Frozen biological specimens in t-butyl alcohol were examined under a low-vacuum environment in a “wet SEM” or “variable pressure SEM (scanning electron microscope)” equipped with a cooling stage and highly sensitive backscattered detector of the YAG type. After fixation with glutaraldehyde and osmium tetroxide, rat tissue blocks (tracheae and kidneys), and cultured human carcinoma cells were dehydrated with a graded series of t-butyl alcohol. The specimens were directly frozen on the cooling stage at -10°C, evacuated to 20Pa in the specimen chamber, and observed by detecting backscattered electrons at accelerating voltages of 5-6kV. The images became clearer 20min after the vacuum reached 20Pa and revealed had good quality by 30min, probably because t-butyl alcohol was sublimated during the time. The cilia of tracheal ciliated cells, end-feet of the podocytes of the renal glomerulus, and processes of cultured cells were clearly observed without any serious preparation artifacts. Since the low-vacuum SEM of t-butyl alcohol frozen samples is both simple and provides high imaging quality, it is expected to be useful in a variety of biological fields such as the rapid pathological diagnosis.

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Ultrastructures of pituitary gonadotropes are known to show a prominent sex-related difference: typical male rat gonadotropes contain both large- and small-sized granules, whereas typical female rat gonadotropes appear to exhibit uniformly small-sized granules. Our preceding studies have demonstrated that two representative granins, chromogranin A (CgA) and secretogranin II (SgII), are separately localized to each type of granule in male rat gonadotropes. To clarify whether or not there is a certain relationship between granin proteins and characteristic features of secretory granules in female rat gonadotropes, we examined the expression levels and immunocytochemical localizations of CgA and SgII in the cells. Northern blot and immunoblot analyses demonstrated that both CgA and SgII were synthesized and stored in the female pituitary, although the amount of CgA was much lower in the female than that in the male pituitary. Immunocytochemical observations clarified that gonadotropes in the female pituitary possessed intermediate secretory granules containing both CgA and SgII, in addition to solely CgA-positive and SgII-positive ones. However, secretory granules containing CgA in the female gonadotropes were much smaller in size and appeared less frequently than those in the male cells, whereas no sexual difference was discerned in SgII-positive granules. Moreover, the size and appearance of CgA-positive secretory granules varied depending on stages of the estrous cycle. These findings suggest that the size and appearance of secretory granules containing CgA are closely associated with the expression and storage levels of CgA in the pituitary.

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We recently reported that Merkel cells in the normal palatine mucosa of adult rodents are highly polymorphic. In order to ascertain whether or not this polymorphism is also evident in the human oral mucosa, palatine mucosae from cadavers without oral diseases and perilesional palatine mucosae of patients with pleomorphic adenoma were examined by immunohistochemistry using an antibody against cytokeratin 20.
Findings showed that Merkel cells in the human normal palatine mucosa were polymorphic, and a number of irregular-shaped Merkel cells (dendritic Merkel cells) with apparent cytoplasmic projections were present among typical oval to round Merkel cells. The mucosa usually contained a small number of oval to round Merkel cells residing in ectopic places such as prickle and granular cell layers. On the other hand, the slightly inflamed perilesional palatine mucosa contained an increased incidence of dendritic Merkel cells. Ectopic Merkel cells were rare in the perilesional palatine mucosa.
Characteristics of dendritic Merkel cells were examined using specimens from perilesional palatine mucosae by means of immunohistochemistry and electron microscopy. It was shown that every dendritic Merkel cell and most roundish Merkel cells in the perilesional mucosa lacked innervation. Electron microscopy suggested that dendritic Merkel cells release secretory granules from the tip of the cytoplasmic process and the basal cytoplasm towards the lamina propria mucosae, in a manner resembling the case of similar cells in rodents.

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Intra-articular injection of hyaluronate (HA) is widely used in the treatment of arthropathies. However, the mechanism of the effects of HA preparations on the arthritic synovium and the relationship between their effects and molecular weights (MW) remains unknown. The objectives of this study were to compare the effects of two hyaluronate preparations, HA84 (MW: 84×10⁴) and HA230 (MW: 230×10⁴), on the synovium of an arthritis model and to examine the mechanism of the effects of HA. The HA preparations were intra-articularly injected in a model of canine arthritis induced by anterior cruciate ligament transection for a total trial of 5 weeks. To define the accessibility of HA preparations to the synovial lining layers, fluorescein-labeled HA84 or HA230 was injected at the last administration. Pathological changes analyzed included increases in volumes and prostaglandin E₂ concentrations in synovial fluids, thickening of the synovial lining layers, vacuolar alterations in the lining cells, and stainability of HA in the synovium. Expression levels of Heat shock protein 72 (Hsp72) were immunohistochemically detected in the tissues to investigate the ability of the cells to survive the degeneration. The pathological changes described above were more significantly suppressed in the HA84-treated than in the HA230-treated groups. In most cases of the HA84-treated group (five cases out of six), fluorescein particles were intensely distributed in the synovial lining layers, but only two cases in the HA230-treated group showed a weak distribution of fluorescein particles in the layers, indicating a certain differnce in the accessibility of HA preparations to the lining cells between the two HA molecules. Moreover, the immunoreactivity for Hsp72 in the lining cells as more intense in the HA84-treated than in the HA230-treated groups. The difference in the accessibility of HA molecules corresponded well with that in the inducibility of Hsp72 in the lining cells. These results suggest that the up-regulation of Hsp72 may offer a new concept concerning mechanism of the effects of HA preparations on the arthritic Synovium.

