Biomedical subjects

N Koibuchi

Publications and source records attributed to N Koibuchi.

At least 37 records · Page 2Linked to original sources

PubMed1999

Promoter-specific regulation of the brain-derived neurotropic factor gene by thyroid hormone in the developing rat cerebellum.

Thyroid hormone (TH) plays a critical role in normal cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. This action could be exerted in part through brain-derived neurotropic factor (BDNF), as cerebellar BDNF messenger RNA (mRNA) expression is lower, and replacement of BDNF partially reverses the abnormal neurogenesis in the hypothyroid rat. The rat BDNF gene consists of four noncoding exons (exons I-IV), each of which is linked to a different promoter, and a protein-coding exon (exon V). To study promoter-specific regulation of the BDNF gene by TH, ribonuclease protection assay of each exon mRNA was performed using total developing rat cerebellar RNA. During cerebellar development, all exon mRNAs were detected, but with different expression patterns; among noncoding exon mRNAs, exon II mRNA was the most abundant. Daily TH replacement induced a 3-fold increase in exon II mRNA on postnatal day (P) 15. On P30, exon II mRNA was still much greater in the TH-replaced animal. Exon I mRNA was detected on P2 and P7. However, in contrast to exon II mRNA, TH treatment suppressed the expression of exon I mRNA on P2. Exon III and IV mRNAs were not detected on P2 and P7, but small amounts were observed starting on P15 in TH-replaced animals. They were not detected by P30 in hypothyroid animals. In contrast, in the cerebral cortex, although all exons are differentially regulated during development, the expression of each mRNA was not significantly altered by TH. These results indicate that TH regulates BDNF gene expression in a promoter-, developmental stage-, and brain region-specific manner, which may play an important role in region- and stage-specific regulation of brain development by TH.

Aging↗

PubMed1999

Behavior of cells in artificially made cell aggregates and tissue fragments after grafting to developing hind limb buds in Xenopus laevis.

During vertebrate limb development, the limb bud grows along the proximo-distal (P-D) direction, with the cells changing their adhesiveness. To know whether the position-related differences in cell adhesiveness are actually utilized by morphogenesis to constitute limb structures, we grafted cell aggregates made of dissociated cells derived from different positions and stages of developing hind limb buds into developing hind limb buds and observed the behavior of the cells. Cell aggregates made of dissociated mesenchymal cells from two different origins were implanted in different positions and stages of limb buds or grafted on limb stumps made by cutting. The two grafted cell populations in the aggregate always sorted out from each other, but their patterning of sorting-out was quite different according to the transplanted regions. In summary, cells in the aggregate that have closer positional identity to the transplanted site were always situated at the boundary between host and donor cells. The pattern of sorting-out seemed to be determined by the relative adhesiveness of surrounding cells to the constituent cells of the aggregates. We also transplanted fragments dissected out from different regions along the P-D axis into st. 50 limb buds. The descendants of grafted cells moved distally to the region corresponding to their positional identity and participated in the formation of more distal structures from that point. These results suggest that the difference in cell adhesiveness may probably play a role in arranging cells along the P-D axis of a developing limb bud.

Animals↗

PubMed1998

Sperm-activating proteins obtained from the herring eggs are homologous to trypsin inhibitors and synthesized in follicle cells.

The activation of sperm motility by the egg is an ubiquitous phenomenon in the animal kingdom, but the molecules by which the egg activates sperm motility have been clarified in only a few invertebrate species. In the Pacific herring, Clupea pallasii, mature unfertilized eggs release the sperm-activating proteins which are prerequisite to successful fertilization. Complementary DNA clones encoding herring sperm-activating proteins were isolated from a herring ovarian complementary DNA library and amino acid sequences were deduced. The herring sperm-activating protein(s) is a secretory product(s) with a strong homology to Kazal-type trypsin inhibitors, such as mammalian acrosin inhibitors. The sperm-activating proteins were globally distributed in the outermost layer of the egg chorion and its gene was found to be expressed in the follicle cells which surround developing oocytes. These results suggest that in the Pacific herring, trypsin inhibitor-like proteins are synthesized in the follicle cells, secreted, accumulated in the egg chorion during oocyte development, and released into the milieu at spawning to activate the motility of spermatozoa at the time of gamete interaction.

