Ultrastructural studies of the synovial membrane were performed on tissue samples obtained from the human lumbar facet joint. Ultrastructural changes in synoviocytes were studied after loading synovial samples with 5-hydroxydopamine (5-OHDA) in an oxygenated Krebs’ solution, prior to fixation.
  Synoviocytes were set loosely in the intimal matrix and classified into type A (phagocytic) and type B (secretory) cells. In general, type A cells populated the surface of the synovial lining, whereas type B cells were located deeper in the tissue, extending a process into the synovial fluid. Type B cells in control samples contained sparse secretory granules. Free nerve endings were not found in the synovial intima.
  In response to incubation in 5-OHDA, a precursor of biogenic monoamines, synoviocytes clustered and established contact. The ultrastructure of type B cells in the loaded group clearly differed from controls. They possessed typical membrane-bound vesicles, containing an electron dense interior surrounded by a lucent space. The size of these dense core vesicles ranged from 100 to 260 nm (on average 180 nm). They were in relation to microtubules and located preferentially in the marginal area of the cytoplasm, close to the Golgi complex. The ultrastructure of type A cells was not significantly altered. The present observations provide morphological evidence for the amine-handling properties of type B cells, indicating that they mtight be added to the list of ‘APUD’ cells of the diffuse neuroendocrine system. A recepto-secretory function for type B cells is discussed.

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CED-9 prevents apoptosis in embryonic cells of Caenorhabditis elegans but not in mammalian cells. We show here that the prevention of apoptosis in mammalian cells requires a CED-3-cleaved form (68-280) of CED-9 which is localized in the inner mitochondrial membrane. The viability of PC12 and HeLa cells was significantly increased after death stimuli when truncated CED-9 was expressed in these cells but full-length CED-9 did not. The truncated CED-9 expressed in these cells was largely localized to the inner mitochondrial and the endoplasmic reticulum membranes, while full-length CED-9 was detected mainly in endoplasmic reticulum fractions. Moreover, truncated CED-9 in purified mitochondria was resistant to trypsin digestion, but full-length CED-9 was not. These results suggest that the CED-3-cleaved form of CED-9 prevents apoptosis in mammalian cells by localizing to the inner mitochondrial membrane.

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The basal laminae of muscle fibers, when treated by denaturing methods including freeze thawing, have been used as conduits for regenerating nerves. In this study, we developed a new method for denaturing skeletal muscle fibers through treatment with a biological detergent, sodium dodecyl sulfate. Laminin and type IV collagen proteins of muscle fiber basal laminae were preserved after the detergent treatment. A segment of detergent-denatured muscle was grafted to a 1-cm defect of the rat sciatic nerve. One week after grafting, regenerating axons immunostained for neurofilaments were seen extending within laminin-positive muscle fiber basal lamina tubes. Four weeks after grafting, numerous myelinated axons at a much higher level than the control unoperated sciatic nerve, were found in the middle of the graft. They were smaller in diameter than those in the control nerve. Distal host nerves were well reinnervated 4 weeks after grafting. These findings suggest that the basal laminae of detergent-denatured muscle fibers provide effective conduits for regenerating axons.

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Secretion of arginine-vasopressin (AVP) from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei is induced by neurokinin B (NKB) and angiotensin. To characterize the mechanisms by which this occurs, we used immunohistochemical techniques to assess the ability of AVP-producing neurons to express NKB, NKB receptor (NK-3 receptor) and angiotensin II type 1 receptor (AT-1 receptor). Double fluorescence immunohistochemistry indicated that AVP-immunoreactive cell bodies in the PVN and SON, as well as their axon varicosities in the posterior pituitary, co-express NKB. Almost all AVP-neuron perikarya also expressed both the NK-3 receptor and AT-1 receptor. Thus, AVP-producing neurons in the PVN and SON, which are regulated by NKB, are themselves a source of NKB. Furthermore, the regulation of AVP release by these neurons by NKB and angiotensin II is mediated by the NK-3 receptor and the AT-1 receptor, respectively.

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Ferric chloride, when boiled with ammonium thiocyanate, ammonia and cacodylic acid, is converted into a fine anionic iron colloid which consists of 1.0-1.5 nm electron dense granules and gives a distinct Prussian blue reaction (OHTSUKA and MURAKAMI, 1986). Light microscopy of tissue sections stained with this fine anionic iron colloid at pH values of 6.0, 7.0 and 8.0 showed that the healthy adult rat brain contains a considerable number of neurons which possess an intensely positively charged perineuronal net. This net was most clearly demonstrable by staining with the anionic iron colloid at a pH value of 8.0, at which ionizations of almost all cationic sites of the tissue elements were obliterated. Transmission electron microscopy of ultrathin sections stained at a pH value of 8.0 showed that the anionic iron colloid was preferentially deposited in the perineuronal tissue spaces. These findings indicate that the intensely positively charged perineuronal net contains some strongly basic substances such as guanidino compounds, and oecupies the perineuronal (perisynaptic) tissue space.

