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Biomedical subjects

S Igarashi

Publications and source records attributed to S Igarashi.

At least 55 records · Page 3Linked to original sources
PubMed2000

Hormonal regulation of estrogen receptor alpha and beta gene expression in human granulosa-luteal cells in vitro.

Estrogen is one of the major sex steroid hormones that is produced from the human ovary, and its actions are established to be a receptor-mediated process. Despite the demonstration of estrogen receptor (ER) expression, little is known regarding the regulation of ER in the human ovary. In the present study we investigated the expression and hormonal regulation of ERalpha and ERbeta in human granulosa-luteal cells (hGLCs). Using RT-PCR amplification, both ERalpha and ERbeta messenger ribonucleic acid (mRNA) were detected from hGLCs. Northern blot analysis revealed that ERalpha is expressed at a relatively lower level than ERbeta. Basal expression studies indicated that ERalpha mRNA levels remain unchanged, whereas ERbeta mRNA levels increased with time in culture in vitro, suggesting that ERbeta is likely to play a dynamic role in mediating estrogen action in hGLCs. The regulation of ERalpha and ERbeta expression by hCG was examined. hCG treatment (10 IU/mL) significantly attenuated the ERalpha (45%; P < 0.01) and ERbeta (40%; P < 0.01) mRNA levels. The hCG-induced decrease in ERalpha and ERbeta expression was mimicked by 8-bromo-cAMP (1 mmol/L) and forskolin (10 micromol/L) treatment. Additional studies using a specific protein kinase A (PKA) inhibitor (adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt) and an adenylate cyclase inhibitor (SQ 22536) further implicated the involvement of the cAMP/PKA signaling pathway in hCG action in these cells. The hCG-induced decrease in ERalpha and ERbeta mRNA levels was prevented in the presence of these inhibitors. Next, the effect of GnRH on ER expression was studied. Sixty-eight percent (P < 0.001) and 60% (P < 0.001) decreases in ERalpha and ERbeta mRNA levels, respectively, were observed after treatment with 0.1 micromol/L GnRH agonist (GnRHa). Pretreatment of the cells with a protein kinase C (PKC) inhibitor (GF109203X) completely reversed the GnRHa-induced down-regulation of ERalpha and ERbeta expression, suggesting the involvement of PKC in GnRH signal transduction in hGLCs. In agreement with the semiquantitative RT-PCR results, Western blot analysis detected a decrease in ERalpha and ERbeta proteins levels in hGLCs after treatment with hCG (10 IU/mL), GnRH (0.1 micromol/L), 8-bromo-cAMP (1 mmol/L), forskolin (10 micromol/L), or phorbol 12-myristate 13 acetate (10 micromol/L). Functionally, we demonstrated an inhibition of progesterone production in hGLCs in vitro by 17beta-estradiol, and this inhibitory effect was eliminated by pretreatment of 10 IU/mL hCG or 0.1 micromol/L GnRHa for 24 h before 17beta-estradiol administration. In summary, we observed a differential expression of ERalpha and ERbeta mRNA in hGLCs in vitro. The demonstration of hCG- and GnRHa-induced down-regulation of ERalpha and ERbeta gene expression suggests that hCG and GnRH may contribute to the control of granulosa-luteal cell function. Furthermore, our data suggest that the effects of hCG and GnRH on ERalpha and ERbeta expression in hGLCs are mediated in part by activation of PKA and PKC signaling pathways, respectively.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

A novel class of inhibitors for human and rat steroid 5alpha-reductases: synthesis and biological evaluation of indoline and aniline derivatives. III.

While searching for novel nonsteroidal inhibitors of human and rat prostatic 5alpha-reductases, we found a new series of indoline and aniline derivatives that showed potent inhibitory activities for both enzymes. Among them, 3-chloro-4-¿[1-(4-phenoxybenzyl)indolin-5-yl]oxylbenzoic acid (2e, YM-36117) showed a more potent inhibitory activity for the human enzyme than ONO-3805 with an IC50 value of 5.3 nM and a reduced rat prostatic dihydrotestosterone (DHT) concentration by oral administration. The synthesis and the structure-activity relationships of these indoline and aniline derivatives are presented.

