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Biomedical subjects

K Ohashi

Publications and source records attributed to K Ohashi.

At least 163 records · Page 9Linked to original sources
PubMed1999

Embryonic lethality and defective neural tube closure in mice lacking squalene synthase.

Squalene synthase (SS) catalyzes the reductive head-to-head condensation of two molecules of farnesyl diphosphate to form squalene, the first specific intermediate in the cholesterol biosynthetic pathway. We used gene targeting to knock out the mouse SS gene. The mice heterozygous for the mutation (SS+/-) were apparently normal. SS+/- mice showed 60% reduction in the hepatic mRNA levels of SS compared with SS+/+ mice. Consistently, the SS enzymatic activities were reduced by 50% in the liver and testis. Nevertheless, the hepatic cholesterol synthesis was not different between SS+/- and SS+/+ mice, and plasma lipoprotein profiles were not different irrespective of the presence of the low density lipoprotein receptor, indicating that SS is not a rate-limiting enzyme in the cholesterol biosynthetic pathway. The mice homozygous for the disrupted SS gene (SS-/-) were embryonic lethal around midgestation. E9.5-10.5 SS-/- embryos exhibited severe growth retardation and defective neural tube closure. The lethal phenotype was not rescued by supplementing the dams either with dietary squalene or cholesterol. We speculate that cholesterol is required for the development, particularly of the nervous system, and that the chorioallantoic circulatory system is not mature enough to supply the rapidly growing embryos with maternal cholesterol at this developmental stage.

Animals↗
PubMed1999

Expression of amylase and glucose oxidase in the hypopharyngeal gland with an age-dependent role change of the worker honeybee (Apis mellifera L.).

Worker honeybees change their behaviour from the role of nurse to that of forager with age. We have isolated cDNA clones for two honeybee (Apis mellifera L.) genes, encoding alpha-amylase and glucose oxidase homologues, that are expressed in the hypopharyngeal gland of forager bees. The predicted amino acid sequence of the putative Apis amylase showed 60.5% identity with Drosophila melanogaster alpha-amylase, whereas that of Apis glucose oxidase showed 23.8% identity with Aspergillus niger glucose oxidase. To determine whether the isolated cDNAs actually encode these enzymes, we purified amylase and glucose oxidase from homogenized forager-bee hypopharyngeal glands. We sequenced the N-terminal regions of the purified enzymes and found that they matched the corresponding cDNAs. mRNAs for both enzymes were detected by Northern blotting in the hypopharyngeal gland of the forager bee but not in the nurse-bee gland. These results clearly indicate that expression of the genes for these carbohydrate-metabolizing enzymes, which are needed to process nectar into honey, in the hypopharyngeal gland is associated with the age-dependent role change of the worker.

Age Factors↗
PubMed1999

The N-terminal LIM domain negatively regulates the kinase activity of LIM-kinase 1.

LIM-kinase 1 (LIMK1, where LIM is an acronym of the three gene products Lin-11, Isl-1 and Mec-3) is a serine/threonine kinase that phosphorylates cofilin and regulates actin cytoskeletal reorganization. LIMK1 contains two LIM domains and a PDZ (an acronym of the three proteins PSD-95, Dlg and ZO-1) domain in the N-terminal half and a kinase domain in the C-terminal half. In this study we examined the role of the extra-catalytic region in the regulation of kinase activity of LIMK1. Limited proteolysis of LIMK1 resulted in the production of the 35-40-kDa kinase core fragments with 3.5-5. 5-fold increased kinase activity. The LIMK1 mutants with deleted LIM domains (DeltaLIM) or conserved cysteines in the two LIM domains replaced with glycines (dmLIMK1) had 3-7-fold higher kinase activities in vitro, compared with the wild-type LIMK1. The C-terminal kinase fragment of LIMK1 bound to the LIM domain but not to the PDZ domain. Furthermore, the LIM fragment dose-dependently inhibited the kinase catalytic activity of the kinase core fragment of LIMK1. Taken together, these results suggest that the N-terminal LIM domain negatively regulates the kinase activity of LIMK1 by direct interaction with the C-terminal kinase domain. In addition, expression of the DeltaLIM mutant in cultured cells induced punctate accumulation of actin filaments, an event distinct from the pattern of actin organization induced by expression of the wild-type LIMK1, suggesting that the LIM domain plays a role in the function of LIMK1 in vivo.

