Eosinophil peroxidase (EPO) is one of the granule proteins in the eosinophil-specific granules. EPO is considered an important effector molecule in the host defense mechanisms against various parasite infections and also contributes to the pathophysiology of allergic diseases. However, the detailed function of EPO in these immune-mediated inflammatory responses has not been fully understood. EPO deficiency has been reported for the first time in humans. Recently, we have found that eosinophils of New Zealand White (NZW) mice lack EPO activity. EPO-deficient mice are advantageous in performing various experiments which cannot be performed upon human subjects. Here we present a brief review on the similarities and differences between human and murine EPO deficiency. NZW mice are useful not only as a murine model of human EPO deficiency, but also as a tool for elucidating the biological role of EPO in particular diseases such as helminth infections and allergies.

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We investigated immunohistochemically the distribution of γ-aminobutyric acid (GABA) and the calcium-binding protein parvalbumin (PV) in postnatal rat retina. At birth, GABA was found in the ganglion cell layer and in some cells in the nuclear layer that differentiate to amacrine cells and horizontal cells, while PV appeared in some cells in the outer portion of the nuclear layer that differentiates to horizontal cells. At P7, when the inner and outer nuclear layers were divided by outer plexiform layer, both GABA and PV were observed in some cells in the ganglion cell layer, amacrine cells, and horizontal cells. As the postnatal day advanced, GABA and PV were observed in some cells in the ganglion cell layer and amacrine cells. PV has been present in the horizontal cells after its appearance, however, the immunoreactivity of GABA was gradually weaker as the postnatal day advanced. Since GABA existed transiently in the horizontal cells in the early postnatal period, it seems that GABA may have some specific function for differentiation of horizontal cells. However, PV has existed in the horizontal cells since they differentiated in the retina, suggesting that PV may be one of the specific marker of horizontal cells.

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Cell growth of rat lens epithelium during galactose-induced cataract formation was examined in the present study using fluorescence cytophotometry or proliferating cell nuclear antigen (PCNA) immunohistochemistry. Six-week old male Sprague-Dawley rats were divided into 4 dietary groups according to galactose supplementation of the diet: 5%, 10%, 15% and 25% galactose diet groups. Whole-mount preparations of rat lens epithelium were prepared. The nuclear content of epithelial cells was measured using fluorescence cytophotometry, and cells undergoing DNA synthesis (PCNA labeled cells) were detected using the PCNA immunohistochemical staining method. The results of fluorescence cytophotometry showed that cells containing tetraploid nuclei (4C cells) gradually increased in number. In the 5% galactose diet fed rats, the number of 4C cells peaked on the 9th day after the start of dietary galactose supplementation, the 7th day in the 10% galactose diet fed rats and on the 5th day in the 15% and 25% galactose diet fed rats. On each day that 4C cells peaked, a large number of PCNA labeled cells were observed in the central zone of the epithelial cell layer. These results suggest that growth stimulation of lens epithelial cells depends upon the concentration of galactose in the diet and precedes changes in lens fiber.

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We developed a new immunohistochemical method to visualize Ki-67 antigen in formalin-fixed, paraffin-embedded sections of bone tumors. The method uses autoclave pretreatment of sections immersed in citrate buffer before incubation with the MIB 1 antibody, which reacts with the Ki-67 gene product. MIB 1 antibody showed strong and discrete staining of cell nuclei in proliferating tumor cells. This method also retrieved antigen in paraffin-embedded sections of alcohol-fixed tissue. Therefore, tissues previously considered unsuitable for immunohistochemical analysis can be studied after autoclave treatment.

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Cyclic nucleotide metabolizing enzymes were studied cytochemically to estimate the changes in these enzymes in the synovial membrane, because one of the functions of this tissue is estimated to clean up the debris from the synovial fluid to facilitate lubrication in the knee joints by filtering up small fragments of cartilage which have been formed as the result of wear and tear from the articular cartilage in osteoarthritis. Since the cyclic nucleotides are assumed to regulate the biological activity of phagocytosis, it may be reasonable to expect changes in this activity during the disability of osteoarthritis. In normal and osteoarthritic synovial membranes, a high adenylate cyclase (ACase) activity was clearly observed in the A cells at the surface of the synovial membrane, positive on the plasma membranes of the microvilli and on the ruffled borders as well as in pinocytotic vesicles underneath. The activity of guanylate cyclase (GCase) was observed to be rather weak; however, a strong 5′-nucleotidase (5′-N) activity was evidenced, indicating the existence of a regulatory function with the cyclic nucleotides in the phagocytotic and pinocytotic activities during osteoarthritis.

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To demonstrate the distribution of endothelin (ET) in ocular tissues, we immunohistochemically elucidated the localization of ETs (ET-1, ET-2, ET-3) and their precursors (Big ETs) using specific antibodies that can distinguish precursor and mature types of each isopeptide. ET-A receptor and ET-1 and ET-3 mRNAs were also examined using immunohistochemical and in situ hybridization methods, respectively. Coincidentally, the positive reactivity for ET-1 and ET-3 mRNAs, Big ET-1, Big ET-2, Big ET-3, mature ETs and ET-A receptor was found in almost the same element of the rat ocular tissues, e. g., the retinal neural cells (ganglion, amacrine, bipolar and horizontal cells), the vessel walls in the retina and choroid, the non-pigment epithelium in the ciliary body and iris, the corneal epithelium (anterior surface) and the endothelium (posterior surface), and the epithelium of the lens. These observations indicated that the various ocular tissues biosynthesize and secrete all the isopeptides of ET which act in an autocrine/paracrine fashion, regulating the neural transmission, intraocular pressure, iris smooth muscle tone and ocular blood vessel tone.

