Biomedical Research
Online ISSN : 1880-313X
Print ISSN : 0388-6107
ISSN-L : 0388-6107
Volume 5, Issue 2
Displaying 1-16 of 16 articles from this issue
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  • SHUNSUKE YAMAMOTO, YASUNORI HIGUCHI
    1984 Volume 5 Issue 2 Pages 91-100
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    Mouse inflammatory macrophages taken from casein-induced pleural exudates (the CANS series) and mineral oil-induced peritoneal exudates (the ONS series) of C57BL/6N (B6) mice (H-2b) were fused with BALB/c mouse (H-2d) derived myeloma cells. After selection of somatic cell hybrids, cloning and prolonged culture, two clones (CANS-196 and ONS-2C lines) strongly chemotactic to bacterial chemotactic factor and C5a were obtained. These lines also showed rosette forming abilities with sheep red blood cells (SRBC) coated with rabbit IgG anti-Forssman antibody (EA) and IgM followed by complement (EAC) and phagocytic activity, and produced lysozyme. By recloning of these clones, chemotactic subclones active for N-formylmethionyl-leucyl-phenylalanine benzyl ester (FMLP), zymosan-activated serum from AKR mice (ZAS(AKR)) and lymphocyte-derived chemotactic factor in addition to bacterial chemotactic factor and C5a, were successfully separated. Parental casein- or mineral oil-induced exudate macrophages responded to all these agents weakly, but significantly. CANS-196 and ONS-2C lines in the early period after cell fusion (8 weeks) displayed no chemotactic activity. They had high chromosome numbers (78 and 76 chromosomes, respectively) and both H-2b and H-2d antigens on their surface, confirming that these lines were hybridized cells. However, when these lines became chemotactic (28 and 20 weeks after cell fusion, respectively) they had low chromosome numbers (42-43 chromosomes) and lost the H-2b antigen, although expression of the H-2d antigen remained unchanged. At the same time, these lines newly expressed certain aged hybridoma specific cell surface antigens which were detectable by BALB/c anti-aged CANS-196 and ONS-2C cell alloantisera. Reasons for the necessity of prolonged culture for the establishment of stable macrophage-like chemotactic hybridomas, and changes in the expression of cell surface antigens are discussed in the context of chromosome loss.

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  • SHINGO HARA, MASAYUKI MIYOSHI
    1984 Volume 5 Issue 2 Pages 101-110
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    Visual cells in the squirrel (Tamius sibiricus) retina were studied by scanning and transmission electron microscopy to examine cell arrangement and occurrence of attachment structures between the visual cells. The squirrel retina, which was previously held to be a cone type, contained two types of visual cells: rods and cones. The two types were morphologically similar to their counterparts in other squirrel species. The two types of the visual cells occurred in a characteristic arrangement as a unit. The rods were sporadically distributed and each of them was surrounded by four to six cones. Gap junctions occurred at the level of the synaptic terminals of the visual cells. The gap junctions were formed between rods and cones, and between cones. However, no junctions were seen between rods. The rods were in contact with all the surrounding cones by gap junctions. The cones were in contact with one or two surrounding visual cells. Occurrence of the gap junctions between the visual cells was closely correlated with the unit-like arrangement of the visual cells. These findings suggest that a rod and its surrounding cones constitute a structural unit.

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  • KAZUO TAIRA, SUSUMU SHIBASAKI
    1984 Volume 5 Issue 2 Pages 111-116
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    Close apposition of mitochondria to the plasma membrane in the rat interscapular brown fat cells was demonstrated by freeze-fracture and thin-section electron microscopy. In thin sections, some mitochondria were seen in close proximity to the plasma membrane, separated only by a thin layer of cytoplasm. Caveolae, endo- or exo-cytotic invaginations of plasma membrane, were not seen in such a region of the plasma membrane. In freeze-fracture replicas, the subplasmalemmal mitochondria formed caveola-free hillocks on the P face, and dimples on the E face of the plasma membrane. These caveola-free domains of the cell surface were preferentially located in the vicinity of the blood capillary, and bulged slightly into the pericapillary space. Apposition of mitochondria to the plasma membrane was consistently observed in the brown fat cells at various stages of postnatal development.

