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Biomedical subjects

R Konno

Publications and source records attributed to R Konno.

At least 55 records · Page 3Linked to original sources

D-amino-acid oxidase is not present in the mouse liver.

Since there are conflict reports on the presence of D-amino-acid oxidase in the mouse liver, this problem was examined. D-Amino-acid oxidase activity was not detected in the homogenates of the mouse liver, lung, or heart, whereas it was detected in the homogenates of the mouse kidney and brain. Western blotting showed that a protein which reacted with the antiserum against pig D-amino-acid oxidase was present in the homogenates of the mouse kidney and brain but not in those of the liver or heart. Northern hybridization using a D-amino-acid oxidase cDNA probe detected a hybridizing signal in poly(A)+ RNAs extracted from the mouse kidney and brain but not in those from the liver, heart, or lung. Reverse transcription-polymerase chain reaction using three primer pairs always amplified D-amino-acid oxidase cDNA fragments of expected sizes in the mouse kidney and brain but very rarely did so in the liver, heart, or lung. The results indicate that D-amino-acid oxidase is not present in the mouse liver in a measurable amount.

Animals↗

Indications for laparoscopic surgery of ovarian tumors.

To investigate the criteria for laparoscopic surgery in ovarian tumors, a retrospective review of indications, preoperative assessments using transvaginal ultrasonography, serum CA 125, surgical methods and diagnosis and post-operative complications were analyzed. Forty-three patients underwent surgical treatment for ovarian tumors, from November 1992 to October 1993. Of the 43 patients with ovarian tumors, 19 (44%) underwent laparoscopic surgery, and 24 (56%) underwent laparotomy. Laparoscopic surgery was performed in 18 of 34 (53%) patients with benign ovarian cysts. Eight patients with malignant ovarian tumors were treated by laparotomy. Coincidence of preoperative assessment and surgical diagnosis was 95%. No major complications were observed in the laparoscopic surgery. We suggest that malignant ovarian tumors and inextricable adhesions are contraindications to laparoscopic surgery. We propose the following criteria for laparoscopic surgery by classification into three groups: A) good indication: benign cystic tumors with good mobility, B) deliberation indication: benign tumors with poor mobility, including some dermoid cysts and endometrial cysts, C) contraindication: malignant tumors and dense adhesions. We conclude that indications of laparoscopic surgery for ovarian tumors should be determined by careful preoperative assessment using transvaginal ultrasonography and tumor markers.

CA-125 Antigen↗

Prevalence of HPV DNA in cervical lesions in patients from Ecuador and Japan.

Cervical cancer is about 6 times more frequent in Ecuador than in Japan. We investigated the association between infection by the human papillomavirus (HPV) and the genesis of cervical cancer in specimens of lesions of the cervical epithelium obtained from patients in Ecuador and Japan. We also examined the results of HPV DNA detection and typing by the polymerase chain reaction (PCR) performed under the same technical conditions in areas with differing rates of cervical cancer. Purified tissue DNA from paraffin-embedded samples was amplified by PCR with universal and type-specific primers. HPV DNA was detected in 8 (20%) of 40 normal cervical epithelial samples from Ecuadorian patients, 19 (45%) of 42 low-grade squamous intraepithelial lesions (LSIL), 16 (50%) of 32 high-grade squamous intraepithelial lesions (HSIL), and 38 (81%) of 47 invasive squamous cell carcinomas (SCC) compared with 3 (10%) of 30 normal cervical specimens from Japanese patients, 107 (51%) of 210 HSILs, and 45 (71%) of 63 SCCs. The prevalence of HPV types 16 and 18 rose significantly with increasing histological grade (p < 0.05). The prevalence of HPV DNA decreased with increasing age in both Ecuadorian and Japanese patients. The detection rate and type-specific distribution of HPV DNA were not correlated with geographic location. Findings suggest that risk factors associated with poverty and underdevelopment may influence the prevalence of HPV infection and the sequence of events after HPV infection culminating in cervical cancer. These factors may help to explain the differing geographic distribution of this disease.

Adult↗

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↗

Anaphylactic reaction to cefazolin in pregnancy.

Anaphylaxis is a rare event in pregnancy, but is one that can have serious implications for both the mother and fetus. We report a case of severe anaphylactic reaction to intravenous (i.v.) cefazolin (CEZ) given during labor with premature rupture of membranes. Maternal shock and prolonged fetal bradycardia occurred. Both the maternal and fetal outcomes were good following treatment with ephedrine, glucocorticoids, and emergency caesarean section.

