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Biomedical subjects

M Sawada

Publications and source records attributed to M Sawada.

At least 127 records · Page 7Linked to original sources
PubMed1998

Differentiation-specific mRNA expression of a mouse bipotential glial cell line.

We have previously reported that a bipotential glial cell line from mouse cerebrum, designated OS3, phenotypically differentiates into oligodendrocytes and astrocytes both in vitro and in vivo. To study the potential mechanisms of differentiation, in this study we investigated mRNA expression of cytokines and developmentally regulated proteins in OS3 during differentiation into oligodendrocytes by semi-quantitative reverse transcription and polymerase chain reaction. In the presence of 10% calf serum OS3 cells expressed IL-1alpha and IL-1beta mRNA. However, when the cells were cultured in chemically defined medium or low serum-containing medium the expression of IL-1alpha and IL-1beta mRNA was down-regulated. Under stimulation of phorbol ester, expression of IL-6 and nerve growth factor mRNA was up-regulated. The capacity for differentiation of OS3 cells into oligodendrocytes in vitro was limited and most OS3 cells ceased their differentiation at the proligodendroblast stage. However, expression of proteolipid protein (PLP) and DM20 mRNA was detectable and was up-regulated in accordance with the differentiation into oligodendrocytes. As a control, primary astrocytes expressed DM20 mRNA but not PLP mRNA and the expression of DM20 mRNA was independent of culture condition. Therefore, OS3 cells will be of use for the study of differentiation of progenitor cells into type-2 astrocytes or oligodendrocytes at the molecular level.

Animals↗
PubMed1998

Biomarkers in long survivors of pediatric acute lymphoblastic leukemia patients: late effects of cancer chemotherapy.

In order to elucidate the late effects of cancer chemotherapy, mutant frequencies (Mfs) at the hypoxanthine phosphoribosyl transferase (hprt) locus were evaluated in pediatric patients with early pre-B acute lymphoblastic leukemia (ALL). Hprt-Mfs were measured at least 2 years after completion of chemotherapy. Ten out of 15 patients were found to have hprt-Mfs exceeding the 99% confidence limits as calculated from observations of healthy controls. Although there was some intraindividual variation, serial measurements of hprt-Mfs with intervals of more than 6 months revealed that hprt-Mfs were fairly stable. Patients with high Mfs tended to have sibling clones as detected by clonality analysis using the T-cell receptor (TCR) rearrangement pattern, but clonality did not have a major effect on the Mfs. On the other hand, Mfs at the TCR locus and sister chromatid exchange frequency were within the normal range in all patients. These data suggest that chemotherapy can cause persistent genotoxicity in vivo in a subset of pediatric ALL patients and that the hprt-Mf is a useful method for measuring such an effect.

Adolescent↗
PubMed1998

Induction of outward current by orexin-B in mouse peritoneal macrophages.

To define effects of novel feeding regulating peptides, orexins, in immunocompetent cells, ion channel activity in mouse peritoneal macrophages was analyzed by the perforated patch-clamp method. Orexin-B (OX-B) induced an outward current at smaller holding potentials than K+ equilibrium potentials. Reversal potentials of OX-B induced current were dependent on external K+ concentrations but not on external Cl- concentration. Orexin-A is less effective than OX-B. Quinine blocked the outward current and tetraethylammonium partially suppressed the current. These results suggest that OX-B can modulate macrophage functions through the activation of Ca2+-dependent K2+ channels.

Animals↗
PubMed1998

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 3. Discovery of a novel series of N-alkyl-N-[(fluorophenoxy)benzyl]-N'-arylureas with weak toxicological effects on adrenal glands.

