Myxoid tissue was studied in the supporting organ of the cat epiglottis (“epiglottic cartilage”). Under the light microscope, myxoid tissue was characterized by stellate cells placed into an avascular acidic extracellular matrix. This extracellular matrix was alcianophilic at pH=2.5, reacting with the colloidal iron stain, and staining metachromatically with toluidine blue O at pH=5.0. Treatment of sections with testicular hyaluronidase abolished these reactions. In addition, staining persisted after methylation/saponification pretreatment, indicating hyaluronic acid as the main acidic component of myxoid extracellular matrix. Under the electron microscope, myxoid extracellular matrix formed flocculent electron dense precipitates. Stellate myxoid cells were characterized by bundles of intermediate (8nm) cytoplasmic filaments. Myxoid cells were devoid of a basal lamina, contained a few small lipid droplets, and stored some glycogen. Bundles of collagen fibrils, 80-120nm in diameter, were seen in myxoid areas. Myxoid cells reacted to S-100 protein, glial fibrillary acidic protein, and neuron specific enolase. Moreover, in adult animals, myxoid cells stained for neurofilament protein 200. All these markers were also present in chondrocytes of elastic and fibrous cartilage, indicating a close relationship between myxoid cells and chondrocytes. This was supported by the observation of continuous transitional forms of myxoid tissue into elastic or fibrous cartilage. In 8-week-old kittens, the supporting organ of the epiglottis was found mainly to consist of myxoid tissue with only a few interspersed islets of chondrocytes. It is therefore concluded that myxoid tissue can serve as a precursor of cartilage.

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The acidic glycoconjugates of mouse ileum Paneth cells were examined with the aid of light and electron microscopy, using cationic colloidal gold (CCG) as a probe. Specimens of mouse ilea were fixed in half-strength Karnovsky's fixative and embedded in Lowicryl K4M resin. Semithin and ultrathin sections were cut for examination with light and electron microscopy, respectively.
Examination of the sections using light microscopy revealed the positive staining of CCG at pH 1.0 and pH 2.5, which was detected at the rim of secretory granules and at the supranuclear regions of the Paneth cells. At pH 4.0, in addition to staining of the secretory granule rim, weak staining was observed in the granule core. At pH 7.2, the cytoplasm other than secretory granules exhibited positive CCG staining.
Examination of the sections using electron microscopy, at pH 1.0, the trans lamellae of the Golgi apparatus, the rim of the secretory granules, and lysosomes were labeled selectively by CCG. At pH 2.5, labeling was also discernible over the same structures in the cells. However, at this pH, the labeling intensity was stronger than that at pH 1.0, due to the dual labeling of sulfated and sialylated glycoconjugates in these structures. At pH 4.0, the Golgi apparatus, rims and cores of secretory granules and ribosomes were labeled. Lysosomes and nuclei were also positively stained. At pH 7.2, the rims of secretory granules were not stained.
The present results indicate that the CCG method gives good resolution and contrast when applied to staining, and therefore is useful for the specific staining of glycoconjugates such as sulfated, sialylated and phosphated glycoconjugates for light and electron microscopy.

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In recent studies of cycloheximide (CHX)-induced apoptosis in sublines of established Burkitt's lymphoma cell lines (BJA-B) both with and without Epstein-Barr virus (EBV) infection, we noticed two distinct types of apoptosis morphology. In the present paper, we have classified these, and further carried out a statistical analysis of their incidence in untreated and CHX-treated EBV-free (EBV (-)) and EBV-infected (EBV (+)) BJA-B cells. Classification: Both types of apoptosis morphology demonstrated typical nuclear and cytoplasmic condensation. However, “Type 1 apoptotic cells” (AP1) maintained a spherical or ovoid shape, but “Type 2 apoptotic cells” (AP2) were typified by the lobulation of their nuclear and cytoplasmic structures to form “clover leaf” shapes. Statistical analysis of incidence: The numbers of AP1 and AP2 cells were analysed using a χ² test, with results as follows: EBV (-) cells underwent AP1 in preference to AP2 (90.5% versus 9.5%) (p<0.001), whilst EBV (+) cells had comparably more AP2, making AP1 and AP2 approximately equal (49.3% versus 50.7%) (p>0.1). In EBV (-) cells, treatment with CHX had little effect on the ratios of differing apoptotic morphology. In contrast, in the EBV (+) cells, cell death was altered from AP2 (50.7%→25.2%) towards AP1 (49.3%→74.8%) (p<0.001). We propose that cellular proteins known to be associated with EBV infection not only protect the cells from apoptosis, but also affect the phenotype of apoptosis. This knowledge may be useful for defining possible mechanisms of apoptosis induction and/or inhibition in specific models.

