Biomedical subjects

K Ishimura

Publications and source records attributed to K Ishimura.

At least 73 records · Page 4Linked to original sources

PubMed1994

Aluminium beta-cyclodextrin sulphate as a stabilizer and sustained-release carrier for basic fibroblast growth factor.

The water-insoluble aluminium salt of beta-cyclodextrin sulphate (Al.beta-CyD-Sul) was used as a stabilizer and sustained-release carrier for recombinant human basic fibroblast growth factor (bFGF). An adsorbate of bFGF with Al.beta-CyD-Sul was prepared by incubating the protein with a suspension of Al.beta-CyD-Sul in water. The mitogenic activity of bFGF released from the adsorbate, as indicated by the proliferation of kidney cells of baby hamster (BHK-21), was almost comparable with that of the intact bFGF. Al.beta-CyD-Sul significantly protected bFGF from proteolytic degradation by pepsin and alpha-chymotrypsin, compared with the water-soluble sodium salt. The in-vitro release of bFGF from the adsorbate was sustained in proportion to a rise in the ratio of Al.beta-CyD-Sul to the protein in the adsorbate. Of the bFGF preparations evaluated, the adsorbate of bFGF with Al.beta-CyD-Sul, when given subcutaneously to the rat, showed the most prominent increase in the formation of granulation tissues, due to the stabilization and slow-release of the mitogen. The limited data presented here suggest that the adsorbate of bFGF with Al.beta-CyD-Sul has a potent therapeutic efficacy for wound healing, and may be applicable to oral protein formulations for the treatment of intestinal mucosal erosions.

Animals↗

PubMed1994

Biochemical and histochemical studies of the effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in mouse brain.

The effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in the mouse brain were studied, and we found that diaphorase activity in the post-mitochondrial fraction of brain homogenate was enhanced by idebenone in a concentration-dependent manner. Histochemical studies also indicated that diaphorase staining was intensified by idebenone at the same concentration. These results suggest that idebenone may stimulate the production of nitric oxide, probably through its direct action on nitric oxide synthase, thus producing its protective action on neurological disorders due to cerebral hypoxia or ischemia as a consequence of dilating the cerebral blood vessels.

Amino Acid Oxidoreductases↗

PubMed1994

Effects of flavonoid compounds on the activity of NADPH diaphorase prepared from the mouse brain.

The effects of flavonoids on NADPH diaphorase activity were studied in vitro, and we found that the enzyme activity was markedly inhibited by quercetin. This inhibitory action was shown to be accompanied by an increase in the apparent Km value of the enzyme for the cofactor NADPH, with a decrease in the Vmax, and an increase in the apparent Km for the substrate nitro blue tetrazolium, without any significant change in the Vmax. These results indicate that quercetin may directly inhibit NADPH diaphorase, thus suggesting the possibility that this compound may be able to inhibit the production of nitric oxide in the brain.

Animals↗

PubMed1994

[Distribution of NADPH diaphorase-positive nerves in human penile tissue].

Recently, nitric oxide (NO) has been thought to be a neuronal messenger to evoke penile erection. NO synthase (NOS)-containing nerve fibers were identified and localized in human penile tissue, but detail distribution of NOS-containing nerve fibers in the human penis was unclear. In the present study we examined their distribution using histochemical staining of NADPH diaphorase (ND), which is a specific marker of neuronal NOS. In the crura penis some various sizes of ND-positive nerve bundles were observed in the cavernous spaces. In the penile shaft large bundles (> 50 microns) decreased in number and were observed only near deep artery. There were abundant ND-positive nerve terminals with fine varicosity innervating both corpus cavernous smooth muscles and deep, dorsal and helicine arteries. In the wall of deep dorsal vein there were also many groups of ND-positive fibers. Endothelium of deep artery and its large branches was clearly ND-positive, but parts of endothelium of deep dorsal vein or corpus cavernous sinus were only faintly stained. Some of the dorsal penile nerve fibers were ND-positive. In corpus spongiosum many ND-positive nerve fibers were observed and urethelium was clearly ND-positive. In conclusion, NO may have an important role in the function of both corpus cavernosum and corpus spongiosum because NOS-containing nerve fibers were widely distributed in both cavernous tissues.

