Biomedical subjects
Publications and source records attributed to T Nagai.
The allele frequencies of 18 dinucleotide repeat marker loci on the X chromosome have been analyzed in 130 Japanese individuals living in Kanagawa by means of multiplex PCR and the ABI PRISM Linkage Mapping Set MD 10 Panel 28, followed by capillary electrophoresis using the ABI PRISM 310 Genetic Analyzer.
One or two in vitro-produced (IVP) Japanese Black (JB) cattle embryos at 8 days after in vitro fertilization were transferred to the contralateral uterine horn of previously bred Japanese Shorthorn (JSH) or JSH-JB cross recipients, and then the occurrence of early embryonic death, abortion during mid- and late gestation, and calving loss were recorded. The survival rate of embryos, including indigenous ones, was not affected by the number of embryos transferred, and a significantly higher twinning rate (68% of pregnant recipients at 80 days after transfer) was achieved when two IVP embryos were transferred, as compared with the rate when one IVP embryo was transferred (24%). In late ET (recipients at 8.5-9.0 days after the onset of oestrus), the embryo survival rate (22%) and the pregnancy rate (42%) at 80 days after ET were significantly lower than those rates in the synchronous ET (recipients at 8.0 days after the onset of oestrus; 47 and 79%, respectively). In the early ET (recipients at 6.0-7.5 days after the onset of oestrus), no significant differences from the synchronous ET were detected in these rates. Twenty-six percent of twin pregnant recipients were aborted during mid- or late-pregnancy, and 39% of twin calves were stillborn. The mean gestation length of the twin-bearing JSH dams (276 days) was 1 week shorter than that of the single-bearing JSH dams, and it was 2 weeks shorter than that of the JB dams bearing a single JB calf derived from the IVP embryos. The longer gestation length of single JB calves derived from IVP embryos resulted in a significantly higher mean birth weight than that of in vivo control calves with the standard length of gestation. In conclusion, the number of embryos to be transferred did not affect the embryo survival rate, and the transfer of two IVP embryos to previously inseminated recipients induced a significantly higher twinning rate during early pregnancy than that of one IVP embryo transfer. The incidence of embryonic losses during early pregnancy increased when Day 8 embryos were transferred to the recipients later in the oestrous cycle (>8.0 days). The results suggested that one cause of the high rate of abortions and stillbirths in twin-bearing dams is the difference in the mean gestation length between the native JSH and JB foetuses derived from transferred IVP embryos.
The long-term effectiveness of zonisamide (ZNS) was evaluated in 11 patients with West syndrome (7 symptomatic) who had cessation of spasms with ZNS monotherapy. During the follow-up period (24 to 79 months, mean = 53 months), this response was maintained in 7 patients (3 symptomatic, relapse rate = 36%), including 2 children in whom ZNS was successfully discontinued. No serious adverse reactions were noted. ZNS may be both effective and well tolerated for the treatment of West syndrome.
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In the present study, the effects of seminal plasma (SP), cumulus-oocyte-complexes (COCs) conditioned medium (CCM) and hyaluronan (HA) on functional changes and in vitro fertilizing ability of porcine spermatozoa were examined. In in vitro fertilization (IVF) experiments, 10% (v/v) of exogenous SP in the fertilization medium prevented sperm penetration (using fresh-extended and frozen-thawed ejaculated spermatozoa). Analysis of frozen-thawed CCM revealed a HA content to levels of 30 ng/mL per incubated COC. Presence of frozen-thawed CCM did not, however, prove effective to increase (furthermore decreasing) oocyte penetration in vitro, and neither did supplementation with exogenous HA at the same concentration as that present in the CCM (secreted by COCs). Analysis of sperm capacitation using the chlortetracycline (CTC) assay showed that frozen-thawed CCM had no elevating effect on 'B-pattern' spermatozoa (implying capacitation-like changes) and that addition of 10% (v/v) SP held spermatozoa in the 'F-pattern' (intact) status. Dose of 500 microg/mL HA and freshly prepared CCM increased, however, the frequency of capacitated spermatozoa (B-pattern) without resulting in increased rates of 'AR-pattern' (acrosome-reacted) spermatozoa, compared with controls. The present results confirm the decapacitating effect of SP and suggest capacitating actions of HA (dose-related) and CCM (freshly prepared) on boar spermatozoa in vitro. The unclear effects of frozen-thawed CCM and a low dose of HA on penetration rates of boar spermatozoa call for further researches of their function in vivo.