5-alpha Reductase Inhibitors↗
PubMed2000

A novel class of inhibitors for human steroid 5alpha-reductase: synthesis and biological evaluation of indole derivatives. II.

In a search for novel nonsteroidal inhibitors of human prostatic 5alpha-reductase, we found a new series of indole derivatives that showed potent inhibitory activities for the human enzyme. Among them, 4-[(1-benzyl-1H-indol-5-yl)oxyl-3-chlorobenzoic acid (2d, YM-32906) showed more potent inhibitory activity than finasteride with an IC50 value of 0.44 nM. 3-Chloro-4-[[1-(4-phenoxybenzyl)-1H-indol-5-yl]oxy]benzoic acid (2m) showed inhibitory activities for both human and rat prostatic 5alpha-reductase with IC50 values of 2.1 and 73 nM, respectively. The synthesis and structure-activity relationships of these indole derivatives are presented.

5-alpha Reductase Inhibitors↗
PubMed2000

Food deprivation depletes gastric mucus glycoprotein in streptozotocin-induced diabetic rats.

Fasting causes gastric mucosal damage in streptozotocin (STZ)-induced diabetic rats, but its pathogenic mechanism remains to be elucidated. The aim of the present study was to investigate the alteration of gastric mucosal mucin, one of the gastric defensive factors against the development of such damage. Diabetes was induced in rats by intravenous injection of STZ (65 mg/kg). The experiments were performed using 4-week STZ-diabetic rats with blood glucose levels above 350 mg/dl. The amount of gastric mucus glycoprotein was determined by gel filtration, and the distribution of neutral and acidic mucins in the stomach epithelium was examined by histochemical analysis. In normal rats, 24-h fasting neither affected the gastric mucin content nor caused any macroscopic gastric mucosal injury. In contrast, starvation significantly reduced the amount of total gastric mucus glycoprotein prior to the formation of mucosal lesions in the STZ-diabetic rats. Nine hours after food deprivation, the gastric damage developed in about 70% of the diabetic rats, the amount of mucus glycoprotein markedly decreased, and both the neutral and acidic mucins diminished in the epithelium. Taken together, in STZ-diabetic rats, fasting by itself depletes gastric mucus glycoprotein, and this depletion may be involved in the pathogenic mechanism of the formation of gastric mucosal lesions.

Analysis of Variance↗
PubMed2000

[A patient with gastric cancer with para-aortic lymph node metastases surviving for 9 years after effective preoperative chemotherapy and radical operation].

A 63-year-old male patient with type 3 advanced gastric cancer was referred to our hospital. Preoperative examination by CT-scan revealed swollen para-aortic lymph nodes and cancer invasion to the pancreas. The patient was treated pre-operatively with intravenous 5-FU, 500 mg/body/day, continuous infusion for 1 week. Immediately after the chemotherapy, the patient underwent total gastrectomy, splenctomy, left-adrenectomy and resection of the body and tail of the pancreas, along with para-aortic lymph node dissection. Microscopic examination revealed that the tumor was a moderately differentiated tubular adenocarcinoma, which displayed invasion to the pancreas with lymph node metastasis up to the level 3 lymph node. Histologically, the effect of preoperative chemotherapy showed a grade 2 effect on the main tumor, but a grade 3 chemotherapeutic effect was observed at the para-aortic lymph nodes. The patient has subsequently remained disease free for 9 years. In this case, it is considered that the preoperative chemotherapy by 5-FU and potentially curative radical operation yielded a good outcome.

Adenocarcinoma↗
PubMed1999

Construction and characterization of mutant water-soluble PQQ glucose dehydrogenases with altered K(m) values--site-directed mutagenesis studies on the putative active site.