Amino Acid Oxidoreductases↗

PubMed1993

Selective on-line enrichment and separation of peptides having aromatic amino acids at their C-termini by column-switching high-performance liquid chromatography using an anhydrochymotrypsin-immobilized precolumn.

A column switching high-performance liquid chromatographic system in which peptides retained on an anhydrochymotrypsin (AHC)-immobilized diol-silica precolumn were selectively transferred to and separated on a reversed-phase analytical column was developed. An investigation of the affinity characteristics of 40 peptides to the AHC precolumn showed that among eleven peptides having Tyr or Phe at their C-termini and more than five amino acid residues, ten were retained almost quantitatively on the precolumn, and two peptides having Trp at their C-termini were less retained. Two peptides having C-terminal PheNH2 were also retained, but the peptide having C-terminal D-PheNH2 was not retained. Among eighteen peptides having no aromatic amino acids at their C-termini, two were retained, one slightly and the other moderately. Calibration graphs for rat atrial natriuretic peptide constructed at various sample sizes were nearly identical, indicating that the peptide could be enriched by this system. The AHC precolumn showed no loss of analytical performance over 1 year, during which about 450 samples were analysed.

Amino Acid Sequence↗

PubMed1993

Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes.

Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, approximately 80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was approximately 70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G0 phase, but that when they formed monolayers, they progressed to the G1 phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions.

Animals↗

PubMed1993

Immunocytochemical localization of prostaglandin endoperoxide synthase in the bovine intestine.

The localization of prostaglandin (PG) endoperoxide synthase in bovine intestine was examined immunocytochemically with polyclonal antibody raised against PG endoperoxide synthase purified from bovine seminal glands. The most intense positive staining reaction for the enzyme was present in mast cells. Mast cells were found to be widely distributed in the intestinal wall, and were particularly numerous in the lamina propria. Most of the mast cells in the lamina propria of the intestinal villi were elongated and oriented with their long axis parallel to the plane of the absorptive epithelium. In whole mount preparations of jejunal villi, mast cells were seen to form a two-dimensional network in the lamina propria. In addition to mast cells, smooth muscle cells of the inner circular muscle layer and muscularis mucosae, nerve cells and fibers, endothelial cells of arterioles, and serosal epithelial cells also showed faint to moderate staining for the enzyme. These results suggested that mast cells are the major source of PGs in the bovine intestinal wall. The characteristic arrangement of mast cells in the intestinal villi may be related to their functions in this portion of the bovine intestine.

Animals↗

PubMed1993

Electron microscopic immunocytochemistry on the colocalization of ATP synthase and cytochrome P-450 of side chain cleavage enzymes in mitochondria of rat and bovine adrenal cortical cells.

To clarify whether ATP synthase and side chain cleavage enzymes (SCC) can coexist in a mitochondrion of the adrenal cortical cell, and whether the functional heterogeneity of mitochondria can occur in an adrenal cortical cell, the immunostainability of these two enzymes in mitochondria was compared by electron microscope using pre- and post-embedding methods. In the pre-embedding method, the inner membrane of most mitochondria in the adrenal cortical cell was positively stained for SCC, 11 beta-hydroxylase, and ATP synthase, but among these immunopositive mitochondria we often observed negatively immunoreacted ones in the same adrenal cortical cell. In the positively stained mitochondria, immunoreaction products were evenly localized along the inner mitochondrial membrane. We therefore think that these differences in the immunostainability are caused by technical artifacts, namely the inadequate penetration of the antibody. In the post-embedding immunocytochemistry, gold particles showing the presence of the enzymes were observed on all mitochondria. Two different antibodies, anti-ATP synthase antibody and anti-cytochrome P-450scc antibody, which were labelled with gold particles of varying diameter (5 nm, 10 nm each) could be observed on all the mitochondria of the bovine adrenal cortical cells. No heterogeneities as to the stainability for both ATP synthase and SCC were detected in mitochondria of the rat and bovine adrenal cortical cells. These results indicate that in the rat and bovine adrenal cortical cells, both ATP synthase and SCC are evenly and simultaneously present on the inner membrane of all mitochondria. Hence, mitochondria in the adrenal cortical cell seem to be homogeneous in their steroid and ATP synthesizing abilities.

