Biomedical subjects

R Konno

Publications and source records attributed to R Konno.

At least 19 recordsLinked to original sources

PubMed2006

Behavioral and biochemical characterization of a mutant mouse strain lacking D-amino acid oxidase activity and its implications for schizophrenia.

D-amino acid oxidase (DAO) degrades D-serine, a co-agonist at the NMDA receptor (NMDAR). Hypofunction of the NMDAR has been suggested to contribute to the pathophysiology of schizophrenia. Intriguingly, DAO has been recently identified as a risk factor for schizophrenia through genetic association studies. A naturally occurring mouse strain (ddY/DAO-) has been identified which lacks DAO activity. We have characterized this strain both behaviorally and biochemically to evaluate DAO as a target for schizophrenia. We have confirmed that this strain lacks DAO activity and shown for the first time it has increased occupancy of the NMDAR glycine site due to elevated extracellular D-serine levels and has enhanced NMDAR function in vivo. Furthermore, the ddY/DAO- strain displays behaviors which suggest that it will be a useful tool for evaluation of the clinical benefit of DAO inhibition in schizophrenia.

Acoustic Stimulation↗

PubMed2006

Alterations in D-amino acid levels in the brains of mice and rats after the administration of D-amino acids.

To mutant ddY/DAO(-) mice lacking D-amino-acid oxidase activity and normal ddY/DAO(+) mice, five D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu and D-Pro) were orally administered for two weeks, and the D-amino acid levels were examined in seven brain regions. The levels of D-Asp markedly increased in the pituitary and pineal glands in both strains. In the ddY/DAO(+) mice, the levels of the other D-amino acids did not significantly change in most of the brain regions. While in the ddY/DAO(-) mice the levels of D-Ser significantly increased in most of the brain regions except for the cerebrum and hippocampus. The levels of D-Ala and D-Leu increased in all regions but the levels of D-Pro did not significantly change. The same five D-amino acids were intravenously injected into Wistar rats and the D-amino acid levels in their brains were examined for 60 min after the administration. The levels of D-Asp markedly increased in the pineal gland 3 min after the administration, while the levels of D-Ser, D-Ala, and D-Pro increased both in the pineal and pituitary glands, the levels of D-Leu increased in all brain regions. These results are useful for the elucidation of the origins and regulation of D-amino acids in the mammalian body.

Amino Acids↗

PubMed2006

C-kit overexpression in neuroendocrine small cell carcinoma of the uterine cervix.

PURPOSE OF INVESTIGATION: Neuroendocrine small cell carcinoma of the uterine cervix (NESCC) grows aggressively, and is resistant to anticancer agents and radiation, having an extremely poor prognosis. The incidence of c-kit proto-oncogene overexpression is high in gastrointestinal stromal tumors (GISTs) and small cell lung cancer, and tyrosine kinase inhibitors have been used effectively to treat GISTs. Few studies have investigated whether c-kit is overexpressed in NESCC. To investigate whether NESCC can be a target for molecular targeted therapy with tyrosine kinase inhibitors, we examined the expression of c-kit in this tumor. METHODS: Twenty-one NESCCs were examined for c-kit expression by immunohistochemical staining using the labeled streptavidin-biotin complex (LSAB) method. The expression of c-kit was regarded as positive (overexpression) and negative when the membrane and cytoplasm of more or less than 25%, respectively, of tumor cells were stained. RESULTS: Nine NESCCs (43%) were c-kit-positive (overexpression). No difference in age or clinical stage was noted. No difference in prognosis was observed between the c-kit-positive and -negative patients. CONCLUSION: The incidence of c-kit overexpression was high in NESCC; therefore, the patients with this tumor may become a future target for molecular-targeted therapy with tyrosine kinase inhibitors.

Adult↗

PubMed2005

Effect of 5-Campestenone (24-methylcholest-5-en-3-one) on Zucker diabetic fatty rats as a type 2 diabetes mellitus model.

We examined the therapeutic effects of dietary exposure to 5-campestenone (24-methylcholest-5-en-3-one), an enone derivative of campesterol, in Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes mellitus. Dietary 0.6 % exposure to 5-campestenone caused marked reduction in hemoglobin A1c (HbA1c), plasma total cholesterol, triglycerides and non esterified fatty acid (NEFA). In particular, plasma triglyceride levels were reduced in the 0.6 % 5-campestenone-fed group to about 25 % of that in the control group. In the oral glucose tolerance test (OGTT) at three and seven weeks after the beginning of treatment, 5-campestenone limited the rise of blood glucose levels by oral administration of glucose dose-dependently. Amounts of adipose tissue in the retroperitoneum and periepididymal area as well as abdominal subcutaneous fat were significantly decreased in animals fed 0.6 % 5-campestenone. The blood leptin concentration on the final day of feeding was significantly in animals administered 5-campestenone. No obvious anomaly due to consumption of 5-campestenone was detected in necropsy or clinical observations.