Based on a PCR mutant enzyme of water-soluble glucose dehydrogenase-harboring pyrroloquinoline quinone as the prosthetic group, PQQGDH-B, a site-directed mutagenesis study was carried out. The substitution of Glu277 residue with Gly resulted in a decrease in the K(m) value for glucose and altered the substrate specificity profile, compared with the wild-type enzyme. Mutational analyses on the neighboring amino acid residues of Glu277 were also carried out and constructed Asp275Glu, Asp276Glu, Ile278Phe, and Asn279His. Considering that Asp275Glu, Asp276Glu and also Glu277Gly showed drastic decreases in EDTA tolerance, this region may construct a PQQGDH-B putative active site, such as a binding site for Ca(2+), which is responsible for the binding PQQ. A series of Glu277 variants, Glu277 substituted by Ala, Val, Asp, Asn, His, Gln, or to Lys, was constructed and they all showed decreased K(m) values and altered substrate specificity profiles. Among them, Glu277Lys showed similar thermal stability with the wild-type enzyme, but its catalytic efficiency increased significantly, compared with the wild-type enzyme. The potential applications of Glu277Lys in analytical use are also discussed.

Amino Acid Sequence↗
PubMed1999

14-3-3 protein eta chain gene (YWHAH) polymorphism and its genetic association with schizophrenia.

Recent genetic analyses have suggested a linkage between schizophrenia and the chromosomal region 22q12-q13. 14-3-3 protein, abundant in the brain, mediates interactions between diverse molecules of biological activities; its gene was recently mapped to chromosome 22q12.1-q13.1. We therefore investigated allele frequencies of a variable number of tandem repeat (VNTR) in the 5'-noncoding region of the 14-3-3 eta chain gene in controls and schizophrenics. The frequencies of the two-repeat allele were significantly higher (P < 0.05) in the schizophrenics, and particularly in those with onset before age 22 (early-onset schizophrenics, P < 0.02), than in the controls. The odds ratio was significantly increased in the early-onset schizophrenics homozygous for the two-repeat allele (OR = 3.3, 95% CI = 1.1-9.7). The 14-3-3 eta chain gene is a potential susceptibility gene for schizophrenia, and particularly for early-onset schizophrenia.

14-3-3 Proteins↗
PubMed1999

Spectrophotometric determination of trace amounts of hemoglobin using the oxidative decomposition reaction of a copper(II)-phthalocyanine complex.

A catalytic effect of hemoglobin was found in the oxidative decomposition of a copper(II)-phthalocyanine complex (Cu-pts) with peroxomonosulfate. Based on this finding, a simple, rapid, highly sensitive and high-precision spectrophotometric method for hemoglobin was developed. In this indicator system, Cu-pts as the indicator and hemoglobin as the catalyst were simultaneously decomposed with peroxomonosulfate. Therefore, the slopes of the absorbance-time curve of the sample solution become equal to that of the blank solution after 5 min. The calibration curve was linear over the range of 12-258 microg/l. Detection limits (3sigma) were 8.13 microg/l, and the relative standard deviation was 2.05% (10 determinations) for 130 microg/l of hemoglobin. The metal ions did not significantly produce an interference in this method. The albumin had some influence but was permissible up to 6 times the weight of hemoglobin. The proposed method was applied to human blood samples with satisfactory results.

Calibration↗
PubMed1999

Immunohistochemical assessment of superoxide dismutase expression in the endometrium in endometriosis and adenomyosis.