Amino Acid Sequence↗

PubMed1998

Thyroid hormone response elements differentially modulate the interactions of thyroid hormone receptors with two receptor binding domains in the steroid receptor coactivator-1.

Ligand-dependent transcriptional activation by nuclear receptors is mediated by interactions with coactivators. Recently, a consensus interaction motif (LXXLL) has been identified in a number of coactivators such as steroid receptor coactivator-1 (SRC-1). SRC-1 contains three such motifs in the central (nuclear receptor binding domain-1, NBD-1) and a single one in the C-terminal (NBD-2) regions. To define the nature and role of the two NBDs in SRC-1, interaction studies between the two NBDs and thyroid hormone receptor (TR) were performed. Although NBD-1 and NBD-2 showed similar ligand- and AF-2-dependent interactions with TR in solution, these two NBDs possessed distinct interaction properties with TR when TR is bound to a thyroid hormone-response element (TRE). Both in vitro and in vivo interaction studies demonstrate that NBD-1, but not NBD-2, exhibits ligand-dependent interaction with TR in the presence of TREs. In addition, a natural isoform of SRC-1, SRC-1E, which lacks NBD-2, preserved TR as well as progesterone receptor-mediated coactivator function on reporter gene expression. Finally, we found that NBD-1 failed to interact with a TR and retinoid X receptor heterodimer complex on a transcriptionally inactive direct repeat +4 TRE in electrophoretic mobility shift assays. These observations indicate that DNA-induced, as well as ligand-induced, conformational change(s) of TR may influence the nature of its binding to SRC-1, and that the two NBDs of SRC-1 may play different roles to regulate ligand-dependent transactivation of TRs.

Animals↗

PubMed1998

Age-related degeneration of the serotoninergic fibers in the zitter rat brain.

The serotonin neuron system was studied using immunohistochemical and neurochemical techniques in zitter mutant rats aged 1-14 months, which are characterized by abnormal metabolism of superoxides. The morphology of the serotoninergic neuron system and the serotonin level in the zitter rat were compared to those of age-matched Sprague-Dawley (SD) rats. Up to age 4 month, the density and distribution of serotoninergic fibers in the zitter rat brain were similar to those of control rats. However, several serotoninergic fibers with abnormal morphology, characterized by swollen varicosities, were observed in the cerebral cortex and caudate putamen of 6-month-old zitter rats. The density and distribution of these fibers in other regions of the brain were similar to those of control rats. The abnormal serotoninergic fibers increased in number and extended into other regions of the brain such as the thalamus, hippocampus, and vestibular nucleus. On the other hand, the density of normal serotoninergic fibers decreased throughout the brain of the 14-month-old zitter rat. Abnormal serotoninergic fibers have also been reported in the brain of normal older (24 months) SD rats. Neurochemical analysis revealed lower levels of serotonin, and 5-hydroxyindoleacetic acid, in all cortical areas (prefrontal, parietal, and occipital cortices), the caudate putamen, and the hippocampus of 12-month-old zitter rats. Levels differed significantly in the parietal cortex and hippocampus between the zitter and SD rats. Based on the morphological and neurochemical similarities, the present results suggest that age-related degeneration of serotoninergic fibers occurs in the zitter rat brain. Furthermore, degeneration of serotoninergic fibers appears to be induced by superoxide species. Thus, the zitter rat may provide a good model for studying the neurotoxic effects of superoxide species on the serotoninergic neuron system.

Aging↗

PubMed1998

Elevated expression of epidermal ornithine decarboxylase mRNA in scleroderma.

Using in situ hybridization techniques, we examined the expression of ornithine decarboxylase (ODC) mRNA in the skin of five patients with systemic sclerosis (SSc) and five normal controls. Sections treated with an anti-sense probe showed concentrated grains exclusively in the epidermis of SSc patients, but not in that of normal controls. Because our subcloned anti-sense probe specifically hybridizes with ODC mRNA, these findings indicate that the expression of ODC mRNA is elevated in SSc epidermis. Possibly polyamines have an important part to play in the skin changes of SSc.