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The present study was performed to examine changes in the arrangement of D-periodic collagen fibrils in the synovium of the growing temporomandibular joint in mice. At 1 week of age, the mandibular condyle was undeveloped, and only a few collagen fibrils were recognizable in the subintimal layer of the synovium. At 8 weeks, the mandibular condyle was structurally developed with an increase of collagen fibrils in the synovium; a fully mature condyle was observed at 6 months of age. The close association of proteoglycans with collagen fibrils in the synovium of the growing joint was examined by both conventional and energy-filtering transmission electron microscopy of cupromeronic blue-stained specimens. Proteoglycans were associated with D-periodic collagen fibrils in the short filamentous form in random fashion at 1 week of age, but in a regular pattern with D-periodicity at 6 months. These associations in the synovium could be correlated with the mechanical character of the temporomandibular joint.

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The ATP-sensitive potassium channel (KATP channel) is an essential ion channel involved in glucose-induced insulin secretion. The KATP channel is composed of an inwardly rectifying potassium channel, Kir6.2, and the sulfonylurea receptor (SUR 1); in the pancreas it is reported to be shared by all endocrine cell types. A previous study by our research group showed that Kir 6.2-knockout mice lacked KATP channel activities and failed to secrete insulin in response to glucose, but displayed normal blood glucose levels and only mild impairment in glucose tolerance at younger ages. In some aged knockout mice, however, obesity and hyperglycemia were recognizable. The present study aimed to reveal morphological changes in pancreatic islets of Kir 6.2-knockout mice throughout life. At birth, there were no significant differences in the islet cell arrangement between the knockout mice and controls. At 14 postnatal weeks glucagon cells appeared in the central parts of islets, and this image became more pronounced with aging. In animals older than 50 weeks insulin cells decreased in numbers and intensity of insulin immunoreactivity; most islets in 70- and 80-week-old mice were predominantly composed of glucagon cells and peptide YY (PYY)-containing cells. Staining of serial sections and double staining of single sections from these old mice demonstrated the frequent coexpression of glucagon and PYY, which is a phenotype for the earliest progenitor cells of pancreatic endocrine cells. These findings suggest that the KATP channel is important for insulin cell survival and also regulates the differentiation of islet cells.

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Retinal projections to the nucleus of the optic tract (NOT) and accessory optic nuclei (AON) were studied in the postnatal hooded rat after monocular injection of cholera toxin B subunit (CTB) into the vitreous chamber of the eye. At all postnatal ages, retinal axons were labeled sensitively; they revealed dense projections to the contralateral, and sparse but distinct projections to the ipsilateral, NOT and AON. The CTB Iabeling enabled the first delineation of the complete morphology of developing retinal axons in the ipsilateral NOT and AON. From postnatal day (P) 1 to P3, axons with complex growth cones were seen, and unbranched collaterals with simple growth cones increased and extended gradually. At P6, complex growth cones disappeared while branched collaterals with simple growth cones as well as small-sized varicosities increased. By P12 (two days before eye-opening) the adult-like pattern of terminal arbors appeared. The branched collaterals with tiny, small-sized varicosities present probably represented developing synaptic boutons. At P16 (after eye opening), the pattern of terminal arbors was well developed, almost to the same extent as in the adult. By contrast, a broadly distributed, transient retinal projection around NOT and AON was gradually eliminated; it started to disappear during the first few postnatal days, and was fully retracted by the time of eye-opening time to a pattern normal for the adult.

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The aquaporin (AQP)-9 gene was recently isolated from human and rat liver cDNA libraries as a member of the water channel family for water and neutral solutes. Although the expression of AQP9 mRNA has been demonstrated in several organs including the liver and testis by Northern blot analysis, the cellular and subcellular localization of the AQP9 protein remains unclear. In the present light and electron microscopic immunohistochemical study, the localization of the AQP9 immunoreactivity was examined in fifteen kinds of rat organs using an antibody against rat AQP9 synthetic peptide. The antibody immunostained a major band of ~33 kDa in the liver by Western blot analysis. Immunoreactivity for AQP9 was found exclusively in the liver and testis among the organs examined. In the liver, positive staining appeared selectively along the space of Disse. Immunoelectron microscopy confirmed the localization of AQP9 on the surface of hepatocyte microvilli facing the space of Disse. In the testis, the plasma membrane of Leydig cells located between seminiferous tubules was conspicuously immunoreactive to the antibody. Intense mRNA expression was detected in the liver and testis but not in other organs by ribonuclease protection assay. These findings suggest a specific role for AQP9 in the transport of water and non-charged solutes in hepatocytes and Leydig cells.

