Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen deficiency.

Genistein, an isoflavone abundantly present in soybeans, has structural similarity to estrogen, suggesting that genistein may act as a phytoestrogen. To examine the possible role of genistein in hemopoiesis and bone metabolism, female mice were either sham-operated or ovariectomized (OVX), and selected OVX mice were administered genistein for 2-4 weeks (0.1-0.7 mg/day) or 17beta-estradiol (E2; 0.01-0.1 microg/day) s.c., using a miniosmotic pump (Alza Corp., Palo Alto, CA). In OVX mice, uterine weight declined but was completely restored by E2 administration. In contrast, genistein did not demonstrate a reversal of the OVX-induced uterine atrophy. The number of bone marrow cells markedly increased, 2-4 weeks after OVX, and most of these were B220-weakly positive pre-B cells. The increased B-lymphopoiesis was completely restored, not only by E2 but also by genistein administration. In OVX mice, the trabecular bone volume of the femoral distal metaphysis, measured by microcomputed tomography scanning and dual-energy x-ray absorptiometry, was markedly reduced; and genistein restored this, as did E2. These results indicate that genistein exhibits estrogenic action in bone and bone marrow, to regulate B-lymphopoiesis and prevent bone loss, without exhibiting estrogenic action in the uterus. Phytoestrogens may be useful for preventing bone loss caused by estrogen deficiency in females.

Induction of cytokines in a human colon epithelial cell line by Shiga toxin 1 (Stx1) and Stx2 but not by non-toxic mutant Stx1 which lacks N-glycosidase activity.

Stx1 and Stx2 produced by Shiga toxin-producing Escherichia coli are cytotoxic due to their N-glycosidase activity on 28S rRNA. In this study, we have shown that proinflammatory cytokine mRNAs, especially IL-8, were induced by Stx1 and Stx2 in Caco-2 cells. A non-toxic mutant of Stxl which lacks N-glycosidase activity did not induce cytokine mRNAs. IL-8 production at the protein level was enhanced by Stx1 and Stx2, but not by the mutant Stx1. These results demonstrate that Shiga toxins induce expression and synthesis of cytokines in Caco-2 cells and their N-glycosidase activity is essential for the induction.

Sphingosylphosphorylcholine induces cytosolic Ca(2+) elevation in endothelial cells in situ and causes endothelium-dependent relaxation through nitric oxide production in bovine coronary artery.

Sphingosylphosphorylcholine (SPC) increased intracellular Ca(2+) concentration ([Ca(2+)]i) and nitric oxide (NO) production in endothelial cells in situ on bovine aortic valves, and induced endothelium-dependent relaxation of bovine coronary arteries precontracted with U-46619. The SPC-induced vasorelaxation was inhibited by N(omega)-monomethyl-L-arginine, an inhibitor of both constitutive and inducible NO synthase (NOS), but not by 1-(2-trifluoromethylphenyl) imidazole, an inhibitor of inducible NOS (iNOS). Immunoblotting revealed that endothelial constitutive NOS, but not iNOS, was present in endothelial cells in situ on the bovine aortic valves. We propose that SPC activates [Ca(2+)]i levels and NO production of endothelial cells in situ, thereby causing an endothelium-dependent vasorelaxation.

A comparison of biological effects of modulated carbon-ions and fast neutrons in human osteosarcoma cells.