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  • KAZUO USHIJIMA, MITSUHIRO KAWATA, TADAO MATSUURA, YOSHIAKI NOJYO, YUTA ...
    1984 Volume 5 Issue 2 Pages 117-124
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    A peroxidase-antiperoxidase (PAP) immunohistochemical procedure using a highly specific anti-caerulein antiserum was employed to investigate the existence and uptake of caerulein in the rat brain. In control rats and in rats that received intramuscular caerulein, no staining was observed. When caerulein was injected into the lateral ventricle, immunoreactive neurons and fibers were observed within 5 min in the cerebral cortex, hippocampus, dentate gyrus, septal area, periventricular regions related to the neuroendocrine system, Purkinje cells, nucleus vestibularis, corpus callosum and fornix. The intensity of immunoreactivity reached its maximum after 15 min; immunoreactivity disappeared within 30 min after the injection. The results indicate that caerulein does not exist physiologically in the rat brain, and that caerulein does not pass the blood-brain barrier. When caerulein is administered intraventricularly, there are some neurons and neuronal fibers which selectively take up caerulein and metabolize it rapidly.

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  • MASANAO KATAGIRI, HIROMASA TOJO, KIHACHIRO HORIIKE, NAOKI KASUGA, KENI ...
    1984 Volume 5 Issue 2 Pages 125-134
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    We have developed an enzyme immunoassay (EIA) system (‘sandwich method’) for a flavoenzyme, D-amino acid oxidase (DAO), employing polystyrene beads as a solid-phase, and Fab’-horseradish peroxidase conjugate. This assay system is accurate and precise in the concentration range of 1.0—100 ng/ml, and up to 26 h at 4°C. At 37°C, the measurable DAO concentration range is considerably decreased. DAO and quasi D-amino acid oxidase (Quasi-DAO) completely crossreacted with the anti-DAO antibody both in the double immunodiffusion test, and in the assay with the EIA system. The system is therefore useful for measuring the total content of DAO and Quasi-DAO. The DAO content in crude samples such as the kidney homogenate and serum can be accurately measured. In hog serum, DAO was not detectable; in hog kidney cortex extract, DAO was estimated to be 2.2 ± 0.6 μg/mg protein. DAO was irreversibly polymerized on iodination by the chloramine T method, and the nonspecific counts were very high, which caused our inability to use a radioimmunoassay in the determination of DAO.

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  • SHOHEI MAEKAWA, KOICHI ISHIGURO, HIROMU MUROFUSHI, HIKOICHI SAKAI
    1984 Volume 5 Issue 2 Pages 135-140
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    Starfish egg fascin was purified by the method previously established for purifying sperm fascin. Both sperm and egg fascins are composed of three isoforms separated by isoelectrophoresis with pI values of 6.0 (α), 6.12 (β), and 6.25 (y). Some differences were detected in the ratios of the amount of the isoforms between sperm and egg fascins. α-Isoform had less affinity for actin filaments than β- or y-isoform.

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  • NOBUKO FUKAMACHI, YASUO WATANABE, BONRO KOBAYASHI
    1984 Volume 5 Issue 2 Pages 141-148
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    Phosphatidylcholine metabolism in blood platelets from the reserpinized rabbit was compared with that in the control platelets. A slight but significant increase in phosphatidylcholine content was observed in platelets from reserpinized rabbits. Incorporation of radioactivity into phosphatidylcholine from [3H]choline, [3H]methionine or [14C]glycerol was significantly stimulated in the reserpinized cells. Thrombin induced a rapid and significant decrease in phosphatidylcholine content and an increase in lysophosphatidylcholine only in the reserpinized platelets, although thrombin rapidly decreased phosphatidylinositol content in both reserpinized and control platelets. These results suggest that the reserpinized platelets accumulate phosphatidylcholine intracellularly through the stimulation of the incorporation of choline moiety, the methylation of phosphatidylethanolamine, and the de nova synthesis from glycerol, and that phosphatidylcholine in the reserpinized platelets is rapidly degraded during thrombin-induced activation. It is possible that the enhancement of thrombin-induced release reaction of reserpinized platelets is associated with the active turnover of phosphatidylcholine.

