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Biomedical subjects

K Ishimura

Publications and source records attributed to K Ishimura.

At least 37 records · Page 2Linked to original sources
PubMed1998

Wound healing of intestinal anastomosis after digestive surgery under septic conditions: participation of local interleukin-6 expression.

This study aimed to evaluate the integrity of anastomotic wound healing after digestive surgery under septic conditions and to observe local interleukin-6 (IL-6) expression around the anastomotic segment. Experimental animals were separated into lipopolysaccharide (LPS) and control groups. Each was injected with LPS or normal saline solution into the peritoneal cavity 24 hours before transection and anastomosis of the colon. The anastomotic bursting pressure (ABP) and tissue hydroxyproline concentration (HP) were measured as indicators of wound healing. Immunohistochemical staining for IL-6 was performed on tissue samples obtained from the anastomotic segment, lung, liver, and kidney. The reactive cells were counted by light microscopy. The ABP and HP were significantly lower in the LPS group than the control group 7 days after the surgery. In the LPS group, IL-6 expression around the anastomotic segment was enhanced 1 and 6 hours after surgery but suppressed 24 hours afterward. In contrast, IL-6 expression in lung, liver, and kidney was enhanced in the LPS group 24 hours after surgery but not in the control group. It is suggested that anastomotic wound healing is impaired after digestive tract surgery under septic conditions, and local IL-6 expression participates in wound healing.

Anastomosis, Surgical↗
PubMed1998

Steroid 5 alpha-reductase type 1 immunolocalized in the adrenal gland of normal, gonadectomized, and sex hormone-supplemented rats.

Steroid 5alpha-reductase in the rat adrenal gland is supposed to play a role in the catabolism of adrenal steroids. We showed immunohistochemically the cellular and subcellular localization of 5alpha-reductase in the rat adrenal gland, using a polyclonal antibody against 5alpha-reductase rat type 1. In the adrenal cortex, positive immunoreaction was found in the cells of the zonae fasciculata and reticularis but was absent in those of the zona glomerulosa. The positive staining was restricted to the cytoplasm but not to the nucleus, Golgi complexes or mitochondria. The staining intensity showed a marked change depending on the steroidal milieu. Gonadectomy for 6 weeks increased the immunoreaction, regardless of the sex. Testosterone replacement for the last 2 weeks considerably reduced the immunoreaction in 6-week-castrated males, and estradiol supplement for the last 2 weeks also resulted in the marked reduction of immunostaining in 6-week-gonadectomized females. In the adrenal medulla, the immunoreaction was localized in the supporting cells and the Schwann cells but not in the chromaffin cells. In these cells, the immunoreaction was not affected by steroidal treatments. These findings suggest that the expression of 5alpha-reductase in the rat adrenal cortex is regulated by sex hormones from the gonads, and the enzyme may participate in the conversion of adrenal steroids depending on the steroidal environment, although the functional significance of the enzyme in the adrenal medulla remains unclarified.

Adrenal Cortex↗
PubMed1998

Evidence that immature rat liver is capable of participating in steroidogenesis by expressing 17alpha-hydroxylase/17,20-lyase P450c17.

Steroid hydroxylase cytochrome P450c17 has been previously purified from microsomal fractions of immature rat livers. In this study, we investigated the expression of P450c17 in rat livers to understand a role of steroidogenesis in the extrasteroidogenic tissue. Upon immunoblot analysis utilizing liver microsomes from rats, P450c17 was detected in 1 and 3 week old rats but not in adult rats. Data from immunohistochemical studies also showed a similar age-dependent expression of P450c17 and indicated that P450c17 detected in immature rat livers is localized in cells surrounding interlobular veins. This age-dependent expression of P450c17 in rat livers was observed in both sexes. Upon enzymatic analysis utilizing microsomal fractions from livers, levels of 17alpha-hydroxylase and 17,20-lyase activity for pregnenolone and progesterone increased by 3 weeks and dramatically reduced at 7 weeks, which is consistent with the expression level of P450c17. These data clearly indicate that P450c17 is expressed in immature rat liver to produce 17alpha-hydroxysteroids and C19-steroids. Based upon immunoblot analysis, the expression level of P450c17 in immature rat livers was approximately one third of that in testis. Compared expression level of P450c17 and total volume of organs between liver and testis, the total amount of steroid metabolites produced by liver P450c17 could be greater than that produced by gonadal P450c17. Because of the absence of P450c17 in rat adrenal glands, rat liver could be the major site for producing 17alpha-hydroxysteroids and C19-steroids in this particular period of life. Although physiological products formed by P450c17 in liver and their roles remain to be elucidated, this study suggests a large capacity of prepubertal rat liver for participating the production of steroid hormones and a putative importance of 17alpha-hydroxysteroids and C19-steroids, such as cortisol and androstendione, which are generally believed to be minor components of steroid hormones in rodents.