The present study was conducted to evaluate the effect of cumulus cells on the in vitro maturation (IVM) and glutathione (GSH) synthesis of porcine oocytes cultured in the presence or absence of cysteamine under different oxygen tensions, and on their subsequent male pronucleus formation after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 45 h in modified TCM-199 supplemented with or without 150 microM cysteamine under a humidified atmosphere of 5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2. When cultured in medium supplemented with cysteamine under 20% O2 tension, the rates of COC maturation to the metaphase II (MII) stage were significantly higher than those of DOs (P<0.05). Regardless of the addition of cysteamine and oxygen tension, the rates of male pronucleus formation in COCs after IVM and IVF were significantly higher than in DOs (P<0.05). The GSH content of oocytes was significantly increased by the addition of cysteamine to the maturation medium (P<0.05), with significantly higher GSH content in COCs than in DOs (P<0.05). However, the GSH content of COCs and DOs was not significantly different when cultured in medium without cysteamine. These results indicate that cumulus cells play an important role in nuclear maturation to MII, GSH synthesis in porcine oocytes cultured in the presence of cysteamine, and subsequent male pronucleus formation after IVF.
To obtain an effective iodine solution, the use of 2-hydroxypropyl-alpha-cyclodextrin (2-HP-alpha-CD) as solubilizer was examined in comparison with alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), potassium iodide (KI), and polyvinylpyrrolidone (PVP). The stability constants for inclusion of iodine with cyclodextrin and KI were ascertained by the solubility method. The apparent stability constants increased in the following order: KI < beta-CD < alpha-CD < 2-HP-alpha-CD. This order was nearly in accordance with that of the stabilization ability. The largest volatile depression effect was exhibited by 2HP-alpha-CD. The measurement of the minimum inhibitory concentration (MC) using Escherichia coli NIH-J-2 and Staphylococcus aureus FDA209P suggested that the bactericidal activity of the iodine/2-HP-alpha-CD system was the same as that of the iodine/alpha-CD, iodine/beta-CD, and iodine/PVP systems. The present results suggest that the combination of 2-HP-alpha-CD and iodine is useful for a stable and effective iodine solution.
The effects of beta-mercaptoethanol (beta-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O(2) supplemented with beta-ME. Addition of beta-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O(2), to almost the same rates when they were cultured in 5% O(2). To investigate whether the growth-promoting effect of beta-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without beta-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without beta-ME. In contrast, addition of beta-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by beta-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by beta-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of beta-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that beta-ME has a protective effect in embryo development against oxidative stress and that the effect of beta-ME is associated with the promotion of cystine uptake of low availability in embryos.
INTRODUCTION: We analyze the efficacy of routine transition zone biopsies in patients undergoing ultrasound-guided systemic prostate biopsies for the first time because of a suspicious digital rectal examination or an elevated serum prostate-specific antigen (PSA) level. PATIENTS AND METHODS: During systemic prostate biopsy two or four additional transition zone biopsies were performed in 192 consecutive patients: in 182 because of a serum PSA concentration >4.1 ng/ml and in 10 because of a suspicious digital rectal examination and a serum PSA level <4.0 ng/ml. RESULTS: The overall prostate cancer detection rate was 37.5% (72/192). In 24 patients (33.3%), cancer was only detected in the peripheral zone, in 3 (4.2%) only in the transition zone, and in 45 (62.5%) in both zones. CONCLUSION: Transition zone biopsies performed at the first time of systemic prostate biopsy seem to have a low efficacy.