PURPOSE: To compare the biological effects of a 135 MeV/u carbon-ion beam and 13 MeV fast neutron beam using human osteosarcoma cells. METHODS AND MATERIALS: We have studied the clonogenic cell survival, recovery of potentially lethal damage (PLD) in plateau phase cells, and spheroid cure in multicellular spheroid after irradiation at various positions in the plateau and spread out Bragg peak (SOBP) of a 135 MeV/u carbon-ion beam and with 13 MeV neutrons. The carbon beam had a 4-cm range in water and a range filter was used to produce a 3-cm extended-peak region. The reference radiation was 137Cs gamma-rays. RESULTS: The relative biological effectiveness (RBE) values for 10% survival level of plateau phase cells for carbon-ions at the position of plateau, proximal peak, midpeak, and distal peak within the SOBP, and neutrons were 1.71, 2.48, 2.63, 3.47, and 2.29, respectively. Corresponding RBE values at 1% level were 1.64, 1.93, 2.06, 2.49, and 2.05. The extent of recovery from PLD was reduced after carbon-ions at proximal peak, midpeak, and distal peak, and neutrons, although not substantially reduced after carbon-ions at plateau. The RBE values for 50% spheroid cure level of spheroids for carbon-ions at the position of plateau, proximal peak, midproximal peak, middistal peak, and distal peak within the SOBP, and neutrons were 1.69, 1.88, 1.87, 1.94, 2.03, and 1.90, respectively. CONCLUSIONS: The biological parameters measured all indicate an approximately comparable biological effectiveness between 75-80 KeV/microns carbon-ions of the SOBP and 13 MeV neutrons in the human tumor model studied in vitro.

A possible multiclonal development in human colonic carcinomas.

In order to define whether all colonic tumors develop from a single transformed cell, we compared two sites each in 40 early-stage colonic carcinomas by K-ras mutation and DNA ploidy pattern. These 40 colonic carcinomas consisted of 26 protruding-type carcinomas and 14 superficial-type carcinomas. The two sites were selected in every tumor tissue and then DNA ploidy pattern and occurrence of K-ras mutation were detected. In cases of "cancer in adenoma", we compared clonality between cancer cells and adenoma cells. In a cytofluorometrical study, it was shown that diploid patterns were predominantly seen in protruding-type carcinomas (76.9%), whereas many superficial-type carcinomas (64.3%) consisted of aneuploid cells. K-ras mutations were more frequently observed in protruding-type carcinomas: 42.3% in protruding-type compared to 21.4% in superficial-type carcinomas. These findings suggested that superficial-type carcinomas have a different mode of carcinogenesis from that of protruding-type carcinomas. Frequently, each pair of sites of the carcinoma tissue carried the same K-ras mutation, and DNA ploidy patterns were essentially the same. However, we encountered some cancers that showed different K-ras mutations in the cancer tissues. In particular, one case of cancer in adenoma demonstrated different K-ras mutations in the cancer and adenoma tissue. These findings suggest that most colonic cancers develop monoclonally, but there are a few cancers (fewer than 5%) that start multiclonally.

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase.

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.

Amino acid-induced responses in CNS neurons of normal mouse and Wriggle Mouse Sagami.

Amino acid-induced responses and their regional heterogeneities in the motor cortical and hippocampal (CA1) neurons of normal mouse and Wriggle Mouse Sagami (WMS) were studied by conventional patch-clamp techniques. There were no differences in the current densities and EC50s of glutamate-, gamma-amino butyric acid (GABA)-, and glycine-induced responses between the two groups of mice. In both types of mice, the GABA-induced current density of cortical neurones was higher and EC50 was lower than in hippocampal neurones, whereas the responses induced by glycine and glutamate did not show any differences.

Simple technique for pancreatogastrostomy and the histological findings of pancreatogastric anastomosis.

The leakage of pancreatic juice is the most serious complication after pancreatoduodenectomy. In an attempt to lessen the incidence of this complication, we have developed a simple technique for the anastomosis of pancreatogastrostomy. Since March 14, 1995, we have already performed surgical procedures on 8 cases of pancreatogastrostomy using this technique and as yet have never experienced fatal complications. We report the autopsy findings of pancreatogastric anastomosis histologically. The specimen taken from the anastomosis reveals good mucosal continuity. This result suggests the importance of reliable sutures between the pancreatic parenchyma and the full thickness of the gastric wall for the anastomosis of pancreatogastrostomy.