OBJECTIVE: To determine the expression of superoxide dismutase (SOD) in the endometrium during the menstrual cycle in endometriosis and adenomyosis. DESIGN: Immunohistochemical identification of SOD in endometrial tissues using the monoclonal antibody. SETTING: Department of obstetrics and gynecology in a university hospital. PATIENT(S): The subjects were divided into three groups: 36 patients with endometriosis, 38 patients with histologically proven adenomyosis, and 47 fertile control subjects. INTERVENTION(S): Endometrium was biopsied throughout the menstrual cycle. MAIN OUTCOME MEASURE(S): Semiquantitative immunostaining (evaluation nomogram) score for endometrial cells. RESULT(S): The analyses revealed phase-dependent changes in the expression of SODs in the glandular and surface epithelia during the menstrual cycle in fertile controls. Specifically, the expression of copper, zinc SOD was weakest in the early and midproliferative phases, then gradually increased, and was most marked in the early and midsecretory phases. The expression of manganese SOD reached a peak in the late secretory phase. The expression of both SODs in endometriosis and adenomyosis was persistently higher than the control levels throughout the menstrual cycle. CONCLUSION(S): The exaggerated expression of both SODs in the endometrium throughout the menstrual cycle suggests that superoxide plays a key role in infertility in endometriosis and adenomyosis.

Adult↗
PubMed1999

Interobserver and intraobserver variation in endoscopic assessment of GERD using the "Los Angeles" classification.

BACKGROUND: A new endoscopic classification of gastroesophageal reflux disease (GERD) has been proposed, and the term mucosal break has been introduced to describe mucosal damage. This new classification was evaluated by endoscopists with different levels of experience. METHODS: Fifty endoscopic photographs for each of 20 randomly selected patients with GERD were assessed retrospectively by three groups of seven endoscopists classified by experience: group 1 (100 to 500 procedures), group 2 (500 to 3000), and group 3 (more than 3000). The new classification was modified by adding grade 0 to describe healed mucosal breaks, so that there were five grades. All photographs were assessed twice at an interval of more than 1 week, and kappa statistics were used to determine observer variation. RESULTS: Interobserver variation within group 3 (kappa = 0.39, n = 21) and between groups 3 and 2 (kappa = 0.36, n = 49) was significantly different (p < 0.01) from that between groups 3 and 1 (kappa = 0.26, n = 49). Intraobserver variation in group 1 (kappa = 0.39, n = 7) was significantly different (p < 0.01) from that in group 2 (kappa = 0.51, n = 7) and group 3 (kappa = 0.54, n = 7). CONCLUSIONS: Observer variation depends on level of endoscopic experience. Only experienced endoscopists should use the new classification for grading of GERD.

Adult↗
PubMed1999

Role of endogenous cathepsin D-like and chymotrypsin-like proteolysis in human epidermal desquamation.

Even though the skin surface is acidic (about pH 5), most in vitro studies on desquamation have been performed at alkaline pH. We demonstrate that the standard in vitro model system, which achieves squame shedding upon incubation of plantar stratum corneum for 1 day in an alkaline buffer that must include a chelating agent, can be extended to a more realistic model in which the incubation is for 4 days, at varying pHs from 5 to 8, without exogenous chelators. Desmoglein I from stratum corneum was degraded by the squames shed at pH 5 as well as at pH 8. Squame shedding was inhibited to varying extents by the addition of proteinase inhibitors, whose specificity suggested that the crucial enzymatic activity at pH 8 was a chymotrypsin-like serine proteinase, while a similar activity at pH 5 was accompanied by an aspartic proteinase activity of comparable strength. Four degradation peaks were observed when the insulin B chain was reacted with shed squames at pH 5. Two of these peptides were suppressed by the addition of phenylmethylsulphonyl fluoride, the other two by pepstatin A; chymostatin inhibited all four, but E-64 and leupeptin showed no effect. The implied specificity was confirmed by reacting the insulin (without squames) with the standard enzymes human liver cathepsin D and pancreatic chymotrypsin, reproducing the expected degradation products. These results suggest that epidermal desquamation at acidic pH requires two proteolytic activities, one of which is an analogue of chymotrypsin and the other of cathepsin D. Endogenous proteinases corresponding to these activities have been previously identified, namely the stratum corneum chymotryptic enzyme and the mature active form of cathepsin D.

