We describe here the immunohistochemical localization of CD44 (hyaluronate receptor) and the ezrin-radixin-moesin (ERM) family (actin binding proteins) in bone cells and discuss possible mechanisms for the organization of their cytoskeletons.
In the cells of osteoblast-lineage, active osteoblasts showed immunoreactivity to CD44 only on their cytoplasmic processes in the bone matrix. They showed degrees of immunoreactivity to radixin and moesin on the cytoplasmic side of their basolateral plasma membrane, but not in their cytoplasmic processes. On the other hand, osteocytes demonstrated intense immunolabeling to CD44, showing scarcely any immunoreactivity to the ERM family. In osteoclasts, intense immunoreactivity to CD44 was detected on their basolateral plasma membranes. The plasma membranes of the clear zone and the ruffled border were not immuno-labeled with CD44. As for the ERM family, the basolateral plasma membrane of the osteoclasts was stained with anti-moesin monoclonal antibody, but not with anti-ezrin or anti-radixin antibody. Thirty minutes after the administration of calcitonin, osteoclasts did not show either clear zones or ruffled borders. CD44 and moesin were colocalized all along their plasma membranes, including the region facing the bone surface.
These findings suggest that: 1) the CD44moesin-actin filament system is involved in the cytoskeletal organization of osteoclasts and their cell-polarity; and 2) other mechanisms, rather than the CD44 and the ERM family, may be involved in the cells of osteoblast-lineage.

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Secretory cell glycoconjugates of the external nasal glands of ruin lizard and seps were investigated by prelectin staining methods (periodic acid-Schiff, alcian blue, high iron diamine) and lectin histochemistry in combination with neuraminidase digestion. Five plant lectins (Canavalia ensiformis, Triticum vulgare, Glycine max, Lotus tetragonolobus and Arachys hypogaea) conjugated with horseradish peroxidase were tested. Two different secretory cell types could be distinguished in the glandular tubules in both species examined. Lectin histochemistry revealed a microheterogeneity of mucins produced by the different secretory cell types of the two lizards regarding to the distribution pattern of glycosidic residues in glycoproteins.

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The expression of bone morphogenetic proteins (BMP) in primary osseous tumors (n=15) with a potential of osteogenesis and/or chondrogenesis was re-evaluated by using a recently characterized monoclonal antibody raised by using rhBMP-2 as an immunogen in a streptavidin-biotin complex immunoperoxidase method. The tumors were histopathologically diagnosed as osteosarcoma (n=6), chondrosarcoma (n=3), osteoma (n=2), and chondroma n=2). In addition, Ewing's sarcoma (n=2) was also evaluated for comparison. Three distinct categories of immunoreactivity for BMP were observed in osteosarcomas. Firstly, no immunoreactivity in the tumor cells and tumor osteoid matrices; secondly, immunoreactivity in the tumor osteoid matrices but not in the tumor cells and lastly, immunoreactivity in the tumor cells but not in the tumor osteoid matrices. The reactivity was, however, found between the stromal cells and in primitive mesenchymal cells. Chondrosarcoma showing proliferating malignant chondrocytes with mitosis revealed immunoreactivity for BMP in their cytoplasm. Adjacent areas containing no mitotic figures in the chondrocytes showed immunoreactive BMP in the the cartilage matrix while the tumor cells were unreactive. The chondrogenic areas in osteosarcoma, chondrosarcoma and chondroma showed peripheral regions of chondroid matrix with immunoreactive BMP. No BMP immunoreactivity was found in Ewing's sarcoma. BMP as a marker may be useful to identify osteogenic or chondrogenic tumor cells but do not necessarily segregate a benign from malignant osteogenic tumors.