Animals↗
PubMed1998

Steroid 5alpha-reductase type 1 immunolocalized in the rat peripheral nervous system and paraganglia.

Steroid 5alpha-reductase is an enzyme that converts a number of steroids with a C-4, 5 double bond and C-3 ketone to 5alpha-reduced metabolites. This enzyme has been suggested to play a role in brain development and myelination in the rat nervous system. In the present study, we examined the cellular and subcellular localization of the enzyme immunocytochemically in the rat peripheral nervous system and paraganglia using a polyclonal antibody against rat 5alpha-reductase type 1. Light and electron microscopical studies localized 5alpha-reductase in the Schwann cells of myelinated and unmyelinated nerve fibres, the satellite cells of the ganglia, the enteric glial cells and the supporting/sustentacular cells of the paraganglia. In the myelinated nerve fibres, immunoreactivity was observed in the outer loops, the nodes of Ranvier and the Schmidt-Lanterman incisures. Subcellularly, the immunoreactivity was localized in the cytoplasm of various glial cells. No immunoreactivity was observed in the myelin membrane, the axon or the neuronal perikaryon. These findings suggest that 5alpha-reductase is widely distributed in glial cells, and that, in addition to myelination, 5alpha-reduced steroids play a role in some glial functions in the peripheral nervous system.

Animals↗
PubMed1998

Effects of point mutations at the flexible loop alanine-145 of Escherichia coli dihydrofolate reductase on its stability and function.

To elucidate the role of a flexible loop (residues 142-149) in the stability and function of Escherichia coli dihydrofolate reductase, alanine-145 in this loop was substituted by site-directed mutagenesis with ten amino acids (Glu, Phe, Gly, His, Ile, Leu, Arg, Ser, Thr, and Val). The amount of three mutant proteins (A145E, A145I, and A145L) in cells was too small to allow the measurement of circular dichroism (CD) spectra and urea unfolding. The CD spectra of other seven mutants were identical with those of the wild-type DHFR, indicating that the native conformation of DHFR was not affected by the mutations. The free energy change of unfolding by urea decreased with an increase in the hydrophobicity of amino acid residues introduced, A145T>A145R>A145G>=A145S>=A145H>A145V++ +>wild-type>=A145F. The steady-state kinetic parameters for the enzyme reaction, Km and ksub, were only slightly influenced by the mutations. These results suggest that site 145 in the flexible loop plays an important role in the stability but has little or no effect on the native structure and function of this enzyme. The characteristics of the mutations are discussed in comparison with those of mutations at site 67 [Ohmae et al. (1996) J. Biochem. 119, 703-710] and at site 121 [Gekko et al. (1994) J. Biochem. 116, 34-41] in two other flexible loops.

Alanine↗
PubMed1998

Light and electron microscopic immunocytochemistry on the localization of 17 alpha-hydroxylase/C17,20-lyase (P450c17) in the rat placenta.