A series of N-alkyl-N-[(fluorophenoxy)benzyl]-N'-arylureas were prepared and evaluated for their ability to inhibit intestinal acyl-CoA:cholesterol O-acyltransferase and to inhibit accumulation of cholesteryl esters in macrophages in vitro. In vivo hypocholesterolemic activity was assessed in cholesterol-fed rats by oral administration as a dietary admixture and/or by gavage in a PEG400 vehicle. Modification of the alkyl substituent on the N'-aryl moiety and on the urea nitrogen significantly influenced macrophage assay in vitro. Toxicological study revealed a distinct relationship between macrophage assay and the toxicity observed in adrenal glands of rabbits treated with representatives of this series of compounds. Investigations utilizing the macrophage assay as an indicator for adrenal toxicity led to the identification of compounds 1g (FR190809) and 1k (FR186485, or FR195249 as its hydrochloride salt) as potent, nonadrenotoxic, orally efficacious ACAT inhibitors irrespective of the administration method.

Acetylation↗
PubMed1998

Synthesis and effects of novel thiazole derivatives against thrombocytopenia.

5-(2-Pyridylsulfonyl)-2-thiazolamine (2) was effective both in mitomycin C (MMC)-induced thrombocytopenia and in an animal model of idiopathic thrombocytopenic purpura (ITP). It also suppressed the increase of autoantibodies against platelets in the ITP model and showed no blood toxicity. Chemical modification of 2 led to the discovery of more potent compounds against MMC-induced thrombocytopenia.

Animals↗
PubMed1998

Brain-specific gene expression by immortalized microglial cell-mediated gene transfer in the mammalian brain.

The intra-arterial injection of immortalized microglia transfected with the lacZ gene, resulted in the expression of beta-galactosidase in the rat brain at 48 h and the activity of -galactosidase was detected for up to 3 weeks post-injection. More than 30-fold higher activity of beta-galactosidase was detected in the brain than in the liver, lung or spleen at 48 h post-injection. This method allows us to easily deliver the gene of interest to the brain without influencing other organs. Our brain-targeting gene delivery system can facilitate gene therapy of several brain disorders, including brain tumor, metabolic disorders, and degenerative disorders, as well as investigation into the roles of particular genes in brain function and development.

Animals↗
PubMed1998

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 2. Identification and structure-activity relationships of a novel series of N-alkyl-N-(heteroaryl-substituted benzyl)-N'-arylureas.

A series of N-alkyl-N-(heteroaryl-substituted benzyl)-N'-arylurea and related derivatives represented by 2 and 3 have been prepared and evaluated for their ability to inhibit acyl-CoA:cholesterol O-acyltransferase in vitro and to lower plasma cholesterol levels in cholesterol-fed rats in vivo. Among these novel compounds, the type 3 series was superior. A pyrazol-3-yl group on the N-benzyl group of this trisubstituted urea (i.e. 3, Ar1 = pyrazol-3-yl) was identified as a heteroaromatic ring providing a good profile of biological activity. As a result of optimization of the combination with the N-alkyl group (R) and N-aryl group (Ar3), compound 3aq (FR186054) was identified as a new, orally efficacious ACAT inhibitor, which exhibited potent in vitro ACAT inhibitory activity (rabbit intestinal microsomes IC50 = 99 nM) and excellent hypocholesterolemic effects in cholesterol-fed rats, irrespective of administration mode (ED50 = 0.046 mg/kg dosed via the diet, ED50 = 0. 44 mg/kg administered by gavage in PEG400 vehicle). Moreover, a toxicological study revealed compound 3aq to be nontoxic to the adrenal glands of dogs when tested at a single dose of 10 mg/kg po.

Administration, Oral↗
PubMed1998

Nitric oxide donors inhibit the acetylcholine-induced Cl- current in identified Onchidium neurons.