Adult↗

Amino acid levels in D-alanine-administered mutant mice lacking D-amino acid oxidase.

D-Alanine was administered orally to mutant mice lacking D-amino acid oxidase (EC 1.4.3.3). The mice had free access to drinking water containing 0.5% D- or L-alanine or 0.1% D-alanine for 2 weeks. The mice were then killed, and levels of the D- and L-enantiomers of free alanine, serine, proline, glutamate, and aspartate were determined in serum, liver, kidney, cerebrum, and cerebellum tissues. D-Alanine content increased by 60-fold (liver) to 110-fold (serum, brain), although the L-alanine level did not change. The increase of serum and brain D-alanine concentrations in animals fed 0.5% D-alanine was approximately five times more than that in animals fed 0.1% D-alanine, ie, the increase was roughly D-alanine dose-dependent in these tissues. The increase due to 0.5% D-alanine administration was reduced by 50% 17 hours after administration of D-alanine was stopped. Administration-induced increases in D-alanine levels in the cerebrum and cerebellum were not less than those in the serum, suggesting that D-alanine passed the blood-brain barrier quite freely. In the liver but not in other tissues, there were slight increases in D-serine and D-proline levels after administration of D-alanine. Administration of D-alanine produced no alterations in free glutamate and aspartate levels. No D-enantiomers of alanine, serine, proline, glutamate, or aspartate were detected in the liver and kidney tissue proteins of any animals, even in the mutant mice that received 0.5% D-alanine.

Alanine↗

Free D-serine, D-aspartate and D-alanine in central nervous system and serum in mutant mice lacking D-amino acid oxidase.

We have examined whether D-amino acid oxidase (DAO) regulates free D-serine content using mutant ddY/DAO- mice lacking DAO activity. We find that the content of D-serine in the serum and cerebellum of mutant mice is much higher than that of normal mice, whereas a slight but significant difference in the cerebral D-serine level is observed between the two strains. These results suggest that, although DAO may participate in the catabolism of D-serine in the cerebellum and periphery, there appears to be other mechanisms for catabolism of endogenous D-serine in the brain.

Alanine↗

Detection and typing of human papillomavirus DNA in the uterine cervix of Japanese women by nonradioactive dot blot and Southern blot hybridization.

HPV infection was examined with Cytobrush in exfoliated cervical cells sampled from 347 women. HPV DNA analysis was conducted in two steps. The presence of HPV DNA was demonstrated by dot blotting and typing of HPV DNA was made by Southern blotting using biotinylated probes. Of 167 cases with cervical intra-epithelial neoplasia (CIN) and cervical carcinoma, HPV DNA was detected and typed in 25 cases (15.0%). HPV 16, 18, and 33 were found mainly, and no HPV 6 or 11 was detected. The frequency of HPV DNA was 7.1% of CIN I, 28.6% of CIN II, 54.5% of CIN III, and 50.0% of invasive carcinoma. HPV occurred in patients newly diagnosed as CIN at 25.8% and in those of diagnosed as CIN in the past and followed up, 7.6%. Of 180 healthy women, the screening test of FO8830 was positive for HPV DNA in four women (2.2%). The present system proved to be useful for facilitating large-scale clinical research.

Blotting, Southern↗

Origin of D-alanine present in urine of mutant mice lacking D-amino-acid oxidase activity.

Urine of mutant ddY/DAO- mice lacking D-amino-acid oxidase contained 13 times more D-alanine than that of normal ddY/DAO+ mice. Because D-alanine is a component of bacterial cell walls, the possibility that the urinary D-alanine came from intestinal bacteria was examined. In ddY/DAO- mice that were made germ free at birth and reared in a germ-free environment, the quantity of urinary D-alanine was found to be at a low level comparable to that of the normal mice. When these germ-free mice were made gnotobiotic by inoculation with gram-negative bacteria (Escherichia coli and Bacteroides vulgatus), the urinary D-alanine increased to a high level. When these gnotobiotic mice were further inoculated with gram-positive bacteria (Bifidobacterium longum and Eubacterium aerofaciens), the urinary D-alanine increased further. These results indicate that most of the urinary D-alanine of the conventionally reared ddY/DAO- mice is of gastrointestinal bacterial origin.