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The development of the lymphatic system in the rat diaphragm was studied from embryonic day 16 to 25 weeks after birth by histochemistry for 5′-nucleotidase, scanning electron microscopy of KOH-treated or intact tissues, and transmission electron microscopy of thin sections. On embryonic day 16, distinct lymphatics were noted in the subpleural space of the diaphragm periphery. The endothelial cells at this stage contained an abundance of rough endoplasmic reticulum, a developed Golgi apparatus and mitochondria, and fewer pinocytotic vesicles than those in adults. The subpleural lymphatics subsequently increased and formed a polygonal network. They possessed many valves, and by postnatal week 6, some thick collecting lymphatics became endowed with smooth muscle cells. On embryonic day 19, some lymphatics appeared in the subperitoneal space. They extended centripetally and had many lateral projections that subsequently became elongated and connected with those from adjacent lymphatics, thus forming a lattice-like network. During the early postnatal days, the subperitoneal lymphatics projected many bulges that subsequently became elongated, and came into contact with the pores among the mesothelial cells, thus forming lymphatic stomata connecting the lymphatic lacunae to the peritoneal cavity. The lymphatic stomata increased until postnatal week 10. The results show that lymphatics appear as early as embryonic day 16 in the subpleural space of the diaphragm periphery, and develop with age by sprouting to form networks in both the subpleural and the subperitoneal spaces, and that the direct connection of the lymphatic lacunae to the peritoneal cavity is fromed after birth.

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We recently produced monoclonal antibodies using macrophagic cells derived from cultured rat glomeruli as the antigen. One of the antibodies, named OS-3, was found to detect a cell population scattered in collecting ducts of the rat kidney as well as macrophages in various tissues. The present study deals with the cellular and subcellular localization of immunoreactivities with OS-3 in the kidney and other organs of rats. Double immunostaining using OS-3 and an antiserum against either calbindin or epidermal cytokeratin showed that OS-3-immunoreactive cells exist exclusively in both the connecting segment and cortical collecting duct, and differ from calbindin- or cytokeratinpositive epithelial cells. Ultrastructurally, OS-3-immunoreactive cells appeared spherical in shape with few cytoplasmic microprojections on the narrow apical surface. Their relatively dark cytoplasm contained numerous mitochondria and a developed tubulo-vesicular system. The intense immunoreactivity was selectively localized in the basolateral membrane exhibiting shallow but complicated infoldings. Distribution and ultrastructural properties of the OS-3-immunoreactive cells showed that they were type B intercalated cells, which are engaged in the regulation of the acid-base balance mainly by secreting HCO₃^-. Another positive staining with OS-3 was found in the macula densa and some epithelial cells of Bowman's capsule, the former monitoring Cl^- concentrations in the urine. Immunoblotting of extracts from the rat kidney demonstrated a protein band immunoreactive to OS-3 at a molecular weight of 43kDa. Aside from the kidney, a specific and intense immunoreactivity with OS-3 was also found in the epithelial cells of the pancreatic excretory duct and in the secretory cells of the salivary, pyloric and duodenal glands, all of which are HCO₃^--secreting cells. These immunohistochemical findings imply that OS-3 is useful for the detection of type B intercalated cells and recognizes a functional molecule involved in the production/secretion of HCO₃^- or transport of Cl^-.

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CAAF1 and CAAF2, newly identified calcium-binding proteins from bovine amniotic fluid, have been revealed to be members of the S100 protein family preferentially produced by fetal squamous epithelial cells, including epidermal keratinocytes. Having previously cloned the cDNA of human CAAF1 protein from the esophageal epithelium, we report here on the characteristic expression pattern of CAAF1 and related S100 proteins in human esophageal epithelial cells. Normal cells of the human esophageal epithelium expressed CAAF1, and also expressed the homologous novel S100 proteins including CAAF2, MRP8, and MRP14, but not S100α. An immunohistochemical study with specific monoclonal antibodies against CAAF1 proteins demonstrated that CAAF1 proteins were produced by the esophageal epithelial cells in the process of cell differentiation. The immature proliferating cells in the epithelium did not produce CAAF1 proteins, but the differentiated cells expressed CAAF1, which overlay the immature cells and were stratifying in the epithelium. These CAAF1-producing cells did not show any proliferating activities. Esophageal carcinoma cells did not express CAAF1, except for the keratinized cells with no proliferating activity. In addition, the forced expression of CAAF1 proteins in the carcinoma cells resulted in a marked decrease in DNA synthesis. These findings indicate that human esophageal epithelial cells express the multiple genes of S100 proteins including CAAF proteins, and that CAAF1 is closely associated with the terminal differentiation of these cells. CAAF1 expression also might play some role in cell growth.

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The embryonic development of the inner ear, especially the sensory epithelia and otoliths in the rainbow trout Oncorhynchus mykiss, was studied by light and electron microscopy.
Light microscopically, the auditory vesicle, saccular otolith and statoacoustic ganglion were first observed by 12 days after fertilization, while the utricular otolith appeared at 15 days after fertilization. Both the saccular and utricular maculae were more developed at 22 days after fertilization, and well developed by 27 days. The crista ampullaris of the horizontal canal was also developed at 27 days after fertilization, while the other cristae were not yet distinguished.
Electron microscopically, vesicular structures and short microvilli were found on the sensory epithelia of the maculae by 15 days after fertilization. At 22 days after fertilization, the saccular otolith possessed 7 incremental layers, and developing cilia, microvilli, and aggregates of secretory materials also appeared on the apical surface of the sensory epithelia. At 27 days after fertilization, the apical surface of each hair cell was covered with a hair bundle consisting of a single kinocilium and a bundle of stereocilia.
These findings are discussed with special regard to the environmental factors on early development in fishes.

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