Administration, Oral↗

PubMed2004

Library screening for D-amino-acid oxidase gene: application of real-time PCR.

Quantitative real-time PCR shows the quantity in addition to the presence of the target sequence. This property seemed very useful for library screening. Then, real-time PCR was employed to screen for lambda phages carrying D-amino-acid oxidase gene from mouse genomic library. Using stepwise dilution screening combined with real-time PCR, positive phages were isolated in a short time.

Amino Acid Oxidoreductases↗

PubMed2003

Hamster D-amino-acid oxidase cDNA: rodents lack the 27th amino acid residue in D-amino-acid oxidase.

The nucleotide sequence of cDNA that encodes hamster d-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,590 nucleotides and a poly(A) tail. It had an open reading frame for a protein consisting of 346 amino acid residues. The number of the amino acid residues is the same as that of the rat DAO. However, the hamster DAO has one residue more than mouse DAO and one residue less than human, pig, rabbit, and guinea pig DAOs. Amino acid sequence of the hamster DAO was highly similar to those of mouse and rat DAOs: 89% and 88% of the amino acid residues were identical between the hamster and mouse DAOs and between the hamster and rat DAOs, respectively. The homology was slightly less between the hamster DAO and the human (81%), pig (78%), rabbit (78%), or guinea pig DAO (82%). It has been proposed that the mouse and rat DAOs lack an amino acid residue corresponding to the 25th residue of the DAOs of other mammals. However, a detailed comparison of the amino acid sequences as well as the underlying nucleotide sequences by inclusion of the hamster ones revealed that the rodent DAOs does not lack the 25th, but the 27th residue.

Amino Acid Sequence↗

PubMed2002

Effects of 5-campestenone (24-methylcholest-5-en-3-one) on the type 2 diabetes mellitus model animal C57BL/KsJ-db/db mice.

We examined the therapeutic effects of dietary exposure to 5-campestenone (24-methylcholest-5-en-3-one), an enone derivative of campesterol, in C57BL/KsJ-db/db mice, which are an animal model of obese type 2 diabetes. Blood glucose levels of db/db mice linearly increased from 270 to 720 mg/dl in 10 weeks, an approximately 7-fold difference from the levels of db/+m mice. The 0.3 % dietary exposure to 5-campestenone caused a marked reduction in blood glucose levels of 330 mg/dl after 10 weeks of feeding with a concomitant inhibition of glucose excretion in urine. Only slight efficacy was observed with 0.1 % dietary exposure to this chemical in db/db mice. Significant decreases of plasma triglyceride and plasma free fatty acid were also observed in db/db mice at a 0.3 % dose. However, feed efficiency and body-weight gain in db/db mice was improved by 5-campestenone. No obvious anomaly due to consumption of 5-campestenone was detected by necropsy or clinical observation.

Animals↗

PubMed2001

Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This D-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for D-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free D-proline in eight brain regions and sera of mutant ddY/DAO- mice, lacking D-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free D-leucine was also investigated. The amount and distribution of D-proline in the brains of ddY/DAO+ mice and ddY/DAO- mice are almost the same, and relatively high amounts of D-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of D-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of D-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO- mice, D-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of L-proline and L-leucine are not significantly different between the two strains. In the serum, the amounts of both free D-proline and d-leucine are significantly higher in the ddY/DAO- mice than those obtained in ddY/DAO+ mice.

Animals↗

PubMed2001

Determination of free D-aspartic acid, D-serine and D-alanine in the brain of mutant mice lacking D-amino acid oxidase activity.

A simple and precise method for the simultaneous determination of free D-aspartic acid, D-serine and D-alanine in mouse brain tissues was established, using a reversed-phase HPLC system with widely used pre-column derivatizing reagents, o-phthaldialdehyde and N-t-butyloxycarbonyl-L-cysteine. With the present method, the contents of these three D-amino acids in hippocampus, hypothalamus, pituitary gland, pineal gland and medulla oblongata as well as cerebrum and cerebellum of mutant mice lacking D-amino-acid oxidase activity were determined and compared with those obtained for control mice. In both mice, extremely high contents of D-serine were observed in forebrain (100-400 nmol/g wet tissue), and the contents were small in pituitary and pineal glands. While, D-serine contents in cerebellum and medulla oblongata of mutant mice were about ten times higher than those in control mice. In contrast, D-alanine contents in mutant mice were higher than those in control mice in all brain regions and serum.