Alanine↗

A single-base-pair substitution abolishes D-amino-acid oxidase activity in the mouse.

Mutant ddY/DAO- mice lacking D-amino-acid oxidase (DAO) activity were examined for the cause of their lack of enzyme activity. Total RNA was extracted from the kidney of the ddY/DAO- mice and cDNA was synthesized. After cDNA encoding DAO was amplified by the polymerase chain reaction it was cloned into a plasmid and sequenced. Comparison of the DAO cDNA sequence with that of normal BALB/c mice revealed the presence of a single-base substitution (G----A) which causes a Gly-181----Arg substitution in the middle of the enzyme molecule. The mutant DAO cDNA was inserted into an expression vector and was expressed in transfected COS-1 cells. The transfected cells synthesized the DAO mutant protein, but they did not show DAO activity. In contrast, when cells were transfected with an expression vector carrying wild-type DAO cDNA, where the substituted base-pair was replaced by a normal base-pair, they showed DAO activity. These results indicate that the single base-pair substitution is the cause of the loss of DAO activity in the ddY/DAO- mice.

Animals↗

The presence of free D-alanine, D-proline and D-serine in mice.

The presence of free D-alanine, D-proline and D-serine was demonstrated in mammalian tissues, using a mutant mouse strain lacking D-amino acid oxidase. In the experiment, free amino acids from the kidney and serum were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) to diastereomers, separated by two-dimensional thin-layer chromatography (TLC), and analysed by reversed-phase high-performance liquid chromatography (HPLC) for the resolution of D- and L-isomers. D/L ratios of alanine, proline and serine were obtained based on the peak areas of HPLC.

Alanine↗

Progression of squamous cell carcinoma of the uterine cervix from cervical intraepithelial neoplasia infected with human papillomavirus: a retrospective follow-up study by in situ hybridization and polymerase chain reaction.

A retrospective study was conducted to reveal the natural history of cervical carcinoma infected with human papillomavirus (HPV) and to document latent persistence of HPV infection. Thirty-eight formalin-fixed, paraffin-embedded hysterectomy specimens of cervical carcinoma and cervical intraepithelial neoplasia (CIN) III were examined for the presence of HPV 6, 11, 16, 18, 31, 33, and 35 DNA by in situ hybridization (ISH) with biotinylated DNA probes. The HPV 6 and 11 were not detected, but HPV 16, 18, 31, 33, and 35 were detected in 63% (24 of 38) of CIN III and cervical carcinoma. The HPV-negative specimens by ISH were subjected to polymerase chain reaction (PCR) for detection of HPV 16 and 52b with high sensitivity. In 11 of 14 specimens, specific amplification of HPV 16 was detected and HPV DNA was demonstrated in 92% (35 of 38). A retrospective examination for the presence of HPV DNA by ISH and PCR was performed on sequential biopsy specimens of eight cases whose hysterectomy specimens were judged as HPV 16/18 positive. In the eight cases, HPV 16 was constantly demonstrated in all biopsy specimens throughout the course, from early dysplastic change to carcinoma. This finding indicates long (up to 10 years) persistence of HPV infection in the natural course of progression to carcinoma of the uterine cervix.

Carcinoma, Squamous Cell↗

Detection of human papillomavirus DNA in normal epithelium and in squamous metaplasia of the uterine cervix by the polymerase chain reaction.

The presence of human papillomavirus (HPV) 16 was studied by using the polymerase chain reaction (PCR) directly on sections histologically defined as normal squamous epithelium and metaplastic squamous tissue of the uterine cervix. Ten specimens of normal epithelium were obtained at hysterectomy from women with uterine leiomyoma. Six specimens of squamous metaplasia were adjacent to the areas of dysplastic epithelium in which HPV 16 DNA had been detected previously by PCR. HPV 16 DNA was amplified specifically and detected in two of 10 normal cervical epithelium specimens and in all of 6 squamous metaplasia specimens adjacent to dysplastic lesions. However, HPV DNA could not be detected in the metaplasia by in situ hybridization. These results suggest that metaplastic squamous tissue adjacent to dysplastic lesions harbors fewer copies of HPV DNA than the dysplastic area and the carcinoma and that the HPV copy number per cell may be relevant to the pathogenesis of cervical carcinoma.

Base Sequence↗

Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice.

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.

Alanine↗