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Using immunoelectron microscopy and a color image analyzer, we quantified the kinetics of synthesis, secretion and reabsorption of thyroglobulin (Tg) in rat thyroid follicular epithelium under thyrotropin (TSH) stimulation. Seven week-old male Wistar rats were injected intravenously with 2IU TSH. Thyroid glands were removed at 5, 30min, 1 and 3 hr after injection, fixed immediately with 1% glutaraldehyde in 4% paraformaldehyde, post-fixed with 1% osmium tetroxide and embedded in LR-White. Immunostaining was performed with anti-rat Tg rabbit antibody and colloidal gold labeled anti-rabbit immunoglobulins. The amount of immunoreactive Tg was evaluated as the density of colloidal gold particles on the follicular lumen and cytoplasmic compartments using the color image analyzer. Area of rough surfaced endoplasmic reticulum (rER) and the mean diameters of subapical vesicles (SV) were measured from electron micrographs magnified ×5, 000 and ×30, 000 respectively using a color image analyzer. Electron microscopy revealed dense, specific labeling of Tg on the follicular lumen and SV, and rather sparse Tg labeling on rER, Golgi apparatus and lysosome in control rats. After TSH stimulation, the density of gold particles on SV increased significantly at 1 and 3 hr after stimulation compared with that of control. The density remained almost unchanged on rER, Golgi apparatus, follicular lumen and colloid droplets (CD). The labeling density on lysosomes gradually increased. Although the density on rER was not changed, the total count of gold particles on rER increased as a result of rER dilation. The diameter and number of SV per cells decreased until 1 hr after stimulation, then gradually increased. These results revealed differential kinetics of Tg in thyroid epithelial cells after TSH stimulation accompanied by morphological changes. The increase in Tg synthesis in rER seemed to be reflected not by the increase in the density of Tg in rER, but by the increase in the area of rER with an unchanged concentration of Tg. A transient decrease in the diameter, number and Tg density in SV suggested that the release of Tg into the follicular lumen followed the mode of regulated secretion.

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The distribution of desmin filaments in neonatal swine muscle was identified by immunoelectron microscopy. As piglets grow from 0 hr to 10 days after birth, the diameter of myofibril gradually increases and the intermyofibril space becomes narrower. The quantitative analysis suggested that the largest number of desmin filaments was identified in 0 hr after birth. Desmin filaments were identified around the immature myofibrils and also distributed in the wide intermyofibril space. These desmin filaments might be involved in the growth and myofibrillogenesis in developing myocytes. Desmin filaments formed an internal framework to connect the myofibrils and cell organelles. The entire array of myofibrils was then anchored to the sarcolemma. Desmin filaments in neonatal skeletal muscle might play a role as like as connective tissue filaments between parenchymal cells. By the quantitative analysis from fetus, neonate to adult, desmin filaments appeared to relocate from the intermyofibril spaces to the Z-lines. As myocytes mature, desmin filaments gradually acted as the integrators between the adjacent myofibrils.

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The phosphates derived from newly synthesized 3′-O-aralkyl-5- (4-biphenylcarboxamido) fluorescein derivatives were examined for the alkaline phosphatase-linked fluorescence assay of the membrane-bound DNA. λDNA was detectable to the amount of 5 fg in the assay using phosphorylated 5- (4-biphenylcarboxamido) -3′-O- (1-naphthyl) methylfluorescein (BNFP). The spots gave distinguishably clear fluorescence without diffusion and nonspecific adsorption. In the Southern blot hybridization, 0.1 pg of DNA could be detected. Detection of two different DNAs on a single blot was successfully performed by using two different fluorogenic phosphates, Phosphrorilated N- (2-biphenyl) -3-hydroxy-2-naphthalenecarboxamide (HNPP) and BNFP. The fluorescein derivatives were also examined as substrates for horse radish peroxidase.