Blotting, Western↗
PubMed1999

Improved fertility through superoxide removal by Toki-shakuyaku-san in mice--a preliminary study.

The effect of a superoxide dismutase inhibitor on pregnancy rate and fetal survival, and the effect of concomitant treatment with Toki-shakuyaku-san, was studied. Twelve week-old IV CS female mice were given diethyldithiocarbamate (DEDC; 0.9, 9, 45, and 225 mg kg(-1)) intraperitioneally, from Day 3 to Day 5 of pregnancy, every 12 h, for a total of six times. Two additional groups received both 45 mg kg DEDC and Toki-shakuyaku-san (50 or 200 mg/100 mL), by water bottle, from 12 h before to 12 h after DEDC administration. For each group, the pregnancy rate, the number of surviving fetuses, and the number of resorbed fetuses was calculated. Pregnancy rates were 95.5, 81.8, 54.5 and 0%, respectively, in the DEDC groups and were 100% in the control group. The fetal resorption rate was 0.9 in the control group, and 1.0, 1.7, and 2.3, respectively, in the 0.9, 9, 45 mg kg(-1) DEDC groups. Administration of 200 mg Toki-shakuyaku-san significantly (P<0.05) improved the pregnancy rate to 81.8%, and improved the degree of reduced intercellular interactions and insufficient decidual reactions found on Day 5 in the endometrium of the DEDC groups. Toki-shakuyaku-san reduces the adverse effects of excess superoxides on the endometrium during implantation.

Animals↗
PubMed1999

Optimal levels of nitric oxide are crucial for implantation in mice.

This study was performed to clarify the critical role of optimal levels of nitric oxide on fecundity in mice during the implantation period. Mature female pregnant mice were treated with either nitric oxide donor molsidomine (3, 15, 60 mg kg(-1)) or nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-name; 0.3, 1.5, 6 mg kg(-1)) every 12 h, seven times from the night of Day 2 to Day 5 of gestation. They were killed on Day 14 of gestation. Pregnancy rates in each group (n = 22) and the number of live or absorbed fetuses in each mouse was calculated. The pregnancy rates in the experimental group were reduced in a dose-dependent manner. The rate in the control group was 100%, whereas those in the 60-mg molsidomine and 6-mg L-name groups were 40.9 and 31.8%, respectively. Histological analysis of uteri on Day 5 of gestation after treatment with 60 mg molsidomine or 6 mg L-name suggested retarded decidualization of stromal cells or defective function of predecidualized cells. In conclusion, optimal levels of nitric oxide are crucial for endometrial function and embryo implantation.

Animals↗
PubMed1999

Transgenic mice harboring a full-length human mutant DRPLA gene exhibit age-dependent intergenerational and somatic instabilities of CAG repeats comparable with those in DRPLA patients.

Dentatorubral-pallidoluysian atrophy (DRPLA) is one among an increasing number of hereditary neurodegenerative diseases determined as being caused by unstable expansion of CAG repeats coding for polyglutamine stretches. To investigate the molecular mechanisms underlying CAG repeat instability, we established three transgenic lines each harboring a single copy of a full-length human mutant DRPLA gene carrying a CAG repeat expansion. These transgenic mice exhibited an age-dependent increase (+0.31 per year) in male transmission and an age-dependent contraction (-1.21 per year) in female transmission. Similar tendencies in intergenerational instabilities were also observed in human DRPLA parent-offspring pairs. The intergenerational instabilities of the CAG repeats may be interpreted as being derived from the instability occurring during continuous cell division of spermatogonia in the male, and that occurring during the period of meiotic arrest in the female. The transgenic mice also exhibited an age-dependent increase in the degree of somatic mosaicism which occurred in a cell lineage-dependent manner, with the size range of CAG repeats being smaller in the cerebellum than in other tissues including the cerebrum, consistent with observations in autopsied tissues of DRPLA patients. Thus, the transgenic mice described in this study exhibited age-dependent intergenerational as well as somatic instabilities of expanded CAG repeats comparable with those observed in human DRPLA patients, and are therefore expected to serve as good models for investigating the molecular mechanisms of instabilities of CAG repeats.