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Light and electron microscopic immunolocalization of taurine, a sulfur-containing free amino acid, was investigated in the photoneuroendocrine pineal organ and the retinal photoreceptor and pigment epithelial layer of the ayu Plecoglossus altivelis, an anadromous fish. Intense immunostaining was found in the outer segments of pineal photoreceptors and retinal cone-like cells. Moderate but definite immunostaining was found in the cytoplasmic processes of pineal supporting cells, the outer segments of retinal rod-like cells, and the apical processes of pigment epithelial cells. Although the electron microscopic immunogold labeling was not completely coincident with the results of light microscopic immunostaining, concentrated immunogold particles appeared in the inner segments of photoreceptor cells, the cytoplasmic processes of pineal supporting cells, and the apical processes of pigment epithelial cells. These light and electron microscopic findings in taurine immunolocalization were discussed in relation to the functions of taurine known mainly from retinal physiology. It was suggested that the abundant taurine localization may be involved at least in the protection of photoreceptor outer segments against harmful factors, and in the transportation of nutrients and metabolites. The immunostaining for taurine is useful for the discrimination of different types of photoreceptor cells in the pineal organ and retina of fish.

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The effect of platelet factor 4 (PF4) on myoblast cultures with or without basic fibroblast growth factor (bFGF) or other growth factors was investigated in the present in vitro experiments, with reference to bFGF binding to myoblast membrane fraction. When PF4 was added to the culture medium 1 day after myoblast cultivation, the nuclei of both myoblasts and myotubes were markedly reduced in number in a dose-dependent manner, whereas the inhibitory effect of PF4 on myoblast development was not observed when PF4 was added to the culture medium 3, 7, or 14 days after myoblast cultivation. In contrast, bFGF significantly increased the numbers of myoblast and myotube nuclei. When bFGF and PF4 were simultaneously added to the culture medium, PF4 abolished the facilitatory effects of bFGF on myogenesis. The real-time biospecific interaction analysis (BIA) core system showed that the myoblast membrane fraction at 1 day after cultivation contains bFGF-binding elements which are blocked by PF4 in a dose-dependent manner. Moreover, [¹²⁵I]-bFGF binding experiments indicated the existence of both high and low affinity binding sites on myoblast membranes, although the high affinity binding sites decreased in number and the dissociation constant increased in value as the culture period was prolonged. Among the six other growth factors examined, acidic fibroblast growth factor and platelet-derived growth factor-BB stimulated myogenesis, and their effects were blocked by PF4 treatment. These findings suggest that: 1) PF4 inhibits myoblast proliferation and myotube formation only for a limited initial period of cultivation, possibly because of the time-dependent down-regulation of high affinity bFGF receptors: and 2) PF4 may be used as a tool to investigate the function of endogenous heparin-binding growth factors upregulated transiently at a certain developmental stage or in case of tissue damage and repair, even though it is not monospecific to bFGF.

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Phagocytic cells disposing of dying granulosa cells in the atretic ovarian follicles were morphologically studied in guinea pig ovaries at various stages of estrous cycle. Epon embedded semithin sections stained with toluidine blue were observed with a light microscope, and ultrathin sections were examined under a transmission electron microscope. Frozen sections were processed for acid phosphatase histochemistry and MR-1 (a monoclonal antibody against guinea pig macrophages) immunohistochemistry.
In animals during the estrus period (days 1 and 2) as well as the second half of the estrous cycle (days 11 and 16), there were numerous mature follicles in which massive groups of granulosa cells were undergoing apoptosis. Two kinds of phagocytic cells were identified in these follicles of the initial stage of atresia: one was intact granulosa cells ingesting neighboring dead granulosa cells, and the other was large round cells identified as macrophages due to their strong acid phosphatase activity and MR-1 immunoreactivity.
Mature follicles of the advanced stage of atresia were frequently recognized during the metestrus period (days 4 and 5). Small stellate cells were regarded as surviving granulosa cells, while large round cells showing intense reactions for acid phosphatase and MR-1 were identified as macrophages.
This study demonstrates that both intact granulosa cells and macrophages participate in the elimination of apoptotic granulosa cells in atretic mature follicles of the guinea pig ovary, and remain even in the advanced stages of atresia.