Humans↗

PubMed1998

Existence of gradient in cell adhesiveness along the developing Xenopus hind limb bud, shown by a cellular sorting-out experiment in vitro.

To examine the possibility of a difference in cell adhesiveness along the developing Xenopus hind limb bud axes, single mesenchymal cells from developing hind limb buds were cultured, allowing them to form an aggregate in a gyratory culture system. By observing the distribution of cells within aggregates, it was found that sorting-out occurred between cells from different positions and different stages. Cells derived from more distal positions tended to be situated interiorly in the aggregates. According to Steinberg's differential adhesion hypothesis, these results support the idea that there is a graded difference in cell adhesiveness along the proximo-distal axis of the developing limb, with adhesiveness increasing distally. Although similar sorting-out was observed between anterior and posterior cell populations, it could not be determined which cell populations were definitely more cohesive. These properties may be correlated with the experimentally demonstrated 'positional value', which should be different among cells located at different positions along the axes of the developing vertebrate limb bud.

Animals↗

PubMed1998

ROR alpha gene expression in the perinatal rat cerebellum: ontogeny and thyroid hormone regulation.

Deficiency of thyroid hormone (TH) during the perinatal period results in severe neurological abnormalities in rodent cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. Of note, a mutant mouse, staggerer, in which the orphan nuclear hormone receptor ROR alpha gene is disrupted, exhibits cerebellar abnormalities similar to those seen in the hypothyroid animals, despite normal thyroid function. We, therefore, speculated that TH (tetraiodo-L-thyronine; T4) may regulate ROR alpha gene expression, which then may regulate genes essential for normal brain development. To test this hypothesis, we studied the changes in ROR alpha gene expression in perinatal hypothyroid rat cerebellum and the effect of TH replacement using Northern blot analysis, ribonuclease protection assay and in situ hybridization histochemistry. During cerebellar development, an approximately 3-fold increase in the cerebellar content of ROR alpha messenger RNA (mRNA) was seen in both propylthiouracil-treated, and propylthiouracil-treated and T4-replaced animals. However, the increase was accelerated when T4 was injected, although the ROR alpha mRNA content was identical, with or without T4, by 30 days after birth (P30). In contrast, T4 treatment suppressed the TH receptor alpha1 and c-erbA alpha2 mRNA content by P30; retinoic acid X receptor-beta mRNA content was not influenced by thyroid status. A significant hybridization signal for ROR alpha mRNA was seen only over Purkinje cells in the cerebellar cortex by in situ hybridization histochemistry. These results indicate that TH alters the timing of expression of the ROR alpha gene in the Purkinje cells of the cerebellar cortex, which may, in turn, influence Purkinje cell differentiation.

Amino Acid Sequence↗

PubMed1997

TRAM-1, A novel 160-kDa thyroid hormone receptor activator molecule, exhibits distinct properties from steroid receptor coactivator-1.

Nuclear hormone receptors (NRs) are ligand-dependent transcription factors that regulate target gene transcription. We report the molecular cloning and characterization of a novel human cDNA encoding TRAM-1, a thyroid hormone receptor activator molecule, a approximately 160-kDa protein homologous with SRC-1/TIF2, by far-Western-based expression screening. TRAM-1 binds to thyroid hormone receptor (TR) and other NRs in a ligand-dependent manner and enhances ligand-induced transcriptional activity of TR. The AF-2 region in NRs has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivators. Surprisingly, TRAM-1 retains strong ligand-dependent interaction with an AF-2 mutant of TR (E457A), while SRC-1 fails to interact with this mutant. Furthermore, we identified a critical TRAM-1 binding site in rat TRbeta1 outside of AF-2, as TRAM-1 shows weak ligand-dependent interaction with a helix 3 ligand binding domain TR mutant (K288A), compared with SRC-1. These results suggest that TRAM-1 is a coactivator that may exhibit its activity by interacting with subdomains of NRs other than the AF-2 region, in contrast to SRC-1/TIF2.

