Biomedical subjects

M Sawada

Publications and source records attributed to M Sawada.

At least 217 records · Page 12Linked to original sources

PubMed1995

Insulin prevents apoptosis of external granular layer neurons in rat cerebellar slice cultures.

Using the slice culture system of 9-day-old rat cerebellum, effects of insulin on cell death of developing granule neurons were examined. Apoptotic cells were observed after 3 days culture by the in situ nick end labeling technique. Insulin deprivation induced apoptosis of granule neurons largely in the external granular layer, but scarcely in the internal granular layer. Proliferation of external granular layer neurons during the early culture period was not affected significantly by the insulin deprivation. These results suggest that insulin may prevent apoptosis of premigratory granule neurons during the development of the cerebellar cortex.

Animals↗

PubMed1995

Simultaneous expression of human CYP3A7 and N-acetyltransferase in Chinese hamster CHL cells results in high cytotoxicity for carcinogenic heterocyclic amines.

To investigate whether several food-derived heterocyclic amines are activated to genotoxic products in human fetal livers, cell lines stably expressing CYP3A7, a human fetus-specific form of cytochrome P450, NADPH-cytochrome P450 reductase, and human monomorphic or polymorphic N-acetyltransferase (NAT1 or NAT2) were established. The expression of CYP3A7 mRNAs and proteins was determined by RNA blot and immunoblot analyses, respectively. The introduction of CYP3A7 cDNA to CR-68 cells which had been transfected with guinea pig NADPH-cytochrome P450 reductase, NAT1, or NAT2 cDNA resulted in increased sensitivity of the cells to aflatoxin B1 compared to parental cells. The cytotoxicity assay for 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) showed that 7P-145 cells, which expressed the reductase, CYP3A7, and NAT2, were approximately 4-, 30-, and 14-fold more sensitive to respective IQ, MeIQ, and MeIQx than parental CR-68 cells. There were no clear differences in sensitivity to these compounds among CHL, CR-68, and the cells which expressed the reductase and CYP3A7 (7R-54), the reductase and NAT1 (CNM-4), the reductase and NAT2 (CNP-40), and the reductase, NAT1, and CYP3A7 (7M-124). From these results, it was suggested that both CYP3A7 and polymorphic NAT2 are required for mutagenic activation of several heterocyclic amines in human fetal livers.

Amines↗

PubMed1995

Programmed cell death of PC12 induced by adenovirus E1A.

PC12 rat pheochromocytoma cells undergo programmed cell death or apoptosis with DNA fragmentation when deprived of serum. Here, we isolated a subclone of PC12, PC12FrR, that was resistant to DNA fragmentation in a serum-free condition. PC12FrR grew slightly faster, and had cells that were slightly larger than parental PC12 cells. Adenovirus E1A gene-transfected PC12FrR cells grew much faster than did parental PC12FrR cells in the presence of serum. In a serum-deprived condition, E1A-transfected PC12FrR cells died with DNA fragmentation, as did PC12 cells under the same conditions. These results suggest that the target(s) of E1A gene products may be involved in the mechanism(s) that regulate growth and death of neuronal cells.

Adenoviridae↗

PubMed1995

Expression of glutamate transporters in cultured glial cells.

Expression of mRNAs for glutamate transporter (GLT-1) and glutamate aspartate transporter (GLAST) was investigated in three different types of purified glial cells by the reverse transcriptase-polymerase chain reaction (RT-PCR). Cultured astrocytes, oligodendrocytes, and microglia expressed mRNAs for GLAST and GLT-1; mRNA for GLAST was expressed more prominently than that for GLT-1 in astrocytes. Oligodendrocytes and microglia expressed mRNAs for both GLT-1 and GLAST equally, but the expression in microglia was not prominent, suggesting glutamate uptake is not essential in microglia. In astrocytes cultured from different brain regions, GLAST mRNA was equally expressed. GLT-1 mRNA was also detected in these astrocytes, but the expression level was lower than that of GLAST.

ATP-Binding Cassette Transporters↗

PubMed1995

cDNA cloning of a thromboxane A2 receptor from rat astrocytes.