Caspase-3 activation and apoptosis induction coupled with the retrograde transport of shiga toxin: inhibition by brefeldin A.

Caspase proteolytic activities, such as caspase-3, -2 and -6, of THP-1 human monocytic cells were markedly increased in a time- and dose-dependent manner by treatment with purified Shiga toxin 1 (Stx1) or Stx2. Caspase-3 activation was strictly correlated with internucleosomal DNA fragmentation and chromatin condensation of the cells. In addition, the specific caspase-3 inhibitor, Ac-DEVD-CHO, decreased the percentage of apoptotic cells. The purified B-subunit of Stx1 did not induce apoptosis in THP-1 cells. Caspase-3 activation, DNA fragmentation and chromatin condensation caused by Stx were completely blocked by pretreatment of cells with brefeldin A, an inhibitor of Golgi functions. The findings suggest that Stx1 as well as Stx2 activate caspase-3, which plays a critical role in apoptosis, and that the apoptotic signals rise after Stx is transported to the Golgi apparatus.

Functional difference in monoamine transmitters in the behaviorally abnormal mouse mutant (wriggle mouse sagami).

A mutant mouse (wriggle mouse sagami, WMS) with neurological disorders was found in a colony of the BALB/c strain. The clinical signs included tremor, dystonia and involuntary movements. The concentrations of the neurotransmitter substances, noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT) and acetylcholine (ACh), were measured simultaneously with their metabolites in dissected brain regions by high-performance liquid chromatography with electrochemical detection. The turnover of 5-HT was significantly higher in the cerebral cortex, hippocampus, hypothalamus, midbrain and pons-medulla of WMS than of the genetic control, BALB/c. The intrastriatal DA and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid were increased. However, there was no evidence to suggest an increase in turnover rate of this neurotransmitter. An increase in concentration of and decrease in turnover rate of NA were observed in the cerebellum of this mutant. These findings suggest that multiple disturbance of the neurotransmitter system was largely responsible for the manifestation of the clinical signs of WMS.

Inhibition of Shiga toxin-induced tumor necrosis factor-alpha production and gene expression in human monocytic cells by CV6209.

Cytokines, in particular TNF-alpha, appear to be necessary to develop the pathological process of Shiga toxin-producing Escherichia coli (STEC) infection. In this study, we investigated whether CV6209, a PAF antagonist, could modulate Shiga toxin (Stx)-induced TNF-alpha production in human monocytic cells. Cells were stimulated by Stx1 or Stx2 (5 ng/ml) with or without CV6209 addition (12-100 microg/ml) for various periods of time. CV6209 significantly suppressed Stx-induced TNF-alpha production in a dose- and time-dependent manner. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that CV6209 suppressed Stx-mediated TNF-alpha mRNA expression. Our results indicated that CV6209 had an important regulatory effect on Stx-induced TNF-alpha production and gene expression.

Observation of the external aperture of the vestibular aqueduct using three-dimensional surface reconstruction imaging.

Observation of the external aperture of the vestibular aqueduct was hitherto possible only in cadavers or dry temporal bones; however, by applying three-dimensional surface reconstruction imaging, it is now possible to observe solid-looking images of this structure in living humans. When the width of the external aperture of the vestibular aqueduct was measured in 58 people, it was found to be significantly narrower in the affected ears of patients with Meniere's disease than in normal ears.

Intramedullary changes of the spinal cord in cervical spondylotic myelopathy.