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Previous studies on the development of the parotid gland in various mammals have demonstrated that terminal clusters and acini contain mucous cells during the early postnatal period. However, little information has been available concerning the exact fate of the secretory granules in the mucous cells, specifically as to whether or not the mucous cells differentiate into serous cells. The present study aimed to determine the time of appearance of the mucous cells in the rat parotid gland as well as the morphological and histochemical changes of their granules.
Light microscopy showed that cells positively stained with periodic-acid Schiff and alcian blue were sparsely distributed in the terminal clusters and acini on day 1 but had multiplied to a maximal level by day 5. They decreased in number on day 8 and were not recognizable at all by day 10.
Electron microscopy revealed that the mucous cells initially contained granules of homogeneous low electron density, and then bipartite and tripartite granules with electron-dense cores were detected. By day 8 granules showing bipartite and tripartite structures and granules of homogeneous high electron density were seen to coexist in single cells. These observations suggest that mucous cells exist in parotid glands for a limited period of time and that, as the gland develops, the mucous granules come to resemble serous granules.
Lectin histochemistry indicated that the secretory granules in the mucous cells were positive for peanut agglutinin, soybean agglutinin and wheat germ agglutinin, suggesting the occurrence of β-D-galactose, α-D-N-acetyl galactosamine and β-D-N-acetyl glucosamine which are the same sugar residues as those found in the granules of mature parotid serous cells.
Immunostaining showed that even the low electrondense granules in the mucous cells were weakly reactive for anti-rat parotid gland amylase; this reactivity gradually increased with development.
These results suggest that mucous cell secretory granules which contain abundant glycoconjugate for a limited period during the development of the gland may change into serous granules.

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Backscattered electron (BSE) imaging of scanning electron microscopy (SEM) was applied to a study on the mineral density of the bone surface. The neonatal and adult mouse parietal bones freed of the periosteum and covering cells were examined in a field emission scanning electron microscope equipped with a high sensitivity BSE detector at 1-30kV accelerating voltages. The mineral density of the bone surface was observable in BSE images at 5kV accelerating voltage while only the topographic structures of the surface were obtained under an accelerating voltage less than 5kV. As the accelerating voltages increased from 5kV, the bright areas were extended, probably due to the imaging of the calcified bone matrix under the uncalcified osteoid.
The bone surface is usually divided into smooth and rough areas according to its irregularities. BSE images at 5kV clearly showed that the smooth areas were further divided into dark and bright areas which apparently corresponded to the uncalcified osteoid and calcified bone matrix, respectively. Bright granules, about 1.0-3.0μm in diameter, were sometimes observed at the border between the osteoid and calcified bone matrix; these granular calcified areas were regarded as the calcifying front forming the calcified bone matrix from the osteoid.
The present study demonstrated that the distribution of the osteoid on the mouse parietal bone surface changes depending on age: the osteoid occupied a large area in the parietal bone surface in neonatal mice, but was small in adult mice. Thus, low accelerating voltage SEM using BSE provides new information on the distribution of the osteoid and the bone matrix calcification under both normal and pathological conditions.

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The presence of subunit proteins, 1H9 for the α-subunit and 2B6 for the β-subunit, of H⁺-K⁺ ATPase and its activity in tubulovesicles and intracellular canaliculi of gastric parietal cells were immunocytochemically and enzyme cytochemically examined. Specimens were taken from healthy human volunteers by endoscopic biopsy in resting, tetragastrin-stimulated and omeprazole-inhibited conditions. H⁺-K⁺ ATPase was present in both intracellular canaliculi and tubulovesicles in these three conditions. Gold particles of the α-subunit decreased in number, and those showing the β-subunit increased under both gastrin-stimulating and omeprazole-inhibiting conditions compared with parietal cells in the resting state. We suggest that the administration of tetragastrin and omeprazole alter the turnover rate of each subunit of H⁺-K⁺ ATPase, resulting in the difference of the proportions of α- and β-subunits. Moreover, the activity of H⁺-K⁺ ATPase was detected strongly beneath the membrane of microvilli and weakly in that of tubulovesicles under these three conditions. After 7 days of daily oral omeprazole intake, H⁺-K⁺ ATPase in parietal cells were detected in intracellular canaliculi and tubulovesicles. However, the H⁺-K⁺ ATPase activity in tubulovesicles was diminished 1h after omeprazole intake, and the activity in intracellular canaliculi was completely inhibited even 3h after omeprazole administration. These results show that omeprazole inhibited the H⁺-K⁺ ATPase activity in both intracellular canaliculi and tubulovesicles.

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