Adrenal Cortex↗

PubMed1992

Kinetic studies on androstenedione production in ovarian microsomes from immature rats.

Androstenedione formation from progesterone by P-450(17 alpha,lyase) was investigated in ovarian microsomes of immature rats treated with pregnant mare serum gonadotropin. Successive monooxygenase reactions in the formation of androstenedione without the intermediate leaving P-450(17 alpha,lyase) were demonstrated by a double-substrate double-label experiment using [14C]progesterone and 17 alpha-[3H]hydroxyprogesterone as substrates and also by specific reduction in the concentration of intermediate 17 alpha-hydroxyprogesterone in the reaction medium by reaction of liposomal P-450C21. A detailed kinetic study on the reactions of P-450(17 alpha,lyase) in microsomes was conducted in the steady state. Kinetic parameters indicated the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone (Km = 80 nM) to be strongly inhibited by progesterone (Ki = 8 nM). In the presence of a high concentration of progesterone, as in the case of in vivo rat ovary, most androstenedione is concluded to be formed directly from progesterone by successive monooxygenase reactions catalyzed by P-450(17 alpha,lyase). 20 alpha-Dihydroprogesterone competitively inhibited the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone with Ki = 23 nM, but had only slight effect on progesterone metabolism to androstenedione. 20 alpha-Dihydroprogesterone, thus, cannot be a regulator for androstenedione formation in rat ovary.

Androstenedione↗

PubMed1992

Increased 5-lipoxygenase activity in massive hepatic cell necrosis in the rat correlates with neutrophil infiltration.

Rats were treated with heat-killed Propionibacterium acnes and subsequent injection of a small amount of lipopolysaccharide after 7 days. After 24 hr most of the rats died of massive liver cell necrosis. Nonparenchymal liver cells were isolated from this liver injury model and incubated with arachidonic acid. Reverse-phase high-pressure liquid chromatography detected the 5-lipoxygenase metabolites (leukotriene B4 and 5-hydroxy-arachidonic acid), whereas these compounds were produced in negligible amounts when the rats were treated with P. acnes only. Immunohistochemical studies with 5-lipoxygenase antiserum revealed that the injured livers contained a large number of positively stained round cells with segmented nuclei, which were rarely found in the livers treated with P. acnes only. These positively stained cells were histologically identified as neutrophils. The results suggested that the increased 5-lipoxygenase activity in the injured rat liver is attributable to the infiltrating neutrophils rather than to nonparenchymal hepatic cells.

Animals↗

PubMed1992

Effects of neonatal asphyxia on the serotonin neuron system in the developing brain studied by immunohistochemistry.

The effects of neonatal asphyxia on the serotonin neuron system were examined using the immunoperoxidase method. Male mice, 2 days of age, were exposed to total asphyxia (100% CO2) for 30 min. Mice that spontaneously survived were perfused transcardially with a fixative at 15, 30 and 60 days of age. Quantitative immunohistochemical analysis at 60 days of age demonstrated a significant decrease in the numbers of serotonin-immunoreactive cell bodies in the nucleus raphe dorsalis, the nucleus raphe pontis, the subpyramidal region, the total raphe system and the whole brain, while no significant reduction in the number of serotonin-immunoreactive cell bodies was observed in the caudal raphe system. Presumably degenerative changes in serotonin-immunoreactive fibers were observed in various parts of the brain of mice subjected to total asphyxia at 15 days of age, and the numbers of degenerated fibers decreased in almost all parts of the brain, the exception being the caudal portion of the brainstem, at 30 and 60 days of age. These results suggested that neonatal asphyxia induced permanent changes in the serotonin neuron system, with regional differences.

Animals↗

PubMed1992

Immunohistochemical studies on the localization of aromatase and 17 alpha-hydroxylase/C17-20 lyase (17 alpha-lyase) in estrous cycling and pregnant hamster ovaries.

