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Biomedical subjects

O Tsutsumi

Publications and source records attributed to O Tsutsumi.

At least 19 recordsLinked to original sources
PubMed2001

Molecular cloning and characterization of mouse EBAG9, homolog of a human cancer associated surface antigen: expression and regulation by estrogen.

We previously identified a human estrogen-responsive gene, EBAG9 (ER-binding fragment-associated antigen9) (Watanabe, T. et al., Mol. Cell. Biol. 18, 442-449, 1998). It was later reported as RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) that induced apoptosis and suppressed the growth of several cells such as activated T cells (Nakashima, M. et al., Nat. Med. 5, 938-942, 1999). Here, we have isolated both cDNA and genomic DNA of mouse EBAG9/RCAS1. Mouse EBAG9 gene spans about 30 kb in genomic DNA and consists of 7 exons. Mouse EBAG9 cDNA encodes a protein that contains the transmenbrane segment and coiled-coil domain. An alignment between the predicted mouse and human EBAG9 shows a high degree of homology at the amino acid level (98%). Northern and Western blot analyses demonstrate that EBAG9 is expressed in several tissues including the heart, brain, spleen, liver, kidney, and testis, and also in developing embryo. In the uterus, a target organ for estrogen, the EBAG9 was shown to be upregulated in vivo by 17beta-estradiol. To determine the biological action of mouse EBAG9, NIH3T3 fibroblastic cells were incubated with recombinant EBAG9 protein, resulting in suppression of cell growth. These findings suggest that EBAG9 is an in vivo estrogen-responsive gene that inhibits the cell growth.

3T3 Cells↗
PubMed2001

Effects of pulsed ultrasound on development and glucose uptake of preimplantation mouse embryos.

The effects of pulsed ultrasound (US) on early mouse embryos were investigated. Two-cell embryos contained in oviducts were irradiated to US (1.875 MHz with an I (SPTA) of 2.96 W/cm(2)) in an experimental unit for either 1 or 5 min (exposure groups). The embryos were cultured to examine the rate of developing to blastocysts, and the uptake of 2-deoxyglucose (2-DG) into blastocysts was measured to evaluate their viability. The rates in the exposure groups were essentially the same, with those of the embryos treated similarly in the unit unless being exposed to US (nonexposure groups). However, they were lower than that of embryos not treated in the experimental unit (a control group). There were no significant differences of 2-DG uptake among the 1-min exposure, 1-min nonexposure, and control groups. The uptake in the 5-min exposure group did not differ from that in the 5-min nonexposure group; however, uptake in both groups was lower than that in the control group. Pulsed US for 1 min did not affect viability of preimplantation mouse embryos.

Animals↗
PubMed2001

Usefulness of long-jaw forceps in laparoscopic cornual resection of interstitial pregnancies.

Although laparoscopic surgery could be an option for treating interstitial pregnancies, it has not gained wide acceptance largely because of difficulty achieving hemostasis. To overcome this, we employed long-jaw forceps in three cases of interstitial pregnancy that were successfully treated by laparoscopic cornual resection. The forceps grasped a relatively large portion of pregnant myometrium without slipping, thus securing hemostasis and facilitating suturing.

Adult↗
PubMed2001

Caspase-2 deficiency prevents programmed germ cell death resulting from cytokine insufficiency but not meiotic defects caused by loss of ataxia telangiectasia-mutated (Atm) gene function.

It is well established that programmed cell death claims up to two-thirds of the oocytes produced during gametogenesis in the developing fetal ovaries. However, the mechanisms underlying prenatal germ cell loss in females remain poorly understood. Herein we report that caspase-11 null female mice are born with a reduced number of oocyte-containing primordial follicles. This phenotype is likely due to failed cytokine processing known to occur in caspase-11 mutants since neonatal female mice lacking both interleukin (IL)-1alpha and IL-1beta also exhibit a reduced endowment of primordial follicles. In addition, germ cell death in wild-type fetal ovaries cultured ex vivo is suppressed by either cytokine, likely via ligand activation of type 1 IL-1 receptors expressed in fetal germ cells. Normal oocyte endowment can be restored in caspase-11 null female mice by simultaneous inactivation of the gene encoding the cell death executioner enzyme, caspase-2. However, caspase-2 deficiency cannot overcome gametogenic failure resulting from meiotic recombination defects in ataxia telangiectasia-mutated (Atm) null female mice. Thus, genetically distinct mechanisms exist for developmental deletion of oocytes via programmed cell death, one of which probably functions as a meiotic quality-control checkpoint that cannot be overridden.