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The distribution of calcium ions in the rat brain synaptic terminals, in their synaptic vesicles, membranes and mitochondria; and in the sarcoplasmic reticula, mitochondria and their endosacs of skeletal or cardiac muscle cells, and in the caveolae of endothelial cells of blood capillaries was investigated with a two-step inorganic chemical precipitation method using potassium oxalate followed by potassium antimonate.
During the process, calcium ions were precipitated with potassium oxalate containing aldehyde fixative as an insoluble precipitate of calcium oxalate under simultaneous computerized microwave irradiation fixation. Then the precipitates were chemically changed to calcium antimonate during the postfixation procedure with an osmium tetroxide fixative containing potassium antimonate. The chemical natures and elemental binding ratios of this precipitate of calcium and antimony in the tube test and in sections were investigated by computerized electron probe X-ray microanalysis using an energy dispersive type X-ray detector (EDX) and electron energy-loss spectroscopy (EELS) imaging. The EDX analyzed chemical nature and calculated values of precipitates, in both atomic binding ratios (AT, %) and molecular weight ratios (WT, %), were exactly the same as those of the theoretical calculated values based upon the chemical formula of calcium antimonate. The data indicated that calcium ions exsisted in the site of precipitate.
Electron energy-loss spectroscopy imaging showed the distribution of digital net calcium images at electron microscopic resolution. Calcium exsisted in the nerve terminals, synaptic vesicles, mitochondria and synaptic membranes; in the skeletal and cardiac muscular endosacs of sarcoplasmic reticula, mitochondria, and in the endocytotic vesicles of cardiac sarcoplasmic membranes, or in the caveolae of blood vessel capillary endothelial cells.
High magnification O-loss images and EELS spectra confirmed differences in the fine precipitates in a section, as most of the precipitates were Ca-antimonate showing a clot of fine needle-like crystals, but in some cases we found antimonic acid showing an electron opaque foamy spot, or just contamination with an irregularly shaped homogeneous opacity. They were found mixed together, and it was not easy to differentiate them under EDX analysis.
Summarized data were reported in the 10th International Congress on Histochemistry and Cytochemistry, Kyoto, Japan, 1996 [24] and XXlth International Congress & International Academy of Pathology, and 12th World Congress on Academy of Enviromental Pathology, Budapest, Hungary, 1996 [26].

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The relation between radical production and acute morphological changes of palisade cells of Saintpaulia induced by a rapid drop in leaf temperature was investigated. The drastic and apoptotic changes were observed immediately after the chilling treatment for 3 sec. OH radical production occurred immediately after the chilling treatment and then increased linearly depending on the time. The radical production might initiate and develop acute irreversible morphological changes resulting in apoptotic cell death of Saintpaulia palisade cells.

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Twenty nucleic acid-specific fluorescent dyes were screened for use in nuclear staining in laser confocal microscopy. Eleven of them, BO-PRO-1, YO-PRO-1, SYBR Green I, PicoGreen, SYTOX Green, TO-PRO-1, POPO-3, propidium iodide, YO-PRO-3, TOTO-3, and TO-PRO-3 gave sufficient fluorescence by in-gel assay. They were also tested for the nuclear staining in cultured cells. When glycerol was used as a mounting medium, it enhanced the fluorescence intensity and retarded the bleaching in most of nucleic acid staining dyes. Among green fluorochromes excited at 488nm, YO-PRO-1, SYTOX Green, and SYBR Green I were suitable for nuclear DNA staining. YO-PRO-1 showed the strongest fluorescent intensity, but it weakly co-stained the cytoplasmic and nucleolar RNAs. SYTOX Green strongly and specifically stained the nuclear DNA. Among red fluorochromes excited at 568nm, propidium iodide stained nuclear DNA with simultaneous staining of RNA. YO-PRO-3 strongly and specifically stained DNA but bleached relatively rapidly. TO-PRO-3 excited at 647 nm was specific for DNA, but its fluorescence was bleached rapidly. Selection of nuclear DNA staining dyes should be done by considering the fluorescence intensity, DNA specificity, and bleaching characteristics.

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We demonstrated a double-labeled immunofluorescent cytochemistry under confocal laser scanning microscope, using antibodies against highly polysialylated isoform of neural cell adhesion molecule (NCAM-H), and Ca⁺⁺ binding astroglial marker in the brain, S100 protein (S100) in the rostral migratory stream (RMS) of the subependymal layer (SEL) of the adult guinea pig forebrain. In the adult olfactory system, NCAM-H is considered to be associated with the continuous generation and growth of granule cells. In the whole length of germinal SEL, varied populations of blast like, beaded, clustered NCAM-H positive elements were densely intermingled with typical astrocytic subpopulations of S100 protein. Some especialized phenotypes of S100 immunopositive ependymoglial tanycytes gave off their basal processes into NCAM-H immunopositive clusters in the rostral extention of subependymal zone (SEZ-re). Our results evinced the complex morphological multitude of neuro-glial (neuronal and neuroglial) coherence in the course of tangential migration of neuronal progenitors in the young adult guinea pig RMS.

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