A cDNA clone for rat thromboxane (TX) A2 receptor was obtained from cultured astrocytes. The cDNA encodes a protein of 341 amino acids with seven putative transmembrane domains. The receptor is homologous with mouse and human TXA2 receptors at 92.7% and 71.8%, respectively. Expression of messenger RNA for the receptor was examined by reverse transcriptase-polymerase chain reaction, showing ubiquitous expression in brain glial cells such as astrocytes, oligodendrocytes and microglia.

Amino Acid Sequence↗

PubMed1995

The bipotential glial progenitor cell line can develop into both oligodendrocytes and astrocytes in the mouse forebrain.

Oligodendrocyte-type 2 astrocyte (O2A) progenitor cells in vivo might differentiate into oligodendrocytes. To examine the influence of the brain micro-environment on the differentiation, a bipotential glial cell line from the mouse cerebrum, designated OS3 cells, was implanted into the telencephalon of infant and adult mice. About a half of the OS3 cells injected into 1-week postnatal brain expressed galactocerebroside (GalC), and even myelin basic protein, which were not observed to be expressed in vitro. By contrast, in the brain over 6 months postnatally, many OS3 cells expressed glial fibrillary acidic protein, and did not express much GalC. These findings suggest that the differentiation of glial cells is controlled by stage specific factors in the brain.

Animals↗

PubMed1995

Fetus-specific CYP3A7 and adult-specific CYP3A4 expressed in Chinese hamster CHL cells have similar capacity to activate carcinogenic mycotoxins.

To assess whether CYP3A4 and CYP3A7 have a similar capacity to activate carcinogenic mycotoxins, we established cell lines stably expressing human CYP3A4 and CYP3A7, which are adult- and fetal-specific forms of cytochrome P450 in human livers, respectively. Each cDNA was introduced into CR-119 cells which had been established by introducing guinea pig NADPH-cytochrome P450 reductase cDNA into Chinese hamster lung cells. The cell lines (4-line and 7-line) stably expressed the mRNA and the protein corresponding to CYP3A4 and CYP3A7, respectively. The concentration-response for aflatoxin B1 (AFB1) cytotoxicity in 4-line and 7-line, respectively, was compared. 4-10 and 7-40 cells were approximately 17- and 20 times more sensitive to AFB1 than the parental CR-119 cells, respectively. In addition, the sensitivities to AFB1 of both 4-10 and 7-40 cells were enhanced approximately seven times by the addition of 10 microM alpha-naphthoflavone, a known activator of CYP3A enzyme, while the sensitivities were suppressed approximately four times by the addition of 100 microM troleandomycin, which forms a metabolite intermediate complex with CYP3A enzyme. Moreover, both cell lines showed approximately 10 and 2 times higher sensitivity to sterigmatocystin and aflatoxin G1 than CR-119 cells, respectively. These results indicate that CYP3A4 and CYP3A7 have essentially similar capacities to activate AFB1, sterigmatocystin, and aflatoxin G1 to produce toxic metabolites.

Adult↗

PubMed1995

cDNA cloning and characterization of a novel receptor-type protein tyrosine phosphatase expressed predominantly in the brain.

Protein tyrosine phosphatase has the potential to control various cellular events by negatively regulating the extent of tyrosine phosphorylation. Here, we report the isolation of a murine receptor protein tyrosine phosphatase, PTPBR7, which is expressed almost exclusively in the brain. Though the cytoplasmic portion of PTPBR7 reveals high similarity to HePTP/LC-PTP and STEP, these are, unlike PTPBR7, non-receptor protein tyrosine phosphatases. Unlike most receptor protein tyrosine phosphatases, PTPBR7 has only one cytoplasmic phosphatase domain, and its extracellular domain reveals no obvious structural similarity to known molecules. Thus, PTPBR7 defines a new subfamily of receptor-type protein tyrosine phosphatases. The putative extracellular domain of PTPBR7 was expressed in COS-7 cells as a chimeric fusion protein with an immunoglobulin Fc portion (PTPBR7-Fc). PTPBR7-Fc was secreted in the culture supernatant, confirming the capability of the extracellular domain of PTPBR7 to translocate across the cytoplasmic membrane. The cytoplasmic portion of PTPBR7 was expressed as a fusion protein in bacteria and was demonstrated to have catalytic activity. The expression of PTPBR7 was detectable in brain and especially in cerebellum but undetectable in liver, lung, heart, kidney, thymus, bone marrow, and spleen. In situ hybridization analysis revealed the most prominent signal in Purkinje cells. The predominant expression of PTPBR7 in the brain suggests that PTPBR7 may have role(s) in neuronal cells.

