Demonstration of maturation-promoting and- inhibiting activities in mouse oocytes

The microinjection of cytoplasm from mature mouse oocytes into immature mouse oocytes induced the resumption of meiosis in the presence of 100 g/ml dibutyryl cyclic AMP or 100 M 3-isobutyl-1-methylxanthine. The volume of cytoplasm injected was critical to bringing about this maturation-promoting activity in mouse oocytes, and 20 pl of cytoplasm seems to be required to overcome the inhibitory effect of cyclic AMP on oocyte maturation. Furthermore, it was demonstrated that the microinjection of cytoplasm from immature mouse oocytes into immature mouse oocytes augmented the inhibitory effect of cyclic AMP on the resumption of meiosis. These results suggest that the appearance and disappearance of maturation-promoting and- inhibiting activities appear to be dependent on the meiotic stages in the mouse oocytes.     

Genetic identification of cinnamon (Cinnamomum spp.) based on the trnL-trnF chloroplast DNA

Genetic identification among cinnamon species was studied by analyzing nucleotide sequences of chloroplast DNA from four species (Cinnamomum cassia, C. zeylanicum, C. burmannii and C. sieboldii). The two regions studied were the intergenic spacer region between the trnL 3'exon and trnF exon (trnL -trnF IGS) and the trnL intron region. We found nucleotide variation at one site in the trnL-trnF IGS, and at three sites in the trnL intron. With the sequence data from analysis of these regions, the four Cinnamomum species used in this study were correctly identified. Furthermore, single-strand conformation polymorphism (SSCP) analysis of PCR products from the trnL-trnF IGS and the trnL intron resulted in different SSCP band patterns among C. cassia, C. zeylanicum and C. burmannii.     

Effects of perinatal exposure to flutamide on sex hormones and androgen-dependent organs in F1 male rats

Flutamide, which has antiandrogenic properties, was administered to pregnant rats, and effects on male offspring were examined. Crj: CD (SD) IGS (SPF) females were administered flutamide (0.15, 0.6, 2.5, 10.0, 100 mg/kg, p.o.) from gestation Day 14 to post parturition Day 3. The number of pups, body weights, clinical features, anogenital distance (AGD), nipple retention, testicular descent, and urogenital malformation in F1 males were examined. Hormone measurement, necropsy and histopathological examination were carried out at post-neonatal Day 4 (PND 4) and PND 60. Sperm analysis was also carried out at PND 60. Decrease in body weight was seen in the 100 mg/kg group and the AGD was decreased at 2.5 mg/kg and above. Retention of nipples, hypospadia, vaginal pouches, penis malformation, unilateral ectopic testis, and decrease of organ weights (prostate, seminal vesicles, levator ani muscle plus bulbocavernosus muscle, testis) were observed at 10 mg/kg and above. Testicular testosterone (T) was increased significantly with 100 mg/kg at PND 4 and tendencies for increase were observed in serum T, LH and FSH at 10 mg/kg and more at the same time point. In contrast, elevated levels of LH and FSH were seen with 100 mg/kg at PND 60. Histopathological examination revealed defects or hypoplastic changes of genital organs (> or = 10 mg/kg), squamous metaplasia (10 mg/kg) or mucification (100 mg/kg) of the urethral diverticulum epithelium and inflammation of genital organs (100 mg/kg). Though only undescended testes lacked spermatogenesis at 10 mg/kg, atrophic change of seminiferous tubules and azoospermia were observed in the 100 mg/kg group, despite testicular descent. Perinatal administration of flutamide affected F1 male rats at 2.5 mg/kg and above. In addition to urogenital malformation, 100 mg/kg flutamide caused high LH and FSH levels at PND 60. This study indicates that the most sensitive parameter is AGD, whereby reduction was observed at 2.5 mg/kg. A clear no-effect level (NOEL: 0.6 mg/kg) was obtained in this perinatal study of an antiandrogenic chemical.     

Different effects of oral administration of synthetic trypsin inhibitor on the pancreas between cholecystokinin-A receptor gene knockout mice and wild type mice

The synthetic trypsin inhibitor camostat has been used for the treatment of acute and chronic pancreatitis in Japan based on the evidences obtained from a rat experimental model. However, rats differ from other rodents and from humans in terms of lacking a gallbladder and no response of pancreatic bicarbonate secretion to cholecystokinin (CCK). In the present study, we determined whether oral administration of camostat showed a trophic effect in mice as observed in rats and whether the trophic effect, if substantial, was mediated via the CCK-A receptor, using CCK-A receptor gene targeting mice. The chow containing 0.1% camostat was fed to 8-month-old mice. Three- and seven-day treatments with camostat did not affect pancreatic wet weight in CCK-A receptor (+/-) mice. After 14-day treatment, the ratio of pancreatic wet weight/body weight was significantly lower in CCK-A receptor (-/-) than (+/+) mice. The protein and chymotrypsin contents were lower and amylase content was higher in CCK-A receptor (-/-) mice, compared to (+/+) mice. No pathological findings were observed by histological examination. Camostat has a trophic effect on the pancreas in mice and this effect is mediated via the CCK-A receptor, but is less potent than in rats.     