Alanine↗

PubMed2001

Exaggerated responses to chronic nociceptive stimuli and enhancement of N-methyl-D-aspartate receptor-mediated synaptic transmission in mutant mice lacking D-amino-acid oxidase.

Formalin-induced nociceptive behaviors and N-methyl-D-aspartate (NMDA) subtype glutamate receptor-mediated excitatory synaptic transmission were analyzed in mutant mice lacking D-amino-acid oxidase, which catalyzes the oxidative deamination of D-amino acids. The second phase of the formalin-induced licking response, a part of which is known to be mediated by NMDA receptors in the spinal cord, was significantly augmented in mutant mice. NMDA receptor-mediated excitatory postsynaptic currents recorded from spinal cord dorsal horn neurons by tight-seal whole-cell methods were significantly potentiated in mutant mice. The present observations provide another line of evidence that D-serine functions as an endogenous coagonist at the glycine site of NMDA receptors, and raise the possibility that D-amino-acid oxidase exerts a neuromodulatory function by controlling the concentration of D-serine in the central nervous system.

Animals↗

PubMed2001

Assignment of D-amino-acid oxidase gene to a human and a mouse chromosome.

A part of D-amino-acid oxidase gene was amplified in the human and mouse by polymerase chain reaction. The amplified fragments were ligated to plasmids and then cloned. The plasmids containing the parts of D-amino-acid oxidase gene were biotinylated and hybridized to human and mouse metaphase chromosomes. The chromosomal slides were treated with fluorescein isothiocyanate (FITC)-conjugated avidin. The hybridized signals were amplified with biotinylated anti-avidin antibody and FITC-avidin. The chromosomes were counter-stained with diamidino-phenylindole for assignment of the signal to a specific band. Using this fluorescence in situ hybridization (FISH), D-amino-acid oxidase gene was assigned to human chromosome 12q23-24.1 and mouse chromosome 5E3-F. Since these regions are syntenic between human and mouse, the present results indicate that the locus for this enzyme has been conserved through evolution.

Animals↗

PubMed2001

Establishment of a rat hepatoma-derived cell line proliferating in D-phenylalanine medium and expressing D-amino-acid oxidase.

A cell line (R-Y121B.DF) has been established from a cell line (R-Y121B) derived from a rat hepatoma line (H4-II-E). The R-Y121B.DF cells have been continuously cultured in a serum-free modified Eagle's minimum essential medium in which L-phenylalanine was replaced by D-phenylalanine. They had D-amino-acid oxidase (DAO) activity which is essential for the growth in the medium containing D-amino acids. The enzyme activity of the R-Y121B.DF cells was approximately one-fourth of that of the rat liver. Northern hybridization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B.DF cells and the rat liver but not in the parental R-Y121B and H4-II-E cells. Reverse transcription-polymerase chain reaction using DAO-specific primers amplified a DNA fragment of the expected size in the R-Y121B.DF cells but not in the R-Y121B and H4-II-E cells. This fragment was confirmed to be DAO cDNA by nucleotide sequencing. Western blotting showed that DAO protein was present in the R-Y121B.DF cells and the rat liver but not in the R-Y121B and H4-II-E cells. Southern hybridization showed that the DAO gene structure was not different among the R-Y121B.DF cells, R-Y121B cells, H4-II-E cells, and the rat liver. These results indicate that the R-Y121B.DF is a unique cell line which proliferates in the medium containing D-phenylalanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.

Animals↗

PubMed2001

A remnant tubal pregnancy after cloacal malformation repair.

OBJECTIVE: Report of a remnant tubal pregnancy after cloacal malformation repair. DESIGN: Case report. SETTING: A university hospital. PATIENT: A woman with cloacal malformation repair. INTERVENTION: Laparoscopic surgery. MAIN OUTCOME MEASURE: Remnant tubal pregnancy. RESULT: Laparoscopic right salpingectomy. CONCLUSION(S): In this patient, ectopic pregnancy was caused by contralateral sperm transmigration to the remnant right tube, which was not connected with the left uterus.

Adult↗

PubMed2001

Retinoid receptors in the human endometrium and its disorders: a possible modulator of 17 beta-hydroxysteroid dehydrogenase.