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To evaluate whether calcitonin influences the lysosomal forming system in osteoclasts, acid phosphatase (ACPase) and glucose-6-phosphatase (G6Pase) were examined in cells with and without hormonal treatment. Many osteoclasts treated with calcitonin (CT) separated from or attached only partly to the bone surface, and the ruffled border (RB) disappeared. In ACPase activity, the reactive products of the enzyme were present in small and large lysosomes, Golgi apparatus, and on the membrane of the RB in CT-free (control) osteoclasts. The reactive products were also present in the same organelles of CT-treated osteoclasts, except RB, but the enzymal activity in the Golgi apparatus was lower than that in control osteoclasts. Additionally, the number of small lysosomes increased strikingly. In G6Pase activity, the reactive products of the enzyme were present in endoplasmic reticulum (ER), nuclear envelope, and vacuoles in CT-treated osteoclasts as well as in control osteoclasts. The vacuoles with the enzyme-positive were distinguished from the lysosomes in size, and the number increased remarkably in CT-treated osteoclasts. From these results, there is a possibility that calcitonin influences the function of Golgi apparatus in the lysosomal forming system of osteoclasts, resulting in the appearance of vacuoles with G6Pase activity.

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The origin and diagnostic criteria of olfactory neuroblastoma are still under dispute. Tosolve these problems, seven cases of olfactory neuroblastomas satisfying diagnostic criteria of Choi and Anderson were analyzed immunohistochemically by using antibodies to markers expressed in developing and mature human olfactory placode-derived neurons such as keratin, a 34 kDa epithelial membrane glycoprotein, luteinizing-hormone-releasing-hormone (LHRH) and to other neural tissue markers. Ultrastructural analysis was also carried out. Five cases were identified as neuroblastomas because neurites were disclosed in them. All of these five cases had widespread immunoreactivity to an antibody to a 34 kDa epithelial membrane glycoprotein (Ber-EP4) in the tumor cell bodies and/or neuritic processes. They also contained keratin positive cells. LHRH was demonstrated in two of these Ber-EP4 positive neuroblastomas. The spectrum of immunohistochemical findings of these five cases was identical to the normal neuroblasts arising from olfactory placode.
Accordingly, it is concluded that neuroblastoma of true olfactory placodal origin does exist. The diagnostic criteria of this tumor is as follows: demonstration of neurites and immunoreactivity to Ber-EP4 and/or LHRH. Keratin is not necessary to regard as an evidence of epithelial differentiation in this tumor.

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It is important to understand the cause of photobleaching and to know how to protect from the bleaching the fluorescent DNA probes stained cells from bleaching in microscopy. We examined the protective effect of sodium azide (NaN₃) from photobleaching on the micrometer and the detection of active oxygen species produced in the probe solution by photoirradiation using an electron spin resonance (ESR) with a trapping agent. It was found that the bleaching of several probes was protected 11-99% by adding 1 mM NaN₃ as a scavenger for singlet oxygen. The NaN₃-pro-tecting efficiency for photobleaching on the microscope corresponded to the typical ESR signal intensity with the spin trapping agent for singlet oxygen. The OH radical produced in the probe-photosensitization was also detected by the ESR measurement with the another trapping agent. From the results, it was found that there were two types (predominantly, singlet oxygen or radicals) of active oxygen species produced at the probe-photoreactions. It was considered that active oxygen species produced from these photoreactions oxidized the probes to decrease the fluorescence intensity of the DNA probes used in the microscopy, and that the NaN₃ protected the photobleaching considerably without selfquenching of fluorescence.

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The pathway of Ca²⁺ absorption across the renal epithelial layer was ultracytochemically investigated in nephrons using an oxalate-pyroantimonate technique. Reaction products were strongly detected in the inter-cellular space in proximal tubules, and in intracellular organelles in distal tubules in the cortex and the outer layer of the outer medulla. These findings are suggestive of paracellular transport of Ca²⁺ in the proximal tubule, and transcellular transport in the distal tubule. The transcellular pathway of Ca²⁺ is of interest, as intracellular Ca²⁺ concentrations must be maintained. Ca-precipitates, in the distal tubule cells, were detected en masse in mitochondria, and to a lesser extent, in smooth endoplasmic reticulum, Golgi apparatus and vesicles. Precipitates were rare in lysosomes and rough endoplasmic reticulum. Calcium containing organelles distributed throughout the cytoplasm. Free precipitates in the cytoplasmic matrix were only rarely seen in the apical portion of the cells. It seems that Ca²⁺immediately after entering a cell, binds to calcium binding proteins, or is sequestered to organelles like mitochondria, Golgi apparatus, smooth endoplasmic reticulum or vesicles. In this way, fluctuations in intra-cellular Ca²⁺ concentrations are avoided. These organelles may be involved in Ca-transport in Ca-absorbing cells.