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We previously demonstrated that lysosomal cysteine proteinases, cathepsins B, H, and L were localized in lysosomes of alveolar macrophages and bronchial epithelial cells in the rat lung, while cathepsin H, a typical aminopeptidase, was additionally distributed in lamellar bodies containing surfactant in type II alveolar epithelial cells (ISHII et al., 1991). The present immunohistochemical study further examined the localization of lysosomal aminopeptidases, cathepsin C, and tripeptidyl peptidase I (TPP-I) in the rat lung. Western blotting confirmed the presence of cathepsin C and TPP-I as active forms in the pulmonary tissue, showing 25 kD and 47 kD, respectively. Immunohisto/cytochemical observations demonstrated that positive staining for cathepsin C and TPP-I was more intensely localized in alveolar epithelial regions than in bronchial or bronchiolar epithelial cells. By double immunostaining using confocal laser microscopy, immunoreactivity for cathepsin H was found to be co-localized with that for cathepsin C or TPP-I in both type II cells and macrophages. Moreover, when doubly stained with anti-cathepsin C and ED2, single-positive type II cells could be clearly distinguished from double-positive macrophages in the alveolar region. Immunoelectron microscopy revealed the gold labeling of cathepsin C or TPP-I in multivesicular and composite bodies, and lamellar bodies of Type II cells. These results showing that lysosomal aminopeptidases such as cathepsin H, cathepsin C and TPP-I are localized in lamellar bodies of type II alveolar epithelial cells strongly argue for the participation of lysosomal aminopeptidases in the formation process of surfactant containing specific proteins.

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The tooth in the gar-pike, Lepisosteus oculatus, an actinopterygian fish, is characterized by the occurrence of both enamel and enameloid, the former covering the tooth shaft and the latter, the tooth cap. Our previous research demonstrated that the enamel in this species was, as in the lungfish, immunoreactive for amelogenin, indicating its homologous nature with the mammalian tooth enamel, whereas the enameloid was completely immunonegative. The present study demonstrates that, during the early maturation stage of the enameloid formation, the inner enamel epithelial cells (IEECs) synthesize through a well-developed Golgi apparatus a fine-granular substance which is intensely immunoreactive for amelogenin. This substance was accumulated in a large saccule extended in a suprabasal zone of the cell; we were unable to find any morphological sign indicating a connection of the substance with the enameloid matrix. The abortive secretion of the enamel matrix-like substance in the IEEC during an enameloid formation was considered to be an instance of rudimental enamel formation. In the gar-pike, the synthesis of amelogenin in the IEEC has been demonstrated to occur independently from that of the enameloid matrix. The present findings demonstrate a prominent difference between the tooth enamel and enameloid.

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We investigate, by the immunogold method, the localization of keratan sulfate (KS) proteoglycan in rat calvaria in order to clarify the detailed process of intramembranous ossification. KS was localized in bone nodules corresponding to calcified nodules, close to the saggital suture of calvaria. The immunoreactivity decreased in fully calcified regions distant from the suture. Electron microscopic observation revealed that KS was distributed in and around matrix vesicles, among collagen fibrils at the initial crystal deposition stage, and then concentrated in bone nodules. According to the progress of mineralization, KS tended to be localized in the peripheral region of the nodules. In addition, these nodules came in contact with collagen fibrils which also showed KS-positive reactivity. In cell organelles of osteoblasts, KS was detected in the Golgi apparatus. These findings suggest that osteoblasts in intramembranous ossification sites actively synthesize KS. KS in the calcified nodules, as well as other glycosaminoglycans in osteoid, may play an important role in additional and/or collagenous calcification by trapping calcium ions through its negative charge.

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The development of astrocytes in the rodent cerebrum has been considered to occur mainly in the perinatal stage after the end of neurogenesis, and the mode of generation and migration of the astroglial precursors in the embryonic cerebrum remains poorly understood. Tenascin-C (TN-C) is an astroglia-derived extracellular matrix molecule, and its expression can be traced to the ventricular germinative zone of the embryonic cerebrum. TN-C expression may therefore be used as a marker of astroglial precursors prior to the expression of the glial fibrillary acidic protein (GFAP), a marker for the late stage of astroglial differentiation. The development of astrocytes in the mouse embryonic cerebrum was examined by in situ hybridization histochemistry for TN-C mRNA as an early marker. Observations showed that TN-C-positive cells were aligned in the ventricular germinative zone and began to migrate radially at E (embryonic day) 15. They were dispersed into the cortex as early as E17, and some were still in the DNA synthetic phase (S phase) even after lodgement in the cortex. TN-C-positive cells differentiated into GFAP-immunopositive astrocytes in the cortical and subcortical areas at the neonatal stage. The distribution of their active generation sites in the ventricular germinative zone was not homogeneous but displayed a restricted pattern. The mode of their migration towards the cortical and subcortical areas also showed a characteristic pattern that correlated to the radial glial arrangement. These findings indicate that TN-C-positive cells represent a new subset of astroglial precursors which arise directly from the ventricular germinative zone, and that astrogliogenesis from this precursor occurs as early as the period of neurogenesis at the embryonic stage.

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