Retinoids have recently been proposed to modulate estrogenic actions in various sex steroid-dependent neoplasms, but little has been studied in human endometrial disorders. Therefore, in this study, we first examined the immunolocalization of retinoic acid receptor alpha, beta, and gamma, and retinoid X receptor (RXR) alpha, beta, and gamma in 20 normal cycling human endometria, 34 endometrial hyperplasia, and 46 endometrioid endometrial adenocarcinomas. We then correlated these findings with other clinicopathological parameters, especially in the correlation between retinoid receptor subtypes and the status of steroid hormone receptors, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and aromatase. We also then examined the effects of retinoic acid on the expression of 17 beta-HSD type 2 in cell lines derived from endometrial carcinoma using Northern blotting analysis to examine the possible roles of retinoids in in situ endometrial estrogen metabolism. Among these six retinoid receptors examined, RXR gamma immunoreactivity was exclusively detected in the epithelial cells of the secretory phase endometrium but not of the proliferative phase, which was well correlated with 17 beta-HSD type 2 immunolocalization. However, in endometrial hyperplasia, RXR gamma was not correlated with 17 beta-HSD type 2. In endometrioid endometrial adenocarcinoma, there was a statistically significant correlation between 17 beta-HSD type 2 immunoreactivity and RXR gamma labeling index (LI) (P < 0.001) and between RXR gamma LI and progesterone receptor LI (r = 0.501, P = 0.003). A significant inverse correlation was also detected between RXR gamma LI and patient age (r = 0.449, P = 0.015). No statistically significant correlation was obtained between LIs of receptors and other clinicopathological parameters including the status of intratumoral aromatase examined by immunohistochemistry. In the endometrial carcinoma cell line, RL95-2, retinoic acid markedly increased the level of 17 beta-HSD type 2 messenger RNA in a time- and dose-dependent manner. These results all suggest that retinoic acids may be involved in modulation of in situ estrogen metabolism in both normal and neoplastic human endometrium possibly through RXR gamma by stimulating the expression of 17 beta-HSD type 2.

17-Hydroxysteroid Dehydrogenases↗

PubMed2001

The analyses of 17beta-hydroxysteroid dehydrogenase isozymes in human endometrial hyperplasia and carcinoma.

Intratumoral metabolism and synthesis of estrogens are considered to play very important roles in the pathogenesis and development of human endometrial adenocarcinoma. The 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isozymes catalyze the interconversion of estradiol (E2) and estrone and thereby serve to modulate the tissue levels of bioactive E2. To elucidate the possible involvement of this enzyme in human endometrial carcinoma, we first examined the expression of 17beta-HSD type 1 and type 2 in 20 normal cycling human endometria, 36 endometrial hyperplasia, and 46 endometrial endometrioid adenocarcinoma using immunohistochemistry, and we then studied immunoreactivity of 17beta-HSD type 2 using immunoblotting analyses, the activity of 17beta-HSD type 1 and type 2 using thin-layer chromatography and their expression using RT-PCR in endometrial endometrioid adenocarcinoma. We correlated these findings with various clinicopathological parameters to examine the biological significance of 17beta-HSDs in human endometrial disorders. 17beta-HSD type 2 immunoreactivity in normal endometrium was present in all cases of secretory phase (n = 14), but not in any endometrial mucosa of proliferative phase (n = 6). In addition, 17beta-HSD type 2 immunoreactivity was detected in 27 of 36 (75%) endometrial hyperplasia and 17 of 46 (37%) carcinoma cases. 17beta-HSD type 1 immunoreactivity was not detected in all the cases examined. In both endometrial hyperplasia and carcinoma cases there were significant positive correlations between 17beta-HSD type 2 and progesterone receptor labeling index (LI). In carcinoma cases, a significant inverse correlation was detected between 17beta-HSD type 2 immunoreactivity and age. In addition, 17beta-HSD type 2 immunoreactivity was also correlated with 17beta-HSD type 2 enzymatic activity, and semiquantitative analyses of 17beta-HSD type 2 messenger RNA. No significant correlations were detected between 17beta-HSD type 2 and estrogen receptor LI, Ki67 LI, amount of aromatase messenger RNA or histological grade. These data indicated that the expression of 17beta-HSD type 2 in hyperplastic and/or neoplastic endometrium may represent altered cellular features through hyperplastic and neoplastic transformation. However, 17beta-HSD type 2 may also play some protective and/or suppressive roles toward unopposed estrogenic effects through inactivating E2 in situ, especially in premenopausal patients.

17-Hydroxysteroid Dehydrogenases↗

PubMed2001

Involvement of granule-mediated apoptosis in the cyclic changes of the normal human endometrium.