Antimicrobial resistance of Staphylococcus aureus isolated from impetigo patients between 1994 and 2000

BACKGROUND: The incidence of strains of Staphylococcus aureus resistant to the antimicrobial agents used in treating impetigo has been increasing. AIM: To determine the antimicrobial susceptibility of S. aureus in impetigo. METHODS: We measured the antimicrobial susceptibility of Staphylococcus aureus isolated from impetigo patients between 1994 and 2000. RESULTS: The MIC50 of gentamicin was always higher than that of other antimicrobial agents until 1999. In isolates obtained since 1996, the MIC90 of gentamicin was over 12.5 micro g/mL, which is markedly higher than that found for other skin infections (folliculitis, furuncles, paronychia, phlegmone, secondary infection of eczema, dermatitis, ulcer and decubitus). There were no strains of S. aureus resistant to vancomycin and fusidic acid. After 2000, we could find only one strain resistant to minocycline and ofloxacin. CONCLUSION: Clindamycin has shown excellent activity against most S. aureus isolates between 1994 and 2000. The incidence of methicillin-resistant S. aureus was always below 20%.     

Energy metabolism and turnover are increased in mice lacking the cholecystokinin-B receptor

Cholecystokinin (CCK) is an important gastrointestinal hormone as well as a neurotransmitter. Two types of CCK receptors, types A and B, have been identified. The CCK-A receptor is involved in satiety, food intake and behavior, whereas the B receptor is involved in anxiety. We recently produced CCK-A, -B and AB receptor knockout mice to study the role of these receptors in energy metabolism. Daily energy intake and expenditure were significantly greater in CCK-BR(-/-) and CCK-AR(-/-)BR(-/-) mice than CCK-AR(-/-) and wild-type [CCK-AR(+/+)BR(+/+)] mice. Relative liver and kidney weights (g/kg body) were significantly greater in CCK-AR(-/-)BR(-/-) mice than in wild-type mice. Energy metabolism and energy turnover were increased in mice with a disruption of the CCK-BR gene, although the underlying mechanism is unknown.     

Physicochemical quality evaluation of natural compounds isolated from crude drugs

Aconite root has high toxicity caused by diester alkaloids, thus it was necessary to define the limiting value of diester alkaloids used in medicine formulation. To give the quality of “Processed Aconite Root” and “Powdered Processed Aconite Root” in the Japanese Pharmacopoeia (14th edn, supplement II), we established the official specification and evaluation methods of standard substances. High qualitative grade diester alkaloids, aconitine, hypaconitine, jesaconitine and mesaconitine, which were useful to evaluate the purity of processed aconite root and powdered processed aconite root, were prepared and evaluated for their stability. We studied the physicochemical specification and evaluation methods of these alkaloids. In addition, an “Aconitum diester alkaloids standard solution for purity”, which was used for the purity test, was prepared, and we also studied its physicochemical specification and evaluation methods. In addition, to evaluate the quality of processed aconite root and powdered processed aconite root, a TLC identification test was established. A monoester alkaloid of benzoylmesaconine hydrochloride was used as the reference standard in the latter test, and we also investigated its physicochemical specification and evaluation methods.     

Detection of human papillomaviruses from histologically normal lymph nodes of Japanese cervical cancer patients by nested polymerase chain-reaction assay

To determine the prognostic significance of human papilloma virus (HPV) DNA in histologically normal lymph nodes, we developed a nested polymerase chain-reaction (PCR) method on HPV16, 18 and 33 DNAs for formalin-fixed, paraffin-embedded tissues. We investigated 370 histologically normal lymph nodes from 15 patients treated for stage-IB/IIB (FIGO) invasive cervical cancer. HPV16 DNA was detected in 7 (47%) and HPV18 DNA in 3 (20%) of the cervical cancers. Examination of histologically normal lymph nodes from these 10 patients by nested PCR revealed HPV DNA in 5 (50%) of them; in all cases HPV type in lymph nodes and tumor was the same. Two of these 5 patients had a recurrence (pelvic cavity or lung) and died of cancer, although all 5 had had pelvic radiotherapy after radical hysterectomy and lymphadenectomy. These findings indicate that nested PCR is useful for evaluating early lymph-node involvement retrospectively in HPV-positive cases.