The present study was undertaken to assess the effects of sodium nitroprusside (SNP) and diethylamine NO (C2H5)2N[N(O)NO]-Na+ (DEA/NO), NO donors, on an acetylcholine (ACh)-induced Cl- current in identified Onchidium neurons using voltage-clamp and pressure ejection techniques. Bath-applied SNP (10 microM) and DEA/NO (5-10 microM) reduced the ACh-induced Cl- current in the neurons without affecting the resting membrane conductance and holding current. The suppressing effect of NO donors were concentration-dependent and completely reversible. Pretreatment with 1H-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-1-one (1 micro M), a specific inhibitor of NO-stimulated guanylate cyclase, and hemoglobin (50 micro M), a nitric oxide scavenger, decreased the SNP-induced inhibition of the ACh-induced current. Intracellular injection of guanosine 3',5'-cyclic monophosphate (cGMP) or bath-application of 3-isobutyl-1-methylxanthine (50 micro M), a non-specific phosphodiesterase inhibitor, inhibited the ACh-induced current, mimicking the effect of NO donors. These results suggest that SNP and DEA/NO inhibit the ACh-induced Cl- current and that this effect is mediated by an increase in intracellular cGMP.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3.

The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http:@www.nite.go.jp.

Chromosomes, Archaeal↗
PubMed1998

Kinetic studies of sequence-specific binding of GCN4-bZIP peptides to DNA strands immobilized on a 27-MHz quartz-crystal microbalance.

Specific protein-DNA interaction was studied quantitatively by using a highly sensitive 27-MHz quartz-crystal microbalance (QCM). Biotinylated DNA double strands (21 bp, having a CRE site of 5'ATGACGTCAT3') were immobilized on an avidin-bound QCM surface, and sequence-specific binding of bZIP 56-mer peptides (having both the basic region for binding and the leucine zipper region for dimerization) to the DNA strand on the QCM was observed. The binding amount (Deltam) at the nanogram level and kinetic parameters such as association constants (Ka) and binding and dissociation rate constants (k1 and k-1) could be obtained from time courses of QCM frequency decreases. A bZIP peptide as a dimer was observed to bind sequence-specifically to one DNA strand having a CRE site. Ka values of ss-bZIP, in which the leucine-zipper region of bZIP was substituted by a Cys-Cys linkage, were largely decreased, and the sequence selectivity also disappeared. Ka values obtained by the QCM method showed good agreement with those obtained from the conventional gel mobility shift assay or from circular dichroism spectrum changes. When the specific sequence of the CRE site of DNA strands was partly changed, Ka values decreased by about a half due to the increase of the dissociation rate constant (k-1) independent of the binding rate constant (k1).

Amino Acid Sequence↗
PubMed1998

Production of interleukin-12 and expression of its receptors by murine microglia.

Production of interleukin-12 (IL-12) by cultured murine microglia and astrocytes was examined, by means of ELISA to detect heterodimeric p70 and RT-PCR to analyze the expression of mRNA encoding p35 and p40. Microglia, but not astrocytes, produced IL-12 p70 in response to lipopolysaccharide and interferon-gamma. The microglial cell line, Ra2, produced only p40, but not p35, upon above stimulation. Thus, it is possible that some population of microglia induce helper 1 type T cell response via producing IL-12 in the CNS. Microglia were induced to express mRNA encoding IL-12 receptors which were exclusively expressed in activated T and NK cells.

Animals↗
PubMed1998

Tryptophan and its metabolite, kynurenine, stimulate expression of nerve growth factor in cultured mouse astroglial cells.

Effects of L-tryptophan and its metabolites were evaluated on expression of nerve growth factor (NGF) in primary culture of mouse astroglial cells. L-Tryptophan produced concentration-dependent increases in accumulation of NGF transcripts in the cells. L-Kynurenine, a metabolite of the kynurenine pathway, markedly increased the levels of mRNAs for NGF, the maximal increases (4-5 fold) occurred at its dose of 1 microM. Kynurenine-induced increase in mRNA levels for NGF occurred as early as 1 h after the addition of the compound, peaked at 4 h and declined thereafter. In contrast to kynurenine, other tryptophan metabolites such as quinolinic acid, kynurenic acid and serotonin had little effect on the levels of NGF mRNA.

Animals↗
PubMed1998

Evaluation of mutant frequencies at the hprt and the T-cell receptor loci in pediatric cancer patients before treatment.