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Cell kinetics of bone and soft tissue tumors was assessed using 12 malignant tumors (3 cases of malignant fibrous histiocytoma, 3 cases of osteosarcoma, 2 cases of liposarcoma, 1 case each of synovial sarcoma, malignant lymphoma, malignant schwannoma and dedifferentiated chondrosarcoma) that were collected during operation in our department and subcultured by transplanting in nude mice. After intraperitoneal injection of iododeoxyuridine (IUdR) and bromodeox-yuridine (BrdU), tumor-bearing mice were sacrificed. Histological sections of the tumors were double-stained with mono-clonal antibodies for these compounds and examined. BrdU-labeling index for the tumors was 5-37%, duration of S phase was 13-34 hr, and potential doubling time was 54-375 hr. To our knowledge, no studies in which these values were measured in malignant tumors of bone and soft tissues have been reported. These values are fundamentally similar to those of carcinomas and leukemias reported before in another fields. In the present study the potential doubling time tended to be short in tumors that were clinicopathologically graded as highly malignant and exhibited poor prognosis. Also, the potential doubling time correlated closely with the tumor volume doubling time obtained by actual measuring in the diameter of tumors. These findings indicated that the potential doubling time of bone and soft tissue tumors may be a useful prognostic indicator beyond the different histologic diagnoses. In addition, they suggested possible and practical utilization of the potential doubling time in anti-cancer drug-sensitivity tests of tumors and application to planning therapeutic strategies against musculoskeletal sarcomas.

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The present study was undertaken to elucidate the age-related changes of DNA synthesis and morphology of tracheal cells in aging mice by light and electron microscopic radioautography. The tracheae of 8 groups of mice from fetal day 18 to 2 years after birth were labeled with ³H-thymidine in vitro. The results demonstrated that the DNA syntheses and morphology of tracheal cells in aging mice changed due to aging. The radioautographic examinations revealed that the DNA syntheses of the nonciliated and basal cells reached their maxima on fetal day 18, then fell from postnatal 3 das onwards. The activity of DNA synthesis of ciliated cell was very low in the fetal stage. Ciliated cells could not synthesize DNA and proliferate in the postnatal stage, which were derived by the division and transformation of basal cells. On the other hand, the DNA synthesis of chondrocytes was the highest on embryonic day 18, and rapidly declined on postnatal day 3. The chondrocytes lost the ability of synthesizing DNA at 2 months after birth. The DNA syntheses of other cells (including fibroblasts, smooth muscle and glandular cells) were the highest on fetal day 18 and fell markedly on the third day after birth. They increased again at 1 week, then decreased progressively due to aging.

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The relationship between extracellular pH and intracellular calcium concentration ([Ca²⁺] ₁) in primary culture of neonatal rat astrocytes with Ca²⁺-indicator dye indo-1/acetoxymethyl ester and a confocal microscope was investigated. In 65 of 86 astrocytes studied, lowering extracellular pH by changing the perfusate solution from pH 7.4 to 6.4 was followed by increases in [Ca²⁺] ₁ in cytoplasm. The increases in [Ca²⁺] ₁ were seen on stimulation and/or during reperfusion. Of all the cells, 45.4% showed a transient pattern and 30.2% manifested a sustained pattern. The results suggest that a short interval but excessive acidic stimulation may be one of the signals that raises [Ca²⁺] ₁ in a certain population of cerebral astrocytes.

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