Our objective is to investigate the involvement of granule-mediated apoptosis in the cyclic changes of the endometrium. We demonstrated the localization of CD56, perforin, granzyme B and caspase-3 in the endometrium by immunohistochemistry. We also confirmed the localization of perforin by immuno-electron microscopy, and demonstrated apoptosis in endometrial glandular cells by TdT-mediated dUTP-biotin nick end labeling (TUNEL) and electron microscopy. Uterine CD56-positive natural killer (NK) cells expressed perforin and granzyme B in its cytoplasm. Uterine NK cells increased significantly in the endometrial stroma during the secretory phase, and peaked during the late secretory phase. These cells started decreasing in number during the menstrual period. In endometrial glandular cells, caspase-3 and TUNEL-positive cells increased significantly from the late secretory phase, with apoptosis reaching a peak during the menstrual period. Using electron microscopy, we observed uterine NK cells with chromatin rich, segmented nuclei and intracytoplasmic granules in the stroma obtained from late secretory phase endometria. These cells extended projections to the lining of endometrial glandular cells and attached to form a cell-to-cell contact. In addition, nuclear chromatin was observed to have already cohered and small cytoplasmic organelles were beginning to disappear, suggesting that these endometrial glandular cells were undergoing apoptosis. Utilizing immuno-electron microscopy, intracytoplasmic granules in uterine NK cells were stained with anti-perforin antibody. The findings of this study suggest that granule-mediated apoptosis in endometrial glandular cells induced by NK cells expressing perforin and granzyme B may be associated with the onset of menstruation.

Adult↗

PubMed2001

The current status of gynecological laparoscopic surgery in educational facilities in Japan.

A questionnaire survey was administered by Tohoku University hospital to investigate the current status of gynecological laparoscopic operations in teaching hospitals in which residents were trained as obstetricians and gynecologists. Thirty-eight senior doctors in 18 hospitals were sent a postal questionnaire. The response rates of facilities and doctors were 94.4% and 76.3% respectively. Diagnostic and operative laparoscopy was performed in all of the hospitals surveyed, however only a few hospitals performed more than 100 laparoscopies per year, and many surgeons did not perform advanced laparoscopic operations including hysterectomy, myomectomy and lymphadenectomy. Half of the doctors performed minilaparotomy, which is a so-called "open" technique. The standard surgical style involved the use of a carbon dioxide (CO2) pneumoperitoneum and an endoscope 10 mm in diameter. Unipolar endocoagulating instruments were generally used for hemostasis and incision. Most of the doctors surveyed thought that operative laparoscopy would become a necessary procedure in gynecological field, and half of them hoped to receive training in the technique. Instruction of expert endoscopic surgeons is necessary, especially in teaching hospitals, for the spread of safe and advanced laparoscopic surgery. An educational and training system for established surgeons as well as for young residents is urgently needed in Japan.

Delivery, Obstetric↗

PubMed2001

[Gene expression profiling of human ovarian epithelial tumors by digo nucleotide microarray].

Gene expression of human ovarian carcinoma cell lines and epithelial ovarian tumors was examined by oligonucleotide microarray for about 6000 human cDNAs. (1) Comparison of gene expression between CDDP-sensitive human ovarian serous adenocarcinoma cell lines and CDDP-resistant cell lines revealed that gamma-glutamylcysteine synthetase, glutathione peroxidase-like protein, dehydrogenase (UGDH), NAD(P)H: quinoneoxireductase, glucose-6-phosphatase, ornithine decarboxylase and dihydrodiol dehydrogenase were associated with a mechanism of CDDP-resistance. Comparison of gene expression between taxol-sensitive human ovarian cell lines and taxol-resistant cell lines showed that up-regulation of 30 kinds of gene expression including MDR and semaphorin E in taxol-resistant cell lines. (2) Comparison of gene expression among serous adenocarcinomas, clear cell adenocarcinomas and non-cancerous ovarian tissues by hierarchical clustering demonstrated that clear difference between carcinomas and non-cancerous ovarian tissues but not obvious difference between serous and clear adenocarcinomas. Genes that were up- and down-regulated specifically in these two types of ovarian carcinomas were further selected by the criteria that difference in the mRNA level by more than 4-fold between tumors and non-cancerous tissues. Tissue type specific alterations of gene expression are likely to play important roles in the carcinogenesis of epithelial ovarian tumors. cDNA microarray is a powerful and high-throughput tool to analyze gene expression of cancer development.

Adenocarcinoma, Clear Cell↗