In order to clarify periodic changes in the localization of enzymes engaged in estrogen biosynthesis during the estrous cycle, immunohistochemical and fine structural studies were performed using estrous cycling and pregnant hamster ovaries. Results showed that ovulation takes place at midnight between Day 4 and Day 1 in the regular 4 day-cycle hamster. Immunoreactivity for aromatase is localized in the granulosa cells of the secondary follicle and granulosa lutein cells during the morning (10:00 am) of Day 1 to the evening (5:00 pm) of Day 4; in the night (9:00 pm) of Day 4, only the granulosa cells of the Graafian follicle showed a strong immunoreaction. As for 17 alpha-lyase, theca interna cells of the secondary follicle are immunopositive throughout Day 2 to the morning (10:00 am) of Day 4. Only a few cells in the theca interna of the Graafian follicles are immunopositive in the evening (5:00 pm) of Day 4. No positive cells for this enzyme were detected in the night (9:00 pm) of Day 4 or morning (10:00 am) of Day 1. The rapid decrease of estrogen biosynthesis occurring just before ovulation is considered to be due to the disappearance of 17 alpha-lyase in the theca interna cells of the ovary. On Day 10 of pregnancy, the granulosa cells of the secondary follicles and both the granulosa and theca lutein cells of the corpora lutea are immunostained with the aromatase antibody, while the theca interna cells of the secondary follicles reacted positively to the 17 alpha-lyase antibody. Only the granulosa and theca interna cells from the large preovulatory Graafian follicle of Day 4 (proestrus) which are positively stained for aromatase as well as 17 alpha-lyase show ultrastructural features typical of steroid secretory cells.

Aldehyde-Lyases↗

PubMed1990

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG, and in pregnant rat ovaries.

Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection. At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts. In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Immunocytochemical and biochemical studies on the localization and changes of 17 alpha-hydroxylase/C17-C20 lyase activity in immature rat ovary treated with PMSG and hCG.

The localization of cytochrome P-450 of 17 alpha-hydroxylase/C17-C20 lyase (P-450(17 alpha, lyase] and the changes of the enzyme activity were studied immunocytochemically and biochemically in the ovaries of immature rats treated with PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin). Immunocytochemically, P-450(17 alpha, lyase) was localized in both the theca interna cells and interstitial gland cells of the ovaries of immature rats treated with PMSG for 48 h. After hCG administration, the immunoreactive cells rapidly decreased in number in the PMSG-pretreated rat ovary. Namely, 6 h after the hCG injection, positive staining for P-450(17 alpha, lyase) was recognized only in a few theca interna cells, while 12 h after the injection to immunostained cells were detected in the ovary. Forty-eight hours after the hGC treatment (96 h after the PMSG injection), most of the theca interna cells and the interstitial gland cells became immunopositive for P-450(17 alpha, lyase) again. The 17 alpha-hydroxylating activity of P-450(17 alpha, lyase) was 0.5, 0.22 and 0.03 nmol/min/mg protein in the ovarian microsomes of PMSG-treated, PMSG + hCG(3 h)-treated and PMSG + hCG(6 h)-treated rats, respectively. Changes of the hydroxylase activities in all the experimental groups are almost parallel to those of P-450 contents in the microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde-Lyases↗

PubMed1990

T cell clones are killed by a thymic stromal cell monolayer following stimulation of T cell receptor with antigen and/or H-2 molecules on the monolayer.

A thymic stromal cell clone, MRL104.8a, expressed class I and class II H-2k antigens after exposure to gamma-interferon (gamma-IFN) and produced thymic stroma-derived T cell growth factor (TSTGF) irrespective of gamma-IFN exposure. Culturing the keyhole limpet hemocyanin (KLH)-specific, I-Ek-restricted 9-16 helper T cell (Th) clone on an Ia (I-Ak and I-Ek)-expressing MRL 104.8a monolayer induced potent proliferation of the 9-16 cells by virtue of the TSTGF produced by the monolayer. In contrast, the addition of KLH to cultures resulted in lethal growth inhibition of the 9-16 Th clone. Such a phenomenon was also observed for various Th as well as cytotoxic T lymphocyte (CTL) clones, and the following were revealed: (i) the growth of the ovalbumin (OVA)-or bovine thyroglobulin (BTg)-specific Th clone on the la-expressing MRL 104.8a monolayer was also inhibited by addition of the relevant antigen. The fact that these Th clones required antigen-presenting cells (APC) capable of processing antigen for the recognition of the respective target antigen suggested the potential of MRL 104.8a cells for antigen-processing; (ii) the lethal growth inhibition of KLH-specific, I-Ak (23-1-8)- or I-Ek (9-16)-restricted Th clone was prevented selectively by anti-I-Ak or anti-I-Ek antibody respectively; (iii) the I-Ek-alloreactive Th clone (2-13) was supported for its growth on a gamma-IFN-unexposed MRL 104.8a monolayer, whereas this clone was killed on an I-Ek-expressing monolayer; and (iv) when I-Ak-reactive CTL clones were cultured on an Ia- or Ia+ monolayer, CTL clones failed to exhibit cytotoxic effect on either the Ia- or the Ia+ monolayer, but were conversely killed by the Ia+ monolayer. Its killing was also prevented by an antibody which inhibits the recognition of Ia antigen on the monolayer by CTL clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Effects of undernutrition on the serotonin neuron system in the developing brain: an immunohistochemical study.