The abundance of neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the carotid body was examined in chronically hypercapnic hypoxic rats (10% O2 and 6-7% CO2 for 3 months), and the distribution and abundance of these four peptidergic fibers were compared with those of previously reported hypocapnic- and isocapnic hypoxic carotid bodies to evaluate the effect of arterial CO2 tension. The vasculature in the carotid body of chronically hypercapnic hypoxic rats was found to be enlarged in comparison with that of normoxic control rats, but the rate of vascular enlargement was smaller than that in the previously reported hypocapnic- and isocapnic hypoxic carotid bodies. In the chronically hypercapnic hypoxic carotid body, the density per unit area of parenchymal NPY fibers was significantly increased, and that of VIP fibers was unchanged, although the density of NPY and VIP fibers in the previously reportetd chronically hypocapnic and isocapnic hypoxic carotid bodies was opposite to that in hypercapnic hypoxia as observed in this study. The density of SP and CGRP fibers was decreased. These results along with previous reports suggest that different levels of arterial CO2 tension change the peptidergic innervation in the carotid body during chronically hypoxic exposure, and altered peptidergic innervation of the chronically hypercapnic hypoxic carotid body is one feature of hypoxic adaptation.
The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.
Mechanical stress induces various hypertrophic responses including activation of mitogen-activated protein kinases (MAPKs) in cardiac myocytes. Here we examined the role of the small GTP-binding proteins of Rho family and reactive oxygen species (ROS) in stretch-induced activation of p38MAPK in cardiomyocytes. Overexpression of dominant-negative mutants of Rac1 (D.N. Rac1), D.N.RhoA and D.N.Cdc42 suppressed stretch-induced activation of p38MAPK. Overexpression of constitutively active mutants of Rac1 (C.A.Rac1) and C.A.Cdc42 increased the p38MAPK activity in the absence of mechanical stress. Pretreatment with N-acetyl-L-cysteine and N-(2-mercaptopropionyl)-glycine (NAC) suppressed stretch-induced activation of p38MAPK. Mechanical stretch increased intracellular ROS generation, which was abrogated by overexpression of D.N.Rac1 and attenuated by overexpression of D.N.RhoA and D.N.Cdc42. An increase in protein synthesis evoked by mechanical stretch was suppressed by overexpression of D.N.Rac1 and pretreatment with NAC. These results suggest that mechanical stress induces cardiac hypertrophy through the Rac1-ROS-p38MAPK pathway in cardiac myocytes.
Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.
Immunohistochemistry of c-Fos protein was performed to study changes in neuronal activity in discrete brain areas of mice repeatedly treated with phencyclidine (PCP) showing enhancement of immobility in the forced swimming test, this behavioral change being considered as avolition, which is one of negative symptoms of schizophrenia. Repeated treatment with PCP significantly prolonged immobility time in the forced swimming test, compared with saline treatment. The c-Fos protein expression of mice showing PCP-induced enhancement of immobility was increased in certain brain regions, such as the retrosplenial cortex, pyriform cortices, pontine nuclei, cingulate, frontal cortex and thalamus, compared with that of PCP-treated, non-swimming and saline-treated, swimming groups. These results suggest that increased c-Fos protein is involved in the expression of PCP-induced enhancement of immobility, and c-Fos expression plays a role in negative symptoms-like behavioral changes.
Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 microg) was intranasally (i.n.) inoculated with influenza vaccine (10 microg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3-14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 microg) was i.n. inoculated with the vaccine (10 microg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27-50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 microg) and influenza HA vaccine (1 microg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 microg) each and diphtheria-pertussis-tetanus (DPT) vaccine (10 microg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 microg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.
The Ca2+-sensitive photoprotein aequorin and the new green fluorescent protein-based fluorescent Ca2+ indicators 'ratiometric-pericam' were selectively expressed in the mitochondria, cytosol and/or nucleus of spontaneously beating ventricular myocytes from neonatal rats. This combined strategy reveals that mitochondrial [Ca2+] oscillates rapidly and in synchrony with cytosolic and nuclear [Ca2+]. The Ca2+ oscillations were reduced in frequency and/or amplitude by verapamil and carbachol and were enhanced by isoproterenol and elevation of extracellular [Ca2+]. An increased frequency and/or amplitude of cytosolic Ca2+ spikes was rapidly mirrored by similar changes in mitochondrial Ca2+ spikes and more slowly by elevations of the interspike Ca2+ levels. The present data unequivocally demonstrate that in cardiac cells mitochondrial [Ca2+] oscillates synchronously with cytosolic [Ca2+] and that mitochondrial Ca2+ handling rapidly adapts to inotropic or chronotropic inputs.