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  • JUN-ICHI KISHI, YOKO HASHIMOTO, HISASHI AOYAMA, YOHEI IZAWA, TARO HAYA ...
    1984 Volume 5 Issue 2 Pages 149-156
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    A fairly high collagenase activity was extracted directly from human post-burn granulation tissues with isotonic sucrose and 4 M urea solutions. Activity of two other enzymes involved in collagen metabolism, i.e., elastase and gelatinase, was also detected in both sucrose and urea extracts along with the collagenase activity. A considerable amount of hydroxyproline, which corresponded to about 10% of the total collagen in the granulation tissues, was found in an isotonic sucrose extract, implying the in vivo degradation of tissue collagen. The collagenase broke down type I collagen preferentially in comparison to type III. A significant correlation (r=0.66; P<0.001) was observed between total collagenase activity and the ratio of type III to type I collagen in post-burn wound tissues. Together with the ratio of type III to type I collagen, activity of all three enzyme in post-burn granulation tissues were decreased significantly 2 weeks after autografting. These results suggest that the preferential degradation of type I collagen by collagenase might be the major event underlying the elevation of the ratio of type III to type I collagen in granulation tissues.

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  • TOSHIAKI SANO, TAKANORI HIROSE, KAZUO HIZAWA
    1984 Volume 5 Issue 2 Pages 157-164
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    Appendiceal mucosa and carcinoid tumors were examined immunohistochemically by the immunoperoxidase method for S-100 protein. Light microscopic examination of serial sections showed sparse extraepithelial subglandular argyrophilic cells surrounded by S-100 protein-positive cells within the lamina propria of normal or slightly inflamed appendices. These pairs of cells closely resembled the ultrastructurally defined enterochromafiin cell-nerve fiber (ECC-NF) complexes in their location and topographical relationship, as well as in their cytological and biochemical properties. All four carcinoid tumors examined contained stellate cells giving a strong reaction for S-100 protein. In three tumors, these immunoreactive cells with long cytoplasmic processes were observed at the margin of tumor nests. They often enveloped smaller tumor nests. Conventional electron microscopy showed that the tumor nests were composed of neurosecretory cells containing enterochromaffin granules and polyaxonal unmyelinated Schwann cells located at the periphery of the nests. Electron immunoperoxidase staining for S-100 protein revealed that these immunoreactive cells were identifiable as Schwann cells. These findings show an intimate relation between appendiceal carcinoid tumors and nerve fibers, and suggest that these tumors develop from ECC-NF complexes.

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  • KOHJI FUKUNAGA, HIDEYUKI YAMAMOTO, ETSUTO TANAKA, EISHICHI MIYAMOTO
    1984 Volume 5 Issue 2 Pages 165-176
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    A Ca2+-calmodulin-dependent protein kinase has been solubilized by Triton X-100 from the particulate fraction of rat brain and purified about 350-fold to apparent homogeneity. The purified enzyme was similar to the cytosol enzyme previously reported (15) with respect to physicochemical and kinetic properties. The titration of the enzyme activity with calmodulin showed that 14 moles of calmodulin are required for full activation of 1 mole of the enzyme. Various endogenous substrate proteins in nuclear, synaptic membrane, and synaptic vesicle fractions were phosphorylated in a Ca2+-calmodulin-dependent manner. The endogenous phosphorylation of proteins in the particulate fraction reached the maximal level within 15 sec and then decreased gradually for 10 min. The addition of the purified enzyme resulted in the phosphorylation with a pattern similar to that of the endogenous phosphorylation.

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  • SUGURU OHSAWA, HISAE HORI, RYU-ICHIRO HATA, YUTAKA NAGAI
    1984 Volume 5 Issue 2 Pages 177-186
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    In order to elucidate the pathogenesis of cartilage destruction in joint diseases, the effect of polymorphonuclear leukocytes (PMNs) on the collagen metabolism in chondrocyte culture was investigated. For this purpose, a simple method for the purification of PMNs from peritoneal exudate cells was developed. The method is based on discontinuous density gradient centrifugation in a Ficoll-Urografin system. Extracts of peritoneal PMNs significantly induced chondrocyte collagenase production, change of the cells to fibroblastic form, and the loss of metachromasia (toluidine blue staining). These effects of PMN extract on the chondrocyte culture were reversible; cells of the fibroblastic form were restored to the original polygonal form by replacing the medium with fresh medium containing no PMN extract, and collagenase production ceased. PMNs from peripheral blood did not show any of this activity, suggesting that PMNs acquire the factor(s) during and/or following infiltration from the blood stream into inflamed tissues. The estimated molecular weight of the factor(s) inducing morphological change and collagenase production of chondrocytes was approximately 15,000—20,000, based on gel filtration. The same effluent fraction induced both collagenase production and morphological change of synovial cells. PMN extract suppressed collagen production in chondrocytes to about 60 % of the control, but not production of non-collagenous proteins. The results suggest that PMNs are involved in pathological destruction of articular cartilage by shifting the balance of collagen metabolism in chondrocytes to the catabolic phase.

