so the method of isolated gap junction observation under a cryo-electron microscope is considered to be very useful in detecting its near-to-native structure.
The localization of 5′-nucleotidase (5′NT) activity on mononuclear cells in the inflammatory focus of the submandibular gland of the adult female non-obese diabetic (NOD) mouse was investigated by an enzymehistochemical technique. At the light microscopical level, the majority of infiltrating mononuclear cells demonstrated intense reaction products on the surface and the reactive cells showed a tendency of forming groups at the center of the inflammatory focus. At the electron microscopical level, mononuclear cells of the NOD mouse could be roughly differentiated into two kinds of cells, those which demonstrated intense reaction products on the plasma membrane (5′NT positive) and those which demonstrated less or no reaction products (5′NT negative). In the acini or granular convoluted tuble (GCT) cells, which lie adjacent to the inflammatory focus, 5′NT positive mononuclear cells were observed and the morphological appearance of these mononuclear cells resembled lymphocytes. In the present study, the possible role and biological significance of 5′NT positive mononuclear cells in the inflammatory focus are discussed.
Sphingolipid activator protein-1 (SAP-1) stimulates enzymatic hydrolysis of sphingolipids including sulfatide. In this study, subcellular localization of the disease-specific inclusions and SAP-1 in cultured skin fibroblasts from a patient with metachromatic leukodystrophy (MLD), sulfatide storage disease, was examined by immunoelectronmicroscopy. When stained with antibody to SAP-1, acid phosphatase positive membrane-bound vesicles were stained in both the controls and MLD cells, indicating that SAP-1 was localized in lysosomes. After sulfatide loading (20μg/ml) for 7 days to MLD cells, 273 lysosomes in a cell were examined regarding the correlation of SAP-1 staining and specific sulfatide inclusions with 6-7nm periodicity. SAP-1 and specific sulfatide inclusions were simultaneously observed in 59% of total lysosome vesicles examined (164 out of 273) and positive staining of SAP-1 was observed in 92% of lysosomes with specific sulfatide inclusion (164 out of 178). These results demonstrated a positive correlation between SAP-1 staining and specific inclusions and suggested the possible induction of a secondary increase of SAP-1 in lysosomes by the pathological storage of sulfatide.
Muscle fibers of flight (pectoralis major) and leg muscles (gastrocnemius) of the adult pigeon were classified based on succinate dehydrogenase (SDH) activity and myosin adenosine triphosphatase (ATPase) activity following preincubation at various pH's. The fiber structure in semithin as well as ultrathin sections was also evaluated. The pectoral muscle was composed almost entirely of the type IIB red fiber, with type IIB white fibers representing only 3.4% of the total fibers; and the average diameter of the type IIB red fibers was 26.8μm±6.3, whereas that of the type JIB white was 50.9μm±7.9. The pectoral muscle also contained a larger amount of fat droplets and mitochondria in the type IIB red fiber, while relatively smaller amounts of fat droplets and mitochondoria were found in the IIB white fiber. These findings suggest that the major part of pigeon pectoral muscle should be categorized as a fast-twitch (alkali-stable ATPase, Type JIB) and fatigue resistant (high oxidative staining). The leg muscle of pigeon was divided into three types, the type I fiber, the type IIA and IIB fiber. Fiber diameter of the three fiber types was not much different, and the fiber population was 23.0% type I, 33.3% type IIA and 43.6% type IIB. Regarding histochemical properties, the flight and leg muscles were very different in their muscle fibers, thus reflecting the different functions of the two muscles.
The localization of Na+, K+-ATPase in human liver was investigated on the light and electron microscopic levels by using anti-sera on the catalytic subunit of Na+, K+-ATPase in human kidney. α-subunit of Na+, K+-ATPase was extracted from the gel of SDS-PAGE as an immune antigen and injected into rabbits in order to produce the antibody. Under light microscopic examination, reaction products were recognized at the bile canalicular, lateral and sinusoidal membranes of hepatocytes and luminal membrane of biliary epithelial cells on the PLP (periodate-lysine-paraformaldehyde) fixed frozen sections. By ultrastructural observation, reaction products were also recognized at the bile canalicular membrane of hepatocytes and luminal membrane of biliary epithelial cells. These results suggest that Na+, K+-ATPase is responsible for bile formation and transport processes on these membranes.