The rat placenta is the primary source of androgens during the second half of pregnancy. Androgens are converted to estrogens in the ovaries and contribute to the maintenance of normal pregnancy. We immunocytochemically characterized the cellular and subcellular localization of cytochrome P450 of 17 alpha-hydroxylase/C 17,20-lyase (P450c17), an enzyme responsible for androgen synthesis, in the rat placenta. We also observed the fine structure of the placenta by electron microscopy. The rat placenta had a different structure from the primate, and contained four zones: labyrinth, basal zone, decidua basalis, and metrial gland. The labyrinth had three trophoblastic layers and fetal endothelium, and P450c17 immunoreactivity was homogeneously localized in the three trophoblastic layers but not in the fetal endothelium. In the basal zone, various types of trophoblasts were observed, and the immunoreaction was localized in small basophilic cells and giant cells. The intensity of staining was heterogeneous among these cells. The decidua basalis showed no immunostaining. Subcellular localization of the enzyme was in the cytoplasm, but not in the nucleus or mitochondria. The present study demonstrated a steroidogenic potency in both the labyrinth and the basal zone, although it was shown only in the basal zone in previous studies.

Animals↗
PubMed1998

[The mechanisms of corticoids biosynthesis revealed by immunohistochemistry and in situ hybridization].

The improvement of immunohistochemistry and in situ hybridization techniques has recently allowed a prominent advancement in morphological research on the mechanisms of steroid-hormone biosynthesis in the adrenal gland. Cholesterol side-chain cleavage enzyme (P450scc) was localized in the inner mitochondrial membrane of all cortical cells. Steroid 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase (3 beta-HSD) and 21-hydroxylase (P450c21) were present in the membrane of smooth endoplasmic reticulum (SER) of all cortical cells. 17 alpha-Hydroxylase/C17-C20 lyase (P45017 alpha) was located in the SER membrane of the zona fasciculata-reticularis cells of human, bovine, swine, guinea-pig, and hamster adrenal gland. This enzyme, however, was absent in the adrenal gland of rat, mouse and rabbit. 11 beta-Hydroxylase (P45011 beta) and aldosterone synthase (P450aldo) were situated in the inner mitochondrial membrane of the zona fasciculata-reticularis cells and in that of the zona glomerulosa cells, respectively. These results indicate that aldosterone is synthesized in the zona glomerulosa cell, and that glucocorticoids such as cortisol and corticosterone are synthesized in the zona fasciculata-reticularis cells. The conversion of cholesterol to pregnenolone and the final steps of corticoids synthesis occur in the mitochondria, while the intermediate steps, leading to the synthesis of deoxycorticosterone or deoxycortisol from pregnenolone, take place in the SER membranes.

Adrenal Cortex↗
PubMed1997

Application of a beta-cyclodextrin sulfate-immobilized precolumn to selective on-line enrichment and separation of heparin-binding proteins by column-switching high-performance liquid chromatography.

A column-switching high-performance liquid chromatography (HPLC) system which consisted of a beta-cyclodextrin (beta-CD) sulfate-immobilized hydrophilic vinyl-polymer gel precolumn and a reversed-phase analytical column was developed for the selective on-line enrichment and separation of heparin-binding proteins. Of 15 proteins investigated, 10 proteins having heparin-binding activity were retained on the beta-CD sulfate precolumn almost quantitatively, in contrast 5 proteins having no heparin-binding activity were not retained. Calibration graphs for basic fibroblast growth factor constructed at various sample volumes were nearly identical, indicating that the protein could be enriched by this system. The system was successfully used for the selective separation of lysozyme in egg white. The beta-CD sulfate-immobilized precolumn showed no loss of analytical performance over 2 years during which about 400 samples were analysed.

Chromatography, High Pressure Liquid↗
PubMed1997

Light and electron microscopic immunohistochemistry of the localization of adrenal steroidogenic enzymes.