Adult↗
PubMed1999

Adenovirus-mediated expression of mutant DRPLA proteins with expanded polyglutamine stretches in neuronally differentiated PC12 cells. Preferential intranuclear aggregate formation and apoptosis.

To investigate the molecular mechanisms of neurodegeneration caused by expanded CAG repeats in dentatorubral-pallidoluysian atrophy (DRPLA), an autosomal dominant neuro degrees enerative disorder caused by unstable expansion of a CAG trinucleotide repeat in the DRPLA gene on 12p13.31, we established an efficient expression system for truncated and full-length DRPLA proteins with normal or expanded polyglutamine stretches in neuronally differentiated PC12 cells and fibroblasts using an adenovirus expression system. Although aggregate body formation was observed both in neuronally differentiated PC12 cells and in fibroblasts expressing truncated DRPLA proteins with Q82, >97% ( n = 3) of neuronally differentiated PC12 cells showed intra-nuclear inclusions, while only 31 21% ( n = 3) of fibro-blasts had intranuclear inclusions at 3 days after infection. The percentage of apoptotic cells was significantly higher in neuronally differentiated PC12 cells expressing the truncated DRPLA protein with Q82 than in fibroblasts, suggesting the possibility that intranuclear aggregate bodies are formed preferentially in neuronally differentiated PC12 cells and that these cells are more vulnerable than fibroblasts to the toxic effects of expanded polyglutamine stretches in the DRPLA protein. When the full-length DRPLA protein with Q82 was expressed, aggregate bodies were found exclusively in the nuclei of the neuronally differentiated PC12 cells, while they were found in the cytoplasm of fibroblasts. Despite the presence of aggregate bodies, apoptosis was not induced by expression of the full-length DRPLA protein with Q82 in either neuronally differentiated PC12 cells or fibroblasts, suggesting that the presence of intranuclear aggregate bodies is in itself not necessarily toxic to cells.

Adenoviridae↗
PubMed1999

A non-familial Huntington's disease patient with grumose degeneration in the dentate nucleus.

We report a non-familial Huntington's disease (HD) patient presenting with increased levels of protein and IgG in his cerebrospinal fluid (CSF), antineuronal antibody in his serum and CSF, Purkinje cell and granule cell degeneration in the cerebellum, and grumose degeneration in the dentate nucleus, in addition to typical HD findings. This patient showed an expanded CAG repeat in the HD gene, and provides new information on the clinical and neuropathologic varieties of HD.

Aged↗
PubMed1999

Pigmented hidrocystoma of the eccrine secretory coil in the vulva: clinicopathologic, immunohistochemical and ultrastructural studies.

A case of pigmented hidrocystoma of eccrine secretory coil is presented. A 47-year-old woman had developed a bluish black small nodule in the anterior portion of the labium minor a few years before entry. Microscopically, the cyst was lined by eosinophilic columnar epithelium with abundant brownish granules. There was a vague suggestion of decapitation secretion focally in the epithelial layer of cuboidal cells. This layer expressed distinct reactivity against CA19-9 with no reactivity for human milk fat globule-1 (HMFG-1). These features demonstrated that the cyst was not of apocrine nature but of eccrine derivation. In addition, positive immunoreaction for cytokeratin (CK)7, CK8 and CK19 defined the cyst as originating from the secretory coil of the sweat gland. Ultrastructurally, melanosomes in various stages were identified in most of the epithelial cells. These findings suggest that the present case was a hidrocystoma of eccrine secretory coil with abnormal melanin accumulation.

Biomarkers, Tumor↗