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  • SATORU KANEKO, SHIGERU OSHIO, TOSHIFUMI KOBAYASHI, HIDEO MOHRI, RIHACH ...
    1984 Volume 5 Issue 2 Pages 187-194
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    Examinations of sperm sedimentation in Percoll density gradients suggested that the sedimentation velocity of X-bearing sperm was faster than that of Y-bearing sperm, despite the fact that the apparent densities of the two types of cells obtained by equilibrium sedimentation were similar. From this, it seems difficult to obtain both types of cells simultaneously by only one procedure. Assuming that the ease with which the cells pass through the density interfaces influences their sedimentation velocity, a discontinuous density gradient with 12 steps (25 to 80% Percoll) was adopted for the selective isolation of X-bearing sperm. Among the conditions examined, centrifugation at 250 g for 30 min gave the optimum result: 23.3 ± 6.3 % of sperm was recovered in the sediment, of which the percentage of Y-bearing sperm was only 6.4 ± 1.8 %; thus the purity of X-bearing sperm was approximately 94 %. Almost all the sperm in the sediment were active and forward motile.

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  • MOTOAKI OHASHI, YOSHIAKI NONOMURA
    1984 Volume 5 Issue 2 Pages 195-210
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    Structural changes during K+-induced isometric contraction of guinea pig taenia coli were investigated under the electron microscope. A marked change was observed in the myofilaments of the smooth muscle cells after the tissue was contracted for more than 30 min. Almost all thick filaments disappeared and thin filaments came to be located around the submembraneous area. Thin filaments became uniformly distributed after the tissue was relaxed by the addition of diltiazem, a calcium antagonist, during prolonged exposure to high K+ solution; however, no thick filaments were restored. When the tissue was incubated in a hypotonic solution, thick filaments disappeared and thin filaments became uniformly distributed. Under the hypotonic condition, carbachol produced strong contraction with the same features as prolonged K+-induced contraction. However, when the solution was hypertonic, thick filaments remained intact and moved to the central part of the cell after prolonged K+-induced contraction. Therefore, two independent phenomena proceed during K+-induced contraction in the smooth muscle cell. One is the disappearance of thick filaments, which is produced by the influx of water into the cell and by the decrease of free magnesium concentration. The other is the rearrangement of the filaments, which occurs during the prolonged isometric contraction. These findings indicate that the smooth muscle contracts even without observable thick filaments.

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  • JUNKO NISHIITSUTSUJI-UWO, MAKIO TAKEDA, HIROYUKI SAITO
    1984 Volume 5 Issue 2 Pages 211-224
    Published: April 01, 1984
    Released on J-STAGE: August 18, 2017
    JOURNAL FREE ACCESS

    A highly potent antiserotonin antiserum was prepared by injecting formaldehydecoupled serotonin-bovine serum albumin conjugate to the rabbit, together with the Freund’s complete adjuvant. Mouse intestinal enterochromaffin cells were stained with this antiserum at the dilution up to 1 : 50,000. The antiserum proved specific against serotonin in situ, though cross-reactivity with related compounds particularly with dopamine was seen in vitro. With this antiserum, serotonin-like immunoreactivity in the cockroach brain-midgut endocrine system was seen in: one large and eight to ten small cells bilaterally located in the pars intercerebralis, two cell groups ventral to the calyx, three pairs of a single cell in the suboesophageal ganglion, one cell in the thoracic ganglia, and one cell group in the terminal ganglion. These neurons extend many fibers widely distributed in the central nervous system, especially in the protocerebral bridge, central body and neuropiles of the deutocerebrum. The frontal ganglion, and the nerves innervating the longitudinal muscle fibers of the midgut showed serotonin-like reactivity, but this was not specific. Some endocrine cells in the midgut showed immunoreactivity to serotonin, but its specificity could not be determined because of an extremely small population of these cells.

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