The human thymuses, the well differentiated carcinoma, the thymoma with spindle epithelial cells and the undifferentiated thymoma have been examined by histochemical techniques [lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), acid phosphatase (AcP), alkaline phosphatase (ALP) and dihydrofolate dehydrogenase (DHFR)] to study the metabolic changes due to the pathological conditions in the epithelial cells (EPCs) and accessory cells. In all three different types of studied thymomas the EPCs showed a more intense LDH and SDH positivity than in normal thymus. These data indicate a possible damage of respiratory chain energy-coupling mechanism. The EPCs presented a different pattern (negative or very strong) of ALP and AcP positivity both in normal and pathological thymuses. These results suggest that the EPCs are a heterogenous population presenting a variety of functional activities both in normal and pathological conditions. The macrophages showed a different pattern (very weak, weak and moderate) in dehydrogenase and in hydrolytic enzymes both in normal and tumour cells. The macrophages were uniformly distributed throughout the thymoma but it is interesting to note that in the well differentiated thymomas these cells were localized in high number only around the neoplastic nodules that were proliferating. These findings suggest that the macrophages are important for defending the organism against the formation of new neoplastic nodules.

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Peritoneal mast cells and eosinophils were incubated with cationized ferritin (CF) an electron dense marker that binds to the plasma membrane. In both cell types, CF was internalized from the plasma membrane in tubular invaginations. Vesicles of various shapes and sizes containing CF were found in the cytoplasm. Trimetaphosphatase (TMPase) positive lysosomes were found near the nucleus and at the cell periphery. In mast cells, the secretory granules were not reactive for TMPase, while in the eosinophils an occasional specific granule contained reaction product. Vesicles containing CF were observed close to the TMPase positive lysosomes and some of these vesicles seemed to fuse with TMPase positive lysosomes. Vesicles containing both CF and TMPase reaction product were also observed. The presence of both CF and positive TMPase reaction in the same granule suggests that they are secondary lysosomes responsible for degradation of endocytosed material. The CF also induced a partial degranulation of the secretory granules and CF was seen associated with the granule matrix.

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We describe the clear resolution of the surface structure and nerve innervation of the end organs of the inner ear using confocal laser scanning microscopy (CLSM). The surface structure of the inner ear organs that were stained by phalloidin-FITC could be clearly observed by CLSM. The nerve endings of the hair cells, stained by a neurofilament marker, could also be detected by CLSM. Type I and II hair cells of the vestibular end organs could be distinguished by observation of the morphology of the nerve endings. CLSM is a very useful approach for the study of the inner ear organs.

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The localization of actin sequestering peptide thymosin β10 in rat testis was analyzed by light and electron microscopic immunohistochemistry using the anti-thymosin β10 antibody. A positive reaction of thymosin β10 was detected in a closely associated portion within the developing spermatid head. In step 15 spermatids, localized deep in the seminiferous epithelium, a prominent positive reaction was detected in the Sertoli cell cytoplasm adjacent to the spermatids. In step 19 spermatids, just before spermiation, thymosin β10 was highly concentrated in the Sertoli cell cytoplasm situated at the concave side of the sickle shaped spermatid head, where the tubulobulbar complex is formed. According to previous ultrastructural observations, actin bundles of ectoplasmic specialization change to electron dense material just prior to spermiation. The localization of thymosin β10 suggests that large amounts of actin monomers accumulate in the region of the tubulobulbar complex just prior to spermiation. These findings suggest that thymosin β10 accumulation is correlated with fragmentation of the ectoplasmic specialization, and strongly support the hypothesis that the tubulobulbar complex is an actin-disorganizing organelle of the Sertoli cell.

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We have previously established a mouse coculture system of osteoblastic cells and spleen cells for examining osteoclast differentiation. In the present study, we examined the morphological features of the cell-cell contact between mouse spleen cells and osteoblastic cells in the co-cultures. Light microscopic investigations revealed that tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated spleen cells appeared in the vicinity of alkaline phosphatase (ALP)-positive osteoblastic cells. Ultrastructurally, spleen cells extended long cytoplasmic filopodia in all directions, by which spleen cells touched adjacent osteoblastic cells. The adjacent osteoblastic cells and spleen cells adhered to each other by forming electron-dense cytoplasmic materials on their inner leaflets of plasma membranes at cell-cell contact sites. Some adjacent osteoblastic and spleen cells made contact on the plasma membranes, forming an extracellular microenvironment. Coated pits were also formed on the plasma membranes facing this microenvironment. These morphological features of the cell-cell contact between osteoblastic cells and spleen cells indicate that there is internalization of organic components, i.e., receptor-mediated endocytosis in the contact sites between the two types of cells.

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Distribution and density of calcitonin gene-related peptide (CGRP), protein gene product (PGP) 9.5, serotonin and chromogranin A immunoreactivity were examined in developing rat respiratory tracts from embryonic day (ED) 15 to postnatal day (PD) 60. On ED 15, the epithelium of the primitive bronchi stained positively for CGRP, PGP 9.5, serotonin and chromogranin A. CGRP, PGP 9.5, serotonin and chromogranin A positive endocrine cells extended distally along the developing bronchial trees. Cell number reached a maximum on ED 19 or ED 17. On ED 21, the clusters of endocrine cells, identified as neuroepithelial bodies (NEBs) stained positively for either CGRP or PGP 9.5 in the bronchioles, and these were observed during all stages examined. CGRP or PGP 9.5 positive nerve fibers appeared during the Perinatal periods and increased in number after birth. However, neither serotonin nor chromogranin A immunoreactivity was observed in NEBs or nerve fibers in the airway at any of the examined developmental stages.