STUDY DESIGN: This study retrospectively reviewed magnetic resonance imaging and delayed computed tomography after myelography of cervical spondylotic myelopathy patients who needed surgical treatment. OBJECTIVES: The purpose of this study is to clarify the meaning of high magnetic resonance intensity areas in cervical spondylotic myelopathy patients. SUMMARY OF BACKGROUND DATA: There is no conclusion whether the high magnetic resonance signal intensity areas can be a predictor for surgical results or not. METHODS: Thirty-one patients with cervical spondylotic myelopathy were examined with magnetic resonance imaging before surgery and delayed computed tomography after surgery. The presence or absence of high intensity areas in the spinal cord was compared with clinical symptoms and surgical outcomes. RESULTS: Twenty-three (74%) of 31 patients showed high intensity areas in the spinal cord on the T2-weighted image. Among these 23 patients, 18 revealed bilateral intramedullary "snake eyes" enhancement in delayed computed tomography. The presence of high intensity areas did not correlate with the surgical outcomes. Patients with multisegmental (linear) high intensity areas frequently manifested muscle atrophy in upper extremities. CONCLUSIONS: High intensity areas on T2-weighted magnetic resonance imaging were not correlated with the severity of myelopathy or surgical outcomes evaluated by the Japanese Orthopaedic Association score. Magnetic resonance imaging or delayed computed tomography in this study could not rule out the presence of white matter changes, including axonal loss or demyelination. Multisegmental (linear) high intensity areas on T2-weighted magnetic resonance imaging were associated with clinical evidence of extensive anterior horn cell and radiographic evidence of gray matter cavitation.

Results of a dental fear survey in Japan: implications for dental public health in Asia.

The purpose of this investigation was to initiate the study of dental fear in Japan. 415 college students, aged 18-22 yr were surveyed. A standardized questionnaire which has been used in the United States was translated into Japanese and was administered to the students. More than 80% of those surveyed reported some dental fear. Six to 14% of the students reported extreme fear of the dentist. The majority of the subjects admitted that they delayed making dental appointments due to fear. Muscle tension was the most common physiological symptom reported. The dental drill and needle were the most fear-provoking stimuli.

Baculovirus-insect cell production of bioactive porcine FSH.

The in vitro and in vivo bioactivity of recombinant porcine FSH (rpFSH) produced from insect cells through use of a baculovirus expression system were studied and compared with those of natural FSH preparations. Determination of in vitro bioactivity, using the rat Sertoli cell aromatase bioassay, indicated that rpFSH is as active as purified pituitary FSH. Determination of in vivo bioactivity, using the mouse uterine weight bioassay, indicated that rpFSH is as active as purified pituitary FSH. Using the mouse Leydig cell testosterone bioassay, it was demonstrated that the intrinsic LH bioactivity of rpFSH is negligible. The increases in ovarian and uterine weight, and the stimulation in follicular growth in immature hypophysectomized rats induced by rpFSH supplemented with hCG were comparable to those induced by natural FSH preparations. Furthermore, rpFSH alone in hypophysectomized mice stimulated preantral follicular growth to preovulatory stages, and the subsequent injection of hCG caused ovulation. These results demonstrate that in vitro and in vivo biological characteristics of rpFSH produced from baculovirus-insect cells are indistinguishable from those of FSH isolated from natural sources.

Induction of fertile estrus in bitches using a sustained-release formulation of a GnRH agonist (leuprolide acetate).

A single subcutaneous injection of a sustained-release formulation of a potent GnRH agonist, leuprolide acetate (LA; [D-Leu6, Pro9NEt]-GnRH), was evaluated as a method of inducing fertile estrus in 12 mature anestrous and 6 prepubertal beagle bitches. The bitches were treated with microencapsulated LA (100 micrograms/kg, s.c.) at 120 or 150 d post partum, or at 1 yr of age, followed by a GnRH-analogue (fertirelin; [Pro9NEt]-GnRH, 3 micrograms/kg, i.m.) on the first day of induced estrus. Signs of estrus were seen within 10.3 +/- 0.9 d after LA administration in all bitches. The interestrous interval in 120- and 150-d post-partum bitches was shortened (P < 0.05) to 191 +/- 3 and 222 +/- 3 d, respectively, compared with 264 +/- 11 d in control bitches. All LA treated dogs demonstrated behavioral estrus and mated. Three of 6 (50%) at 120 d post partum, 6 of 6 (100%) at 150 d post partum and 5 of 6 (83%) of prepubertal (1-yr old) bitches then became pregnant and produced a mean litter size of 4.1 +/- 0.8 pups. A normal circulating estrogen and progesterone response pattern was observed in mature anestrous bitches. A prepubertal bitch that failed to become pregnant had a similar estrogen response pattern but an insufficient progesterone profile. The results suggest that microencapsulated LA can be useful in inducing fertile estrus in the domestic dogs.