Mutant frequencies (Mfs) at the two genetic loci, the hypoxanthine phosphoribosyl transferase (hprt) gene and the T-cell receptor (TCR) gene were evaluated in pediatric cancer patients before starting chemotherapy or radiotherapy. The study population consisted of 27 patients with various solid tumors (mean age +/- SD; 5.5 +/- 5.1 years, range; 0.2-14.5 years), 5 patients with acute leukemia (10.3 +/- 6.1, 1.3-17.0 years), and 26 healthy controls (11.6 +/- 4.0, 4.4-22.2 years). Although the age distributions were different, the mean Mf values of the hprt and the TCR loci were comparable among these three groups. On an individual basis taking the age factor into consideration, the hprt-Mfs of 3 patients with solid tumors, i.e., two patients with Hodgkin's disease and one patient with Askin tumor, were found to be well above the 95% confidence limit. There were no patients with a TCR-Mf exceeding the 95% confidence limit. These data suggest the possibility that some patients with solid tumors may be predisposed to mutational susceptibility before treatment. The assay of the hprt-Mf appears more sensitive than the TCR-Mf assay in distinguishing these patients.

Adolescent↗
PubMed1998

Activation of outward current by pituitary adenylate cyclase activating polypeptide in mouse microglial cells.

In order to investigate the interaction between the nervous and immune systems, we have analyzed the effect of one of the neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP), on microglia cells by the patch-clamp method. Puff application of PACAP38 onto mouse microglial cells induced an outward current in a dose-dependent manner. Reversal potentials of the outward current were dependent on external K+ concentrations ([K+]0) and independent of [Cl-]0. Ion channel blockers of potassium currents, quinine (1 mM), tetraethylammonium (TEA, 20 mM) and 4-aminopyridine (4-AP, 5 mM), suppressed the outward current with a potency order of quinine>TEA>4-AP. PACAP27 also induced outward current less effectively than PACAP38. A fragment of PACAP38 [PACAP(6-38)], known as an inhibitor for PACAP38, suppressed the outward current. These data suggest that PACAP38 activates a quinine-sensitive K+ outward current and modulates activities in microglia. They indicate that the immune system in the brain can be modulated by neurotransmitters, the mediators of neurons.

Animals↗
PubMed1998

Role of protein tyrosine phosphorylation in etoposide-induced apoptosis and NF-kappa B activation.

When a human myeloid cell line, U937, was incubated with etoposide (10 micrograms/mL), morphologically apoptotic cells first appeared at 3 hr and increased with time to 50% at 6 hr. Pretreatment of U937 cells for 30 min with a potent tyrosine kinase inhibitor, herbimycin A (10 microM), significantly suppressed the appearance of apoptotic morphological changes. Concomitantly, herbimycin A pretreatment prevented both high molecular weight and internucleosomal DNA fragmentation induced by etoposide. Two major bands at 30 and 66 kDa with enhanced tyrosine phosphorylation inhibited by herbimycin A were detectable after 30 min of incubation with etoposide. In addition, herbimycin A prevented etoposide-induced NF-kappa B activation. The expressions of Bcl-2 and Bax, on the other hand, were not affected by herbimycin A pretreatment. Herbimycin A was also found to inhibit 1-beta-D-arabinofuranosylcytosine-induced apoptotic changes and NF-kappa B activation. These results suggest that activation of tyrosine kinase(s) may play an important role in apoptotic processes induced by a variety of anti-cancer drugs.

Apoptosis↗
PubMed1998

Synthesis, X-ray crystal structure, and biological activity of FR186054, a novel, potent, orally active inhibitor of acyl-CoA:cholesterol O-acyltransferase (ACAT) bearing a pyrazole ring.

The synthesis, single crystal X-ray structural analysis, and biological activity of FR186054 (2c), a new, potent, orally efficacious inhibitor of acyl-CoA:cholesterol O-acyltransferase (ACAT), containing a pyrazole ring are described. This compound displayed excellent in vitro efficacy, irrespective of dosing method, indicating superior characteristics compared to other ACAT inhibitors.

Administration, Oral↗