Amino Acid Sequence↗

PubMed1997

Local disturbance of neuronal migration in the S-100beta-retarded mutant mouse.

Homozygotes of a mouse strain with genetic polydactyly (Pdn) show disrupted cortical lamination and a significant decrease of S-100beta-immunoreactive elements in a particular area of the brain. In order to understand the abnormal cortical formation at the cellular level, the migration of cortical neurons and the development of glial cells were studied using bromodeoxyuridine (BrdU), S-100beta, and glial fibrillary acidic protein (GFAP) immunohistochemistry. Homozygous mice (Pdn/Pdn) displayed a variable pattern of abnormalities. Irregular GFAP-positive radial glial cells and disturbance of neuronal migration were found in a circumscribed area of the caudo-dorsal cortex of newborn Pdn mouse. The number of S-100beta-positive cells was reduced in this area. The present results suggest that abnormal cortical lamination closely correlates with disturbance of neuronal migration and abnormalities of glial cells, especially a significant decrease of S-100beta-immunoreactive cells.

Animals↗

PubMed1996

Ontogenic changes in the expression of cytochrome c oxidase subunit I gene in the cerebellar cortex of the perinatal hypothyroid rat.

The thyroid hormone plays a critical role in normal development of the mammalian central nervous system. This study was designed to examine the effect of perinatal hypothyroidism on ontogenic change in cytochrome c oxidase subunit I (COX I) gene expression in the rat cerebellum by using quantitative in situ hybridization histochemistry (ISH). Newborn rats were rendered hypothyroid by continuous administration of methimazole in the mothers' drinking water. The pups were then killed by decapitation on 1, 5, 10, 15, 20, and 30 days after birth (P1, P5, P10, P15, P20, and P30). Their cerebella were removed, and frozen sections were cut and processed for ISH with 35S-labeled RNA probe for COX I messenger RNA. After hybridization, emulsion autoradiography was performed. The numbers of grains within the external granule cell layer, molecular layer, and internal granule cell layer were then counted. A significant decrease in grain density was detected in the hypothyroid animal in all these areas on P5, P10, and P15. On P15, in the molecular layer, a greater hybridization signal was detected in the inner portion than in the outer portion in the euthyroid animal. No such difference was seen in the hypothyroid animal. Daily T4 treatment for 15 days restored the effect of methimazole treatment. The significant effect of perinatal hypothyroidism on COX I gene expression was not detected after P20. These results indicate that altered thyroid states affect the COX I gene expression in the cerebellar cortex during development, suggesting that the COX I gene is one of the key genes regulated by the thyroid hormone and plays an important role in the morphogenetic changes observed in the perinatal hypothyroid cerebellum.

Aging↗

PubMed1996

Regional comparison of prolactin gene expression in the human decidualized endometrium in early and term pregnancy.

Prolactin (PRL) is known to be expressed in the decidualized human endometrium and secreted into amniotic fluid. Although the site of synthesis of endometrial PRL is known to be the decidual cells, the difference in PRL gene expression within each area of decidua, i.e. decidua basalis, decidua parietalis and decidua capsularis, during pregnancy is not clear. We have applied an in situ hybridization histochemistry technique using a radiolabeled RNA probe to compare the difference in expression of PRL gene within each area of the decidualized endometrium. Specific hybridization signals were distributed over the decidual cells in early and term pregnancy. More intense hybridization signals were always detected in the tissues of early pregnancy than in those of term pregnancy. In the decidua capsularis of early pregnancy, labeled cells were concentrated close to the amniotic cavity, whereas cells were concentrated close to the maternal surface of the fetal membrane in term pregnancy. In the decidua parietalis, almost all decidual cells were labeled, but no specific labeling was seen in the endometrial glands or capillary endothelium in both groups. In the decidua basalis, most decidual cells showed hybridization signals whereas no hybridization signal was seen over the trophoblast cells. These results show that there are regional and periodic differences in PRL gene expression in the decidual cells during pregnancy.