The effects of undernutrition during the first 3 weeks of postnatal life on the serotonin neuron system were investigated using a peroxidase-antiperoxidase immunohistochemical method. Undernourishment was induced by increasing the litter size from 6 to 18 mice during the first 3 weeks of postnatal life. At 15 days of age a morphological change (large swelling of serotonin-immunoreactive axons) was noted in various parts of the brain of the undernourished mice with no remarkable changes in dendrites and cell bodies. Quantitative immunohistochemical analysis at 60 days of age showed a significant decrease in serotonin-immunoreactive cell bodies in the nucleus linearis intermedius, the nucleus raphe obscurus, the total raphe system and the subpyramidal region. These results suggest that undernutrition during early postnatal life affected the serotonin neuron system with regional differences.

Animals↗

PubMed1989

Capacities of a newly established thymic stromal cell clone to express Ia antigens and to produce interleukin-6, colony-stimulating factor, and thymic stroma-derived T-cell growth factor.

Thymic stromal cell lines, termed MRL104 and MRL28, have been isolated from long-term liquid cultures of thymic stromal cells from MRL/l mice. The capacities of these parental lines and derived clones to express Ia antigens and to produce cytokines involved in T-cell proliferation and/or differentiation were investigated. Parental lines and their clones did not exhibit a typical fibroblastic, macrophage-like, or epithelial appearance in electron as well as phase-contrast micrographs. These thymic stromal cells seemed to differ from established fibroblast lines in that these thymic stromal cells expressed Ia antigens after exposure to interferon-gamma (IFN-gamma), whereas fibroblast lines did not. They also appeared to differ from macrophage cell lines in that they lacked the expression of Mac-1 antigens on their cell surface and produced no detectable level of interleukin-1 (IL1) before or even after exposure to lipopolysaccharide. When these parental lines and its clones were tested for their ability to produce various types of cytokines, it was revealed that they were capable of producing colony-stimulating factor (CSF), IL6, and thymic stroma-derived T cell growth factor (TSTGF), which was recently described, but were unable to generate other lymphokines and IFNs. Thus these cell lines and clones represent unique features in that they have potentials to express Ia antigens and to produce CSF, IL6, and TSTGF. The biological significance for the expression of these features is discussed in the context of intrathymic T-cell maturation and T-cell repertoire selection.

Animals↗

PubMed1989

Ultrastructural and immunocytochemical studies on the cytoskeleton in the anterior pituitary of rats, with special regard to the relationship between actin filaments and secretory granules.

As previously reported, in anterior pituitary cells of the rat, secretory granules are linked with adjacent granules, cytoorganelles, microtubules, and plasma membrane by thin filaments, 4-10 nm in diameter. The quick-freeze, deep-etching method revealed that some of the filaments linking adjacent secretory granules show 5 nm-spaced striations on their surface which are known to be characteristic of actin. Immunocytochemistry showed that actin is localized in the cytoplasm beneath the plasma membrane, and around or between secretory granules. The heavy meromyosin decoration method demonstrated that actin filaments are mainly located in the cytoplasm beneath the plasma membrane, while some actin filaments are connected with the limiting membrane of the secretory granules. The actin filaments associated with the secretory granules are considered to be involved in the intracellular transport of the granules, while those localized in the peripheral cytoplasmic matrix might control the approach of the secretory granules to the plasma membrane and their release.

Actins↗