Actins↗
PubMed1999

Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase.

The actin cytoskeleton undergoes extensive remodeling during cell morphogenesis and motility. The small guanosine triphosphatase Rho regulates such remodeling, but the underlying mechanisms of this regulation remain unclear. Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIM-kinase. Cofilin was phosphorylated in N1E-115 neuroblastoma cells during lysophosphatidic acid-induced, Rho-mediated neurite retraction. This phosphorylation was sensitive to Y-27632, a specific inhibitor of the Rho-associated kinase ROCK. ROCK, which is a downstream effector of Rho, did not phosphorylate cofilin directly but phosphorylated LIM-kinase, which in turn was activated to phosphorylate cofilin. Overexpression of LIM-kinase in HeLa cells induced the formation of actin stress fibers in a Y-27632-sensitive manner. These results indicate that phosphorylation of LIM-kinase by ROCK and consequently increased phosphorylation of cofilin by LIM-kinase contribute to Rho-induced reorganization of the actin cytoskeleton.

Actin Cytoskeleton↗
PubMed1999

The effect of lithium on methamphetamine-induced regional Fos protein expression in the rat brain.

Lithium has been used widely for the treatment of manic states. Since amphetamines produce effects in humans similar to the symptoms of idiopathic mania, amphetamine administration to animals has been proposed as a model of this condition. To investigate the neurobiologic substrates of the antimanic effects of chronic lithium administration, we investigated its effects on methamphetamine-induced regional Fos protein expression in the rat brain. Chronic lithium administration (14 days; serum lithium concentration, 0.41+/-0.02 mEq/l) significantly reduced the number of neuronal nuclei showing immunoreactivity induced by methamphetamine (2mg/kg) in the prefrontal cortex, caudate/putamen, nucleus accumbens, and central nucleus of the amygdala. These results indicate the structural basis in CNS which is responsible for the antimanic effect of lithium.

Animals↗
PubMed1999

Bovine leukaemia virus envelope peptides cause immunomodulation in BALB/c mice.

Immunomodulatory activity of two bovine leukaemia virus envelope (BLVEnv) derived peptides were examined in BALB/c mice. One is peptide homologous to CKS-17 which is known as a 17-amino acid peptide derived from p15E of feline leukaemia virus (CKS-17/BLV), and the other is an 18-amino acid synthetic peptide of BLV Env 61-78 (pep61). Priming with CKS-17/BLV in vitro, as well as CKS-17, significantly suppressed the mitogen-induced proliferative responses of spleen cells in naive BALB/c mice. In addition, priming of spleen cells with pep61 in vitro and in vivo resulted in suppression of lipopolysaccaride-induced B-cell proliferative response. This suppression was partially due to the basic amino acid sequence in the peptide because if the pep61-derived peptide lacking Arg was used, this inhibitory activity was partially restored. In contrast, pep61 enhanced both concanavalin A-stimulated proliferative response and IL-2 production. These findings showed that pep61 may contribute to the modification of the host immune responses in the course of BLV infection.

Adjuvants, Immunologic↗
PubMed1999

In vivo antiviral effect of interleukin 18 in a mouse model of vaccinia virus infection.