Decidua↗

PubMed1996

Acute inhibitory effect of excess iodide on ornithine decarboxylase in the thyroid of propylthiouracil-treated rats.

Polyamines such as putrescine, spermidine and spermine have been thought to play an important role in thyroid growth induced by goitrogens. Reduced biosynthesis of these polyamines might play a role in the antigoitrogenic effects of excess iodide. This study was designed to examine the effect of potassium iodide (KI) on ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. Thyroidal ODC activity, protein content and mRNA were increased in rats made hypothyroid by 10 days of propylthiouracil treatment. The increase in ODC activity was suppressed after subcutaneous injection of KI (13mg/kg body weight); the apparent half-life of ODC activity after the treatment was estimated to be 19 min and the maximum suppression (90%) was seen 60 min after the treatment. On the other hand, administration of iodine-containing compounds including L-thyroxine, L-di-iodotyrosine,amiodarone, iopanoic acid and erythrosine showed no significant effect on ODC activity. The inhibitory effect of excess iodide was not reversed by pretreatment with dibutyryl cAMP and theophylline. The amount of immunoreactive ODC protein was reduced by iodide treatment (40%). However, the decrease was not as great as the decrease in ODC activity (90%). No significant change in thyroidal ODC mRNA content was seen 1 and 3 h following KI treatment. These results suggest that excess iodide reduces ODC activity in the rat thyroid gland by a post-transcriptional mechanism.

Animals↗

PubMed1996

[Expression of prolactin receptor gene in human decidua of early pregnancy].

UNLABELLED: This study was designed to examine the expression of the prolactin receptor (PRL-R) gene in the human decidualized endometrium and to determine the localization of endometrial cells expressing the PRL-R gene. METHOD: Decidua and trophoblast tissues of normal and ectopic pregnancy were obtained by curettage from patients undergoing artificial abortion at 8 -10 weeks of gestation. Total RNA was extracted to perform northern blot hybridization with a radiolabeled human PRL-R cDNA probe. Some tissues were cut and processed for in situ hybridization histochemistry with a radiolabeled RNA probe. RESULTS: 1. Northern blot hybridization: Approximately 9.0, 3.6 and 2.8kb size bands were detected in decidua in normal and ectopic pregnancy. No hybridization signal was detected in the chorionic villi. 2. In situ hybridization: Hybridization signals were detected in the cytoplasm of the decidual cells not only in normal pregnancy but also in ectopic pregnancy. No hybridization signal was detected in the trophoblast cells or endometrial glands. CONCLUSION: The human PRL-R gene is expressed in the decidual cells in early pregnancy not only in normal pregnancy but also in ectopic pregnancy. And it has been reported that the decidual cells produce PRL. These results suggest that PRL may act directly on the decidual cells by paracrine and/or autocrine mechanisms.

Blotting, Northern↗

PubMed1995

Localization of prolactin and its receptor messenger RNA in the human decidua.

Prolactin (PRL) is known as an anterior pituitary hormone. On the other hand, PRL is also produced in the human decidualized endometrium. The physiological role and site of action of endometrial PRL have not yet been clarified. This study was designed to investigate the localization of PRL receptor (PRL-R) gene-expressing cells in the human decidualized endometrium using in situ hybridization histochemistry. Sense and antisense 35S-labeled RNA probes for human PRL-R mRNA were hybridized with cryostat sections of human decidua, which were obtained from patients undergoing therapeutic abortion at 8-10 weeks of gestation. Hybridization signals for PRL-R mRNA were seen over the decidual cells. No labeled cells were seen in the chorion, amnion, or trophoblast. Comparing the localization of PRL-R gene-expressing cells to that of PRL gene-expressing cells using adjacent sections, their distributions were quite similar. These results indicate that not only PRL but also PRL-R transcripts are located in the decidual cells.

Decidua↗

PubMed1995

Localization of D-amino acid oxidase mRNA in the mouse kidney and the effect of testosterone treatment.