Animals↗
PubMed2001

Evidence for the expression of interleukin (IL)-18, IL-18 receptor and IL-18 binding protein in the human endometrium.

To see whether the interleukin (IL)-18 system is operative in the endometrium, we examined the expression of IL-18, IL-18 receptor (IL-18R) and IL-18 binding protein (IL-18BP), the substance known to neutralize IL-18 activity, in this tissue. Reverse transcription-polymerase chain reaction analyses showed that IL-18, IL-18R and IL-18BP mRNA were constitutively expressed without significant fluctuation throughout the menstrual cycle. When epithelial cells and stromal cells were cultured separately, the expression levels of IL-18 mRNA in epithelial cells were about 18-fold higher compared to those in stromal cells. Furthermore, the IL-18 precursor protein was detected by Western blot analysis in cultured epithelial cells but not in stromal cells. Recombinant human IL-18 stimulated the secretion of interferon (IFN)-gamma by resident bone marrow-derived cells in the endometrium. On the other hand, IFN-gamma up-regulated the IL-18BP expression both in cultured epithelial cells and stromal cells. Thus, we have presented evidence for the presence of the IL-18 system in the human endometrium. In light of its immunomodulatory roles in a variety of tissues, this system may afford protection against pathogenic micro-organisms and provide a regulatory mechanism for controlled trophoblast invasion by modulating a local cytokine network.

Bone Marrow Cells↗
PubMed2001

[Establishment of an optimum bowel preparation method before gynecologic laparoscopic surgery].

OBJECTIVE: To establish a new method for preoperative bowel preparation that facilitates nursing care and minimizes the patient's discomfort during the clinical pathway of laparoscopic surgery. METHOD: A randomized controlled trial was conducted for the following two preparation methods. Twenty cases were assessed with Method 1 and 18 cases with Method 2. Method 1 (the conventional procedure): oral magnesium citrate is given in the afternoon of the day before surgery, followed by a glycerin enema in the night of the day before surgery and in the morning of the day of surgery. Method 2 (a new procedure): oral magnesium citrate is given in the afternoon of the day before surgery, followed by oral picosulfate in the night before the day of surgery and a bisacodyl suppository in the morning of the day of surgery. To evaluate the two methods we sent questionnaires to the surgeons (blinded to the method used), nurses, and patients. RESULTS: No statistical difference existed between the two methods in their effectiveness as a preoperative treatment. Facilitation of nursing care was significantly better in Method 2, and patients had considerably reduced discomfort with Method 2. DISCUSSION: Patients who received oral picosulfate and a bisacodyl suppository experienced much less discomfort and nursing care was easier when compared with the conventional method of administering a glycerin enema. Since an enema is disliked by young women and an effect comes out with discomfort very shortly after the administration, the degree of discomfort of patients would have become high. Picosulfate is an oral medicine and thereby the effect comes out mildly. That would be the reason why the degree of discomfort of patients was low. In the nursing care, an enema requires time for preparation and administration, while picosulfate is easy to administer, making the nursing care easier. Therefore, Method 2 was chosen as a preoperative bowel treatment for the clinical pathway. Thus, we could establish a new evidence-based method useful for the preoperative bowel preparation in the clinical pathway of laparoscopic surgery.

Adult↗
PubMed2001

Evidence for the presence of keratinocyte growth factor (KGF) in human ovarian follicles.