Interleukin-18 (IL-18), originally called interferon-gamma (IFN-gamma)-inducing factor is a novel cytokine which exhibits pleiotropic immunomodulatory activities such as the activation of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). In this study, the efficacy of IL-18 on viral infection in mice was investigated. IL-18 treatment significantly suppressed pock formation on the tails of BALB/c mice inoculated intravenously with vaccinia virus when the cytokine was administered intraperitoneally on days 0, 2 and 4 after infection. Sequentially, NK and CTL activity of the infected mice were significantly augmented by IL-18 injection. The in vivo anti-vaccinia virus activity of IL-18 was only partially inhibited by treating the infected mice with anti-asialo GM1 antibody. When infected mice were injected with anti-IFN-gamma antibody only, severe deterioration of health and significant body weight loss were observed, suggesting that IFN-gamma is very important in protecting mice against vaccinia virus infection. Interestingly, IL-18 injection visibly improved the severe vaccinia virus-induced symptoms in mice treated with anti-IFN-gamma antibody, even though a pivotal involvement of IFN-gamma in IL-18-mediated anti-vaccinia virus effect is not yet determined. Taken together, these results indicate that the IL-18-elicited anti-vaccinia virus effect in the acute phase of infection would be raised by the sum of various host defence mechanisms including NK cells and CTL, and not from a specific immunocompetent cell population or effector molecule.

Animals↗
PubMed1999

Oncogenic osteomalacia presenting as bilateral stress fractures of the tibia.

We report on a patient with bilateral stress fractures of the tibia who subsequently showed classic biochemical features of oncogenic osteomalacia. Conventional radiographs were normal. MR imaging revealed symmetric, bilateral, band-like low-signal lesions perpendicular to the medial cortex of the tibiae and corresponding to the only lesions subsequently seen on the bone scan. A maxillary sinus lesion was subsequently detected and surgically removed resulting in prompt alleviation of symptoms and normalization of hypophosphatemia and low 1,25-(OH)2 vitamin D3. The lesion was pathologically diagnosed as a hemangiopericytoma-like tumor. Patients with oncogenic osteomalacia may present with stress fractures limited to the tibia, as seen in athletes. The clue to the real diagnosis lies in paying close attention to the serum phosphate levels, especially in patients suffering generalized symptoms of weakness and not given to unusual physical activity.

Adult↗
PubMed1999

Re-isolation of Marek's disease virus from T cell subsets of vaccinated and non-vaccinated chickens.

To know the effect of Marek's disease (MD) vaccines, we analyzed the distribution of MD virus (MDV) among T cell subsets from chickens vaccinated or non-vaccinated with MD vaccine and subsequently challenged with a virulent MDV. The challenged MDV was reisolated preferentially from CD4+ T cells, and the average titers of challenged MDV rescued were significantly lower in vaccinated chickens compared to that of non-vaccinated chickens. In addition, it was also shown that different serotypes of MDV, CVI988 and SB-1, have remarkable difference in recovery rates of viruses from CD4+ and CD8+ T cells, though both CVI988 and SB-1 can reduce the infection rates of virulent MDV to splenocytes.

Animals↗
PubMed1999

Anti-viral and anti-tumor effects induced by an attenuated Marek's disease virus in CD4- or CD8-deficient chickens.

By vaccination with an attenuated Marek's disease virus (MDV), strain CVI988, chickens are protected from the development of T cell lymphoma caused by an oncogenic MDV. To clarify the role of T lymphocyte subsets in the protection mechanisms of this vaccine, vaccinated chickens were depleted of T cell subsets by neonatal thymectomy and injections of monoclonal antibodies specific to chicken CD4 and CD8 molecules, and then challenged with an oncogenic MDV, strain Md5. The MDV titers rescued from CD8(+) T cells, which are the main targets for latent infection and subsequent transformation by MDV, was much higher in the CD8-deficient vaccinated chickens than in untreated vaccinated chickens at the early stage of the latent phase. However, the neonatal vaccination prevented lymphoma formation by strain Md5 even in either CD4(+) or CD8(+) T cell-depleted chickens. These results suggest that specific CD8(+) T cell responses induced by the MD vaccine play a crucial role in the prevention of MDV infection during the latent phase, but may not be essential for the prevention of lymphoma formation by an oncogenic MDV.