D-Amino acid oxidase (DAO), which catalyzes oxidative deamination of D-amino acids, is known to be highly expressed in the kidney. This study was designed to examine the localization of DAO mRNA in the mouse kidney using in situ hybridization histochemistry (ISH). For comparison, ISH for mRNA of ornithine decarboxylase (ODC), which is also highly expressed in the mouse kidney, was simultaneously performed. Adult, male mice which received 1 mg of testosterone propionate or vehicle injection, were sacrificed 14 h after injection and their kidneys were removed and processed for ISH. Hybridization signals for both mRNAs were exclusively located over the epithelial cells of the proximal tubule in the vehicle-treated animals. Signals for the DAO mRNA were observed at nearly the same hybridization intensity throughout the proximal tubule, whereas hybridization signals for the ODC mRNA were observed exclusively in the pars convoluta. Following testosterone treatment, ODC mRNA in the pars convoluta was expressed with a stronger intensity than that in the vehicle-injected animals. ODC mRNA was also expressed in the pars recta with a weaker intensity than in the pars convoluta. On the other hand, DAO mRNA expression was little affected by testosterone treatment. These results indicate that, although both genes are possibly expressed in the same cells, the expression of these genes is regulated by different mechanisms.

Amino Acids↗

PubMed1995

Effect of perinatal hypothyroidism on expression of cytochrome c oxidase subunit I gene, which is cloned by differential plaque screening from the cerebellum of newborn rat.

Early development of the central nervous system is influenced by several hormones including thyroid hormone. This study was designed to clone the gene whose expression is changed in association with perinatal hypothyroidism in the rat cerebellum. Rats were sacrificed at 15 day-old postnatal age (P15) and their cerebella were removed. Poly (A)+ RNA was extracted to construct a cDNA library using lambda gt 10 cloning vector. Differential plaque screening was then performed using 32P-labeled antisense cDNA synthesized from poly (A)+ RNA of the methimazole-treated (hypothyroid) P15 rat cerebellum (hypothyroid probe), and of the euthyroid P15 rat cerebellum (euthyroid probe). The clones, which hybridized strongly to the euthyroid probe and weakly or not at all to the hypothyroid probe, were isolated. Sequence analysis of these clones revealed that all isolated clones encode cytochrome c oxidase subunit I (COX I), which is located in the mitochondrial DNA. The decrease in COX I gene expression was not seen in the animals, which received methimazole treatment and daily replacement of thyroid hormone. In situ hybridization detection showed not only overall decrease in COX I gene expression but also change in distribution of hybridization signal in the cerebellar cortex of hypothyroid rat. Such change was not observed in the T4-replaced animals. Based on the evidence that thyroid hormone greatly influences brain development, the results of the present study indicate that the terminal enzyme of mitochondrial respiratory chain, COX I is one of the important target molecules regulated by thyroid hormone in the newborn rat cerebellum.

Animals↗

PubMed1995

In situ hybridization histochemistry of c-erbA alpha 2 mRNA in the hypothalamus and its surrounding structures in the adult male rat.

In order to detect the localization of c-erbA alpha 2 mRNA in the hypothalamus and its surrounding structures, in situ hybridization histochemistry was performed in the adult male rat. Sections through the hypothalamus were hybridized with a [3H]-labeled RNA probe complementary to c-erbA alpha 2 mRNA and then coated with photographic emulsion. Autoradiograms were then developed and localization of the hybridization signal was detected under a light microscope. Hybridization signal was detected throughout the hypothalamic and extrahypothalamic structures such as the paraventricular hypothalamic nucleus (PVN), ventromedial nucleus (VMN), arcuate nucleus (Arc), amygdala (Am), habenula (Hb), paraventricular thalamic nucleus (PVT) and hippocampus (Hipp). No hybridization signal was detected in the section which was hybridized with the sense probe. These results not only confirm previous findings in the PVN, Hb, PVT and Hipp, but also extend the finding to the VMN, Arc and Am, in which cells expressing c-erbA alpha 2 primary transcript were concentrated. These new findings may provide an important aid to the understanding of the roles of thyroid hormone in the function of the central nervous system.

Animals↗