Recent immunohistochemical studies have revealed the precise localization of the enzymes involved in adrenal steroidogenesis. Light microscopical investigations showed that cytochromes P450 of cholesterol side-chain cleavage enzyme (P450scc) and of 11 beta-hydroxylase (P45011 beta), 3 beta-hydroxysteroid dehydrogenase/ delta 5-4 isomerase (3 beta HSD), and 21-hydroxylase (P450C21) are localized in all the adrenocortical cells, especially in those of the zona fasciculata-reticularis. 17 alpha-Hydroxylase/C17-C20 lyase (P45017 alpha,lyase) is present in the zona fasciculata-reticularis cells of human, bovine, pig, and guinea-pig adrenals, but absent in the adrenals of some rodents such as rat, hamster, and mouse. Aldosterone synthase (P450aldo) is contained only in the zona glomerulosa cells. In the rat adrenal, P45011 beta, which catalyzes the conversion of deoxycorticosterone to corticosterone, is localized in the zona fasciculata-reticularis cells. Electron microscopic investigations demonstrated that P450scc and P45011 beta are colocalized in the matrix side of inner mitochondrial membrane including cristae, while 3 beta HSD, P450C21, and P45017 alpha, lyase are present in the membranes of smooth endoplasmic reticulum (SER). These results clearly indicate that aldosterone, the most potent mineralocorticoid, is synthesized in the zona glomerulosa cells, and glucocorticoids, such as corticosterone and cortisol, are produced in the zona fasciculata-reticularis cells. The conversion of cholesterol to pregnenolone and the final steps of corticosteroid synthesis occur in the mitochondria, while the intermediate steps, leading to the synthesis of deoxycorticosterone or deoxycortisol from pregnenolone, take place in the SER membranes.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Induction of cyclooxygenase-1 in a human megakaryoblastic cell line (CMK) differentiated by phorbol ester.

Human megakaryoblastic cells (CMK line) are known to differentiate to mature megakaryocyte-like cells by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). There are two isozymes of prostaglandin-forming cyclooxygenase enzyme. Constitutive cyclooxygenase-1 and inducible cyclooxygenase-2 were followed during differentiation of CMK cells. Treatment of the cells with 0.1 microM TPA for 4 days resulted in a 5-20-fold increase in cyclooxygenase activity. Northern and Western blot analyses revealed that cyclooxygenase-1 mRNA and protein increased in parallel with the enzyme activity. In contrast, cyclooxygenase-2 mRNA was detected only at 3 h. Furthermore, most of the increased cyclooxygenase activity was immunoprecipitated with anti-cyclooxygenase-1 antibody, and was not affected by a cyclooxygenase-2-specific inhibitor, NS-398. These results indicated that cyclooxygenase-1 rather than cyclooxygenase-2 was predominantly induced depending on TPA. The enzyme thus induced was localized by immunoelectron microscopy in nuclear envelope and endoplasmic reticulum of the CMK cells.

Arachidonic Acid↗
PubMed1997

Subcellular localization of arachidonate 12-lipoxygenase and morphological effect of its overexpression on murine keratinocytes.

Arachidonate 12-lipoxygenase enzyme oxygenates the position 12 of arachidonic acid and produces 12-hydroperoxy-arachidonic acid. Mouse keratinocytes were transiently transfected with an expression vector of human platelet 12-lipoxygenase cDNA. The cells were homogenized, and the subcellular localization of the enzyme was examined by differential centrifugation. The 12-lipoxygenase activity was detected predominantly in the particulate fractions. In contrast, immunoelectron microscopy detected the enzyme mainly in the cytoplasm of the transfected cell, but not in the nucleus, subcellular organelles or plasma membrane. To explain the discrepancy between these findings, we performed an electron-microscopic examination of the 176000 g pellet of the keratinocyte homogenate. The pellet contained mainly insoluble proteins such as keratin but not membrane structures such as the plasma membrane. Thus, it is possible that the enzyme was localized originally in the cytoplasm of the keratinocyte, and found in the particulate fractions due to its association with insoluble proteins during fractionation procedures. Unique structural changes were observed in the transfected keratinocytes. The nucleus had very scant karyoplasm and coarse fibrillary structures. When the keratinocytes were transfected with a mutant 12-lipoxygenase cDNA or a vector without 12-lipoxygenase cDNA, these structural changes were not observed.

Animals↗
PubMed1997

Possible involvement of intracellular Ca2+ in hyposmosis-evoked catecholamine release from adrenal chromaffin cells.