Animals↗
PubMed1999

Effects of clarithromycin on the metabolism of omeprazole in relation to CYP2C19 genotype status in humans.

BACKGROUND AND PURPOSE: A triple therapy with omeprazole, amoxicillin (INN, amoxicilline), and clarithromycin is widely used for the eradication of Helicobacter pylori. Omeprazole and clarithromycin are metabolized by CYP2C19 and CYP3A4. This study aimed to elucidate whether clarithromycin affects the metabolism of omeprazole. METHODS: After administration of placebo or 400 mg clarithromycin twice a day for 3 days, 20 mg omeprazole and placebo or 400 mg clarithromycin were administered to 21 healthy volunteers. Plasma concentrations of omeprazole and clarithromycin and their metabolites were determined before and 1, 2, 3, 5, 7, 10, and 24 hours after dosing. CYP2C19 genotype status was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: Subjects were classified into three groups on the basis of PCR-RFLP analyses for CYP2C19: homozygous extensive metabolizer group (n = 6), heterozygous extensive metabolizer group (n = 11), and poor metabolizer group (n = 4). Mean area under the plasma concentration-time curves from 0 to 24 hours (AUC) of omeprazole in the homozygous extensive metabolizer, heterozygous extensive metabolizer, and poor metabolizer groups were significantly increased by clarithromycin from 383.9 to 813.1, from 1001.9 to 2110.4, and from 5589.7 to 13098.6 ng x h/mL, respectively. There were significant differences in the mean AUC values of clarithromycin among the three groups. CONCLUSION: Clarithromycin inhibits the metabolism of omeprazole. Drug interaction between clarithromycin and omeprazole may underlie high eradication rates achieved by triple therapy with omeprazole, amoxicillin, and clarithromycin.

Adult↗
PubMed1999

CYP2C19 genotype status and effect of omeprazole on intragastric pH in humans.

OBJECTIVE: Omeprazole is metabolized by genetically determined S-mephenytoin 4'-hydroxylase (CYP2C19) in the liver. This study aimed to determine whether the effect of omeprazole on intragastric pH depends on CYP2C19 genotype status. METHODS: CYP2C19 genotype status for 2 mutations associated with the poor metabolizer phenotype was determined by a polymerase chain reaction-restriction fragment length polymorphism method in 16 healthy volunteers. Helicobacterpylori status was determined by serology and the [13C]urea breath test. After a single oral administration of 20 mg omeprazole or a placebo, intragastric pH values were recorded for 24 hours. Plasma levels of omeprazole and its 2 metabolites and gastrin were measured before and 1, 2, 3, 5, 7, 10, and 24 hours after administration. RESULTS: Fifteen of the 16 subjects were H pylori negative. Five of the 15 subjects were homozygous extensive metabolizers, 4 were heterozygous extensive metabolizers, and 6 were poor metabolizers. After omeprazole administration, significant differences in mean intragastric pH values and plasma levels of gastrin, omeprazole and its metabolites were observed among the 3 groups, whereas no significant differences in these parameters were observed with the placebo administration. CONCLUSIONS: The effect of omeprazole on intragastric pH significantly depends on CYP2C19 genotype status. The genotyping test of CYP2C19 may be useful for an optimal prescription of omeprazole.

Adult↗
PubMed1999

Two-color fluorescence staining of lectin and anti-CD46 antibody to assess acrosomal status.