The presence of keratinocyte growth factor (KGF) in human follicular fluid (FF) was investigated in a total of 145 FFs obtained during oocyte retrieval for in vitro fertilization (IVF) from 29 patients with no apparent endocrine disorders. The concentrations of KGF, estradiol, progesterone, testosterone and human chorionic gonadotropin (hCG) in FF were measured by enzyme-linked immunosorbent assay. FF samples contained relatively higher amounts of KGF (2194+/-87 pg/ml), whereas its concentrations in serum were below assay limit (<31.2 pg/ml). Concentrations of KGF in FF were positively correlated with both progesterone (r=0.311, p<0.0005) and testosterone (r=0.230, p<0.01) concentrations in FF. However, KGF concentrations were not significantly correlated with estradiol and hCG concentrations. KGF in FF was detected as a broad band (26-29 kD) by immunoblotting, the size being reduced by 7kD after N-glycosidase treatment. In an in vitro experiment, KGF suppressed the basal and hCG-stimulated progesterone production by cultured human luteinized granulosa cells. summary, we demonstrated the presence of KGF in human ovarian follicles, suggesting its possible role as a local factor in regulating human ovarian functions.

Adult↗
PubMed2000

An estrogen receptor beta isoform that lacks exon 5 has dominant negative activity on both ERalpha and ERbeta.

An alternatively spliced isoform of human estrogen receptor beta (ERbeta) has been isolated from normal human testis mRNA that is coexpressed with wild-type ERbeta by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis of the ERbeta isoform PCR product reveals the absence of 139 bp that corresponds to the entire exon 5 of wild-type ERbeta, which predicts to lack part of the hormone-binding domain. The transient expression of the exon 5-deleted isoform of ERbeta (ERbetaDelta5) had no effect on basal transactivation activity of an estrogen-responsive luciferase reporter gene. This finding was in contrast to the previous reports that the exon 5-deleted isoform of ERalpha (ERalphaDelta5) acts as a dominant positive receptor, increasing basal gene transactivation itself. Moreover, when ERbetaDelta5 was cotransfected with the wild-type ERalpha or ERbeta, it behaved as a dominant negative receptor that inhibited not only estradiol-stimulated transactivation by ERbeta but also that by ERalpha. The ligand-independent nuclear localization of ERbetaDelta5 was confirmed by immunohistochemistry, and the coexpression of the isoform and the wild-type receptors could be observed in a single cell that transfected with both receptor cDNAs. These findings indicate that ERbetaDelta5 has a potential as a dominant negative receptor that blocks both ERalpha and ERbeta signaling pathways, suggesting some physiological roles of this isoform as an "ER inhibitor".

Alternative Splicing↗
PubMed2000

Human NB-2 of the contactin subgroup molecules: chromosomal localization of the gene (CNTN5) and distinct expression pattern from other subgroup members.

NB-2 is one of the neural recognition molecules in the contactin subgroup, which belongs to the immunoglobulin superfamily. In rat, the six molecules in this subgroup that have been reported to date are contactin, TAG-1, BIG-1, BIG-2, NB-2, and NB-3. We have isolated cDNAs encoding the two splicing isoforms of human NB-2. The long isoform of human NB-2 consists of 1100 amino acids residues that are 91% homologous to rat NB-2 at the amino acid sequence level. The short isoform lacks 74 amino acid residues between residues 19 and 93 of the long isoform. Among various regions of the adult human brain, high-level expression of NB-2 was detected in the amygdala and occipital lobe, whereas expression was low in the corpus callosum, caudate nucleus, and spinal cord. Although there were some differences, the expression pattern of NB-2 was the most similar to that of BIG-1 in the brain. Likewise, contactin and BIG-2 exhibited similar expression patterns. The expression of TAG-1 showed the least regional differences. The human NB-2 gene (CNTN5) was mapped to chromosome 11q21-q22.2 by fluorescence in situ hybridization. Our results suggest that the NB-2 gene may contribute to human neurological disorders.

Alternative Splicing↗
PubMed2000

Promoter analysis and chromosomal mapping of human EBAG9 gene.

The human EBAG9 was previously identified as an estrogen responsive gene using CpG-genomic binding site cloning (Watanate et al., (1998) Mol. Cell. Biol. 18: 442-449). Recently it was revealed that the EBAG9 is identical with RCAS1 which is a cancer cell surface antigen implicated in immune escape. Here, we isolated and analyzed the 5'-flanking region of human EBAG9 gene. We determined transcription initiation site, which has a homology with an initiator element YYCAYYYY, and found that TATA motif was absent. Deletion analysis of the 5'-flanking region using MCF-7 breast cancer cells indicated that the sequences -86 to -36 containing the ERE had the basal level of promoter activity and the upstream GC-rich region positively regulated the activity. EBAG9 promoter luciferase reporters containing the ERE could respond to estrogen, and electrophoretic mobility shift assay showed that ERalpha bound to the ERE. Moreover, fluorescent in situ hybridization analysis has shown that the human EBAG9 gene is located at chromosome 8q23 which is frequently amplified in tumors. These findings suggest that the human EBAG9 might be involved in carcinogenesis as an estrogen responsive gene.