The influence of hyposmotic conditions on catecholamine release was studied using cultured adrenal chromaffin cells. Incubation of the cells in hyposmotic solution led to the enhancement of catecholamine release in a manner dependent on the reduction of osmolarity. Hyposmosis-evoked catecholamine release was similarly observed in the presence or absence of extracellular Ca2+, and was not significantly affected by organic and inorganic Ca2+ entry blockers. These results indicated that the hyposmosis-evoked release might be associated with a rise in the intracellular Ca2+ concentration. Further studies showed that neither ryanodine nor thapsigargin caused any significant effect on hyposmosis-evoked catecholamine release, whereas pretreatment of chromaffin cells with carbonyl cyanide m-chlorophenyl hydrazone significantly enhanced the hyposmosis-evoked release. Catecholamine release evoked by exposure to hyposmotic medium is therefore thought to be mediated through intracellular Ca2+, which may be mainly sequestered by the mitochondrial pools. Neither caffeine- nor inositol 1,4,5-trisphosphate-sensitive Ca2+ pools seems likely to be involved in hyposmosis-evoked catecholamine release, although the Ca2+ pools that contribute to the elevation of intracellular Ca2+ observed under hyposmotic conditions are not yet completely identified.

Adrenal Medulla↗
PubMed1997

Localization of NADPH diaphorase and vasoactive intestinal polypeptide-containing neurons in the efferent pathway to the rat corpus cavernosum.

OBJECTIVE: We examined the coexistence of nitric oxide synthase (NOS) and vasoactive intestinal peptide (VIP) in the efferent pathway to the rat corpus cavernosum. METHODS: We used NADPH diaphorase (ND) histochemical staining, a specific marker of neuronal NOS, in combination with retrograde axonal transport of True Blue (TB) and VIP immunohistochemistry. RESULTS: Many neurons were labeled by TB in the cavernous nerve and the body of the major pelvic ganglion (MPG), and fewer neurons in the pelvic nerve. In the cavernous nerve, about 90% of TB-labeled efferent neurons were ND positive, and about 80% in the body of the MPG and pelvic nerve. Besides, 60-80% of TB-labeled efferent neurons projecting to the corpus cavernosum showed VIP immunoreactivity. VIP was colocalized with NOS in 70-80% of neurons. CONCLUSIONS: These findings suggest that most of the ND-positive nerves and terminals in the corpus cavernosum come from the MPG via the cavernous nerve and that NO plays an important role; it may act in combination with NO because most NOS-containing neurons simultaneously showed VIP immunoreactivity.

Animals↗
PubMed1996

Immunohistochemical presence of 5 alpha-reductase rat type 1-containing cells in the rat brain.

We showed immunohistochemically the localization of 5 alpha-reductase-containing cells in the rat brain, using a rabbit antibody generated against 5 alpha-reductase rat type 1. The antibody was produced by injecting the synthetic peptide corresponding to the amino acids 38-53 of 5 alpha-reductase rat type 1, conjugated to keyhole limpet hemocyanin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Western blot analysis revealed that this antiserum recognized the protein with a molecular weight of 29,000 Da. The immunoreactive cells were distributed throughout the brain and they were preferentially located in the white matter rather than in the grey matter. These cells were mostly small and round and had a few fine processes. The immunoreaction was confined to the cytoplasm and processes. These findings indicate that 5 alpha-reductase rat type 1-containing cells are widely distributed in the rat brain and are located preferentially in the white matter rather than in the grey matter.

Animals↗
PubMed1996

Immunohistochemical localization of estrogen receptors within aromatase-immunoreactive neurons in the fetal and neonatal rat brain.

We elucidated the anatomical relationship between estrogen receptors and aromatase, the enzyme converting androgens to estrogens, in the fetal and neonatal rat brain by means of double immunohistochemical labeling, using antibodies against rat estrogen receptors and human placental aromatase cytochrome P450. Numerous aromatase-immunoreactive neurons were found in the medial preoptic area, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus. Estrogen receptors were also abundant in these areas. Most of the aromatase-immunoreactive neurons showed immunoreactivity for estrogen receptors in the medial subdivision of the bed nucleus of the stria terminalis and in the posterodorsal, division of the medial amygdaloid nucleus. There were also many double-labeled cells in the ventromedial nucleus. However, in the medial preoptic area the localization of aromatase-immunoreactive neurons was distinct from that of neurons containing estrogen receptors. These results suggested that estrogens, which are converted from androgens in aromatase-containing neurons, are involved in the sexual differentiation of the brain through estrogen receptors within aromatase-immunoreactive neurons in the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus, but through estrogen receptors in aromatase-immunonegative neurons in the medial preoptic area.

Animals↗