OBJECTIVE: To examine potential methods for distinguishing between the acrosome reaction and acrosomal loss. DESIGN: Prospective randomized study. SETTING: Department of Obstetrics and Gynecology, Osaka University Hospital, Suita, Japan. PATIENT(S): Five healthy volunteers and 34 patients with normozoospermia who were participating in an IVF program. INTERVENTION(S): Semen samples were collected from the volunteers before the hamster egg penetration assay and from the patients at the time of IVF. MAIN OUTCOME MEASURE(S): The numbers of oocytes penetrated and spermatozoa bound were determined with the hamster egg penetration assay. Acrosomal status was assessed with two-color fluorescence staining using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and MH61 (anti-CD46 monoclonal antibody) with Texas red-conjugated antimouse immunoglobulin G antiserum. RESULT(S): The MH61 monoclonal antibody inhibited the penetration of human spermatozoa into hamster oocytes but did not reduce the number of spermatozoa bound to the zona-free hamster oocytes. Two-color fluorescence staining revealed four staining patterns of the acrosomal region. The percentage of PSA-negative/CD46-positive spermatozoa increased to a greater extent than that of PSA-negative/CD46-negative spermatozoa with an increase in the incubation time. CONCLUSION(S): Two-color fluorescence staining with FITC-PSA and the anti-CD46 monoclonal antibody may be useful for distinguishing between the acrosome reaction and acrosomal loss.

Acrosome↗
PubMed1999

Up-regulation of tumor necrosis factor alpha mRNA is associated with bovine-leukemia virus (BLV) elimination in the early phase of infection.

Protective immune responses were analyzed in eight sheep vaccinated with BLV envelope peptides and experimentally infected with bovine-leukemia virus (BLV). Five of eight peptide-immunized sheep showed a high T-cell proliferative response to the BLV peptides and all of these were protected from the infection. The other three peptide-immunized sheep showed no T-cell proliferative responses to any BLV antigens similar to control sheep, though they also exhibited resistance to BLV challenge. To investigate other mechanisms which suppress BLV expansion in these non-responding sheep, we measured the levels of the cytokine expressions before, and after, BLV challenge using competitive reverse-transcriptase polymerase chain-reaction systems. It was revealed that the expression of tumor necrosis factor alpha (TNFalpha) was higher in BLV-resistant sheep than in BLV-susceptible sheep. Thus, TNFalpha expression rather than specific T-cell activity may play an important role in the protective mechanism against BLV infection, at least during the primary viremia phase.

Amino Acid Sequence↗
PubMed1999

Establishment of a persistent mutant of canine distemper virus.

We produced a B95a lymphoid cell line persistently infected with canine distemper virus (CDV), in which virus-specific antigens were present in nearly 100% of cells without causing cytopathic effect. The virus recovered from this cell line was able to infect fresh B95a cells persistently, indicating that a persistent CDV was established.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and sequence analysis of the phosphoprotein (P) gene of the lapinized rinderpest virus.

We determined the nucleotide sequence of the coding region for the phosphoprotein (P) gene of the L strain of rinderpest virus (RPV). The gene encodes two overlapping open reading frames of 1521 and 531 nucleotides. Use of the first ATG would produce a P polypeptide of 507 amino acids, while use of the second ATG would produce a C polypeptide of 177 amino acids. In addition, the insertion of an extra G residue at the editing site generates an alternative mRNA potentially encoding the V protein of RPV. Homology comparisons of the P, C and V proteins among various viruses suggest that RPV is closer to measles virus (MV) than to canine distemper virus (CDV). Alignment of the sequences unique to the V protein revealed that the cysteine residues are well conserved among RPV, MV and CDV, and form a "zinc finger"-like motif.

Amino Acid Sequence↗
PubMed1999

Molecular remission in adult T cell leukemia after autologous CD34+ peripheral blood stem cell transplantation.

This report describes a patient with acute-type adult T cell leukemia/lymphoma (ATLL) successfully treated by autologous CD34+ peripheral blood stem cell transplantation after fractionated total body irradiation and high-dose cytarabine and cyclophosphamide. A newly established inverse polymerase chain reaction method was used to demonstrate the disappearance of ATLL clonal cells. The patient achieved a sustained molecular remission after transplantation, but died from opportunistic infection 4 months after transplantation. Thus, autologous CD34+ peripheral blood stem cell transplantation is promising for this type of malignancy. However, a prudent clinical attitude toward immunological fragility after transplantation is needed for better outcome.

Antigens, CD34↗