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In order to elucidate the causes of ossification in spinal ligaments, ultrastructural alteration and immunolocalization of transforming growth factor-β (TGF-β), as well as that of the TGF-β receptor were examined during the chondrification of ligamentous fibroblasts, by inducing ossification in ligamenta flava of murine lumbar spines, employing recombinant human bone morphogenetic protein-2 (rhBMP-2). Normal ligamenta flava, consisting of flattened fibroblasts, showed no immunolocalization of TGF-β isoforms. From the second week following BMP-administration, cartilage gradually replaced the ligaments. Cells in the central portion of the ligament contained abundant Golgi apparatus as well as enlarged endoplasmic reticula. Proliferative or hypertrophic chondrocytes in this area showed immunoreactivity to TGF-β₃ latency-associated peptide, while calcified hypertrophic chondrocytes accompanied by both matrix vesicle- and collagen-calcification were noticed at the insertion site to spinal lamina, where there was no immunolocalization of this peptide. In addition, the TGF-β type I receptor was localized on the proliferative and hypertrophic cells of the central portion. Therefore, as regards the pathological chondrification of ligamentous fibroblasts induced by exogenous BMP-2, TGF-β is speculated to promote matrix-formation and chondrocytic maturation, under the autocrine/paracrine system, in concert with exogenous BMP-2.

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The hepatic lobular localization of alcohol dehydrogenase (ADH) activity was examined histochemically in mice, rats, hamsters and guinea-pigs with the nitro blue tetrazolium method. In the mouse liver, the ADH activity was localized mainly in the centrilobular zone. In the rat and hamster livers, it was observed throughout the lobule, with increased centrilobular staining. Interestingly, in the guinea-pig liver, it was found to be evenly localized in both the centrilobular and periportal zones, with little or no midlobular staining. These findings show that the distribution pattern of ADH activity in the hepatic lobule of the guinea-pigs differs strikingly from that of the mice, rats and hamsters.

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We developed a novel auto-micromanipulation system for microinjection of a low molecular weight fluorescent probe into a target cell to analyze gap junctional intercellular communication in a three-dimensional perspective in combination with vital staining of cell membranes. This computerized system consisted of an ultrasonic linear motor-driven manipulator (OMMS) which could be precisely controlled along 4 axes and a confocal laser scanning microscope (CLSM) having excitation waves of 488nm and 515nm plus a specimen stage equipped with a piezotranslator that could be accurately leveled up or down. After vital staining with PKH26, a target cell in cultured neonatal rat cardiac myocytes overlapping each other was microinjected with Lucifer Yellow or fluo-3 to evaluate whether this system was practical. The results revealed that the three dimensional coordinates of the target cell were recognizable after vital staining and the fluorescent probe microinjected into the target cell with this system portrayed reliable distribution. This system is therefore useful for the analysis of intercellular communication in three-dimensional cells and organ structures.

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We cultured 3-day-old mouse livers for organotypic slice culture using a roller-tube technique to maintain parenchymal cells for a long term. The sliced tissues in medium without growth factors gradually spread and flattened for 3 weeks. Parenchymal cell formed a trabecular structure and albumin-immunoreactivity was seen in them in light and ultrastructural immunoperoxidase studies. Cholangiolar cells formed glandular structures which had microvilli on the inner facing membrane and basement membrane on the basal area. There were also well-developed tight junctions and few cytoplasmic organella. These findings suggest that our organotypic slice culture of neonatal livers developed with the roller-tube technique could preserve hepatocytes and cholangiolar cells for at least 3 weeks. This culture system may be a useful tool not only to maintain parenchymal cells but also to study structural organization of the liver in ontogenesis.

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For a better performance of nonradioactive in situ hybridization for specific RNA sequences, signal amplification is sometimes required especially in the use of clinical specimens processed under suboptimal conditions and in the analysis of gene expression with very low reiteration. In the present study, we examined the usefulness of catalyzed signal amplification (CSA) or catalyzed reporter deposition system with biotinylated tyramine to amplify colorimetric in situ hybridization signals. Our CSA protocol included the use of biotinylated tyramine after the reaction of thymine-thymine (T-T) dimerized DNA with horseradish peroxidase (HRP) labeled anti-(T-T dimer) antibody, followed by the reaction with HRP-labeled streptavidin. When thymine-thymine dimerized λ phage DNA was spotted onto a nitrocellulose filter at 1pg-10ng and then detected by the direct immunoperoxidase method, the indirect immunoperoxidase method or the CSA method, the CSA method gave the highest sensitivity with about 100-fold increase compared to that of the direct method. Next, in the frozen sections of rat brain, which was fixed with 4% paraformaldehyde, 28S rRNA staining by in situ hybridization with only 10ng/ml of T-T dimerized oligonucleotide probe complementary to rat 28S rRNA was compared among the direct method, the HRP-labeled avidinbiotin complex method and the CSA method. Again, intense 28S rRNA signal was obtained only with the CSA method. Therefore, we confirmed the usefulness of the catalyzed deposition system with biotinylated tyramine in nonradioactive colorimetric in situ hybridization for specific RNA sequences in tissue sections.

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