Base Sequence↗
PubMed2000

Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos.

The effects of bisphenol A, a xenoestrogen widely used in industry and dentistry, were studied in early preimplantation mouse embryos. Two-cell mouse embryos were cultured with 100 pM to 100 microM bisphenol A with or without 100 nM tamoxifen and evaluated at 24-h intervals for their development to eight-cell and blastocyst stages. At 72 h, blastocysts were cultured for another 48 h without bisphenol A, and surface areas of trophoblast spread were measured. At 24 h, more embryos exposed to 3 nM bisphenol A than to controls had reached the eight-cell stage. At 48 h, more embryos exposed to 1 nM and 3 nM bisphenol A than to controls had become blastocysts. At 100 microM, bisphenol A decreased frequency of development to blastocysts. Tamoxifen counteracted both stimulatory and inhibitory effects of bisphenol A on blastocyst formation. Although bisphenol A did not alter blastocyst morphology or cell number, early exposure to 100 microM bisphenol A increased subsequent trophoblast areas. These findings suggest that bisphenol A may not only effect early embryonic development via estrogen receptors even at low, environmentally relevant doses, but also exert some late effects on subsequent development of these embryos.

Animals↗
PubMed2000

Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.

Hormone-sensitive lipase (HSL) is known to mediate the hydrolysis not only of triacylglycerol stored in adipose tissue but also of cholesterol esters in the adrenals, ovaries, testes, and macrophages. To elucidate its precise role in the development of obesity and steroidogenesis, we generated HSL knockout mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutant HSL allele (HSL-/-) were superficially normal except that the males were sterile because of oligospermia. HSL-/- mice did not have hypogonadism or adrenal insufficiency. Instead, the testes completely lacked neutral cholesterol ester hydrolase (NCEH) activities and contained increased amounts of cholesterol ester. Many epithelial cells in the seminiferous tubules were vacuolated. NCEH activities were completely absent from both brown adipose tissue (BAT) and white adipose tissue (WAT) in HSL-/- mice. Consistently, adipocytes were significantly enlarged in the BAT (5-fold) and, to a lesser extent in the WAT (2-fold), supporting the concept that the hydrolysis of triacylglycerol was, at least in part, impaired in HSL-/- mice. The BAT mass was increased by 1.65-fold, but the WAT mass remained unchanged. Discrepancy of the size differences between cell and tissue suggests the heterogeneity of adipocytes. Despite these morphological changes, HSL-/- mice were neither obese nor cold sensitive. Furthermore, WAT from HSL-/- mice retained 40% of triacylglycerol lipase activities compared with the wild-type WAT. In conclusion, HSL is required for spermatogenesis but is not the only enzyme that mediates the hydrolysis of triacylglycerol stored in adipocytes.

Adipocytes↗
PubMed2000

Basal leptin concentrations in women with normal and dysfunctional ovarian conditions.

OBJECTIVE: To determine whether leptin is involved in ovarian function. METHODS: Fasting serum samples were obtained from 20 women with normal menstrual cycles who were either obese or non-obese: 12 non-obese patients with polycystic ovary syndrome (PCOS), 8 obese patients with PCOS, 10 patients with stress-related hypothalamic amenorrhea, and 8 patients with weight loss-related hypothalamic amenorrhea. RESULTS: Serum leptin levels were strongly related to body mass index (BMI) in each group, but there was no difference in the mean serum leptin levels among the BMI-matched study groups. A significant difference in the mean serum leptin levels was found between the non-obese and obese control groups (P<0.001) and between the non-obese and obese PCOS groups (P<0.001). CONCLUSIONS: These findings indicate that circulating leptin levels in women with normal menstrual cycles and those with ovarian dysfunction are strongly related to BMI. Leptin does not appear to be primarily involved in regulating ovarian function.

Adult↗