Recombinant porcine follicle stimulating hormone produced in baculovirus-insect cells induces rat ovulation in vivo and gene expression of tissue plasminogen activator in vitro.

Superovulatory responses in cattle are known to be highly variable. In the present study, a recombinant porcine follicle stimulating hormone (rpFSH) produced in baculovirus-insect cells was utilised to evaluate the role of this recombinant FSH in control of the ovulatory process. Immature hypophysectomised rats were implanted with oestrogen pellet (10 mg diethylstilbestrol) and then primed with pregnant mare serum gonadotropin (PMSG, 17.5 IU, sc). Fifty-two hours later, 100 microg rpFSH or saline was injected (sc) to induce ovulation. All rats that received rpFSH ovulated with about eight ova rat(-1), whereas none of the control animals did. Ovulation induced by rpFSH was associated with an increase in the ovarian activity and message levels of tissue-type plasminogen activator (tPA), a protease important in the preovulatory degradation of the follicle wall. Furthermore, addition of rpFSH to the cultured rat granulosa cells resulted in a significant increase in tPA enzyme activity. These results demonstrate that rpFSH produced in baculovirus-insect cells has biological potency in ovulation as well as gene expression of tPA, providing a large advantage of this massive expression system in the reproduction of domestic animals.

Influence of altered middle ear pressure on cochlear microphonics.

Patients with otitis media with effusion often have impaired hearing loss caused by bone conduction. Among several possible causes for this hearing loss, the influence of middle ear pressure upon hearing was examined. Cochlear microphonics (CM) of guinea pigs were recorded under positive or negative pressure created simultaneously in the middle ear and in the external ear canal. CM was recorded from each turn (basal, second, third and apical). Whereas under negative pressure CM decreased suddenly but soon became stable, under positive pressure CM normalized gradually after the initial drop. Thus, in the stable state, CM amplitude was low under negative pressure but was slightly subnormal under positive pressure. One possible explanation is that perilymphatic efflux appears to cause such recovery of CM under positive pressure. it is postulated that perilymphatic inflow does not seem to occur under negative pressure, since CM and perilymphatic pressure were stable.

The effect of injection of high K+ solution into scala media.

Thirty guinea pig ears were studied to investigate the effect of endolymphatic hydrops on the cochlea. High K+ solution was injected into the scala media, and cochlear microphonics (CM) and endocochlear potential (EP) were observed before, during and after the injection. The CM amplitude decreased rapidly after injection, ending in a depressed plateau value. By contrast, EP remained almost unchanged. By changing the composition of the solution it was suggested that the effect of the injection was mechanical one, rather than biochemical. In three ears, spontaneous recovery of CM was observed during a relatively long interval after the injection. The meaning of these findings for the hearing loss in Meniere's disease is discussed.

The effect of injection of high k(+) solution into scala media.

Thirty guinea pig ears were studied to investigate the effect of endolymphatic hydrops on the cochlea. High K(+) solution was injected into the scala media, and cochlear microphonics (CM) and endocochlear potential (EP) were observed before, during and after the injection. The CM amplitude decreased rapidly after injection, ending in a depressed plateau value. By contrast, EP remained almost unchanged. By changing the composition of the solution it was suggested that the effect of the injection was mechanical one, rather than biochemical. In three ears, spontaneous recovery of CM was observed during a relatively long interval after the injection. The meaning of these findings for the hearing loss in Meniere's disease is discussed.