Amino Acid Sequence↗

PubMed1995

Nitric oxide induces an increased Na+ conductance in identified neurons of Aplysia.

The ionic mechanism of the effects of micropressure ejections of hydroxylamine (HOA) and sodium nitroprusside (SNP), nitric oxide (NO) generators, on the membrane of identified neurons (R9-R12) of Aplysia kurodai was investigated with conventional voltage-clamp, micropressure ejection, and ion-substitution techniques. Micropressure ejection of HOA and SNP onto the neurons caused a marked depolarization in the unclamped neurons. Clamping the same neurons at their resting potential level (-60 mV) and reejecting HOA and SNP with the same dose produced a slow inward current (Ii(HOA) and Ii(SNP), 3-7 nA in amplitude, 15-60 s in duration) associated with an increase in input membrane conductance. Bath-applied hemoglobin (50 microM), a nitric oxide scavenger, almost completely blocked Ii(HOA) and Ii(SNP), and 3-isobutyl-1-methylxanthine (IBMX, 50 microM) prolonged and enhanced both Ii(HOA) and Ii(SNP). An intracellular injection of cyclic guanosine 3',5'-monophosphate (cGMP) into the same neurons produced a slow inward current (Ii(cGMP)) which resembled the responses to HOA and SNP, and this current was enhanced in IBMX. Bath-applied methylene blue (10 microM), an inhibitor of guanylate cyclase, significantly reduced Ii(HOA) and Ii(SNP). The inward currents induced by HOA, SNP and cGMP were sensitive to changes in the external Na+ concentration. These results suggest that extracellular NO can induce a slow inward current associated with an increase in Na+ conductance, mediated by an increase in intracellular cGMP.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Efficacy of combination therapy with interferon and azidothymidine in chronic type C hepatitis: a pilot study.

The effects of interferon are seen in only a limited number of patients with hepatitis C virus (HCV) of the K1 type, indicating that a combination therapy with other antiviral drugs may be essential to obtain better results. In the present pilot study, the effects of combination therapy with interferon (IFN) and an antiviral drug azidothymidine (AZT) were analyzed. The combination therapy was conducted in 22 patients with chronic hepatitis C after obtaining their informed consent (combination group). Three or six million units of natural IFN alpha was administered daily for 3 weeks and then three times a week for 21 weeks. Combination therapy was initiated at the beginning of the 8th week of IFN treatment, 500 mg of AZT per day being given for 8 weeks. As a control, changes in HCV-RNA were also analyzed in patients treated with interferon alone (IFN-alone group). At the end of the treatment, blood was negative for HCV in 32.5% of the IFN-alone group and in 50.0% of the combination group, the difference not being significant. However, in patients with HCV-K1, HCV-negative rates were 14.2% in the IFN-alone group and 45.5% in the combination group, showing a significant difference. In patients with other HCV genotypes, HCV-negative rates did not different between the two groups. These results suggest that combination therapy with IFN and AZT may be an effective treatment for chronic type C hepatitis caused by the K1 type virus, although further studies on larger number of patients will be needed to obtain definite conclusions.

Adolescent↗

PubMed1995

Biphasic pressor responses to norepinephrine in humans.

We investigated pressor responses to intravenous bolus infusion of norepinephrine in seven healthy volunteers. Norepinephrine (1, 2, and 4 micrograms/kg) elevated blood pressure in a concentration-dependent manner and decreased heart rate. The pressor response to norepinephrine was biphasic (early and late). Intravenous administration of phentolamine (10 mg) completely abolished the pressor response to norepinephrine, and prazosin (5 mg, given orally) inhibited the early and late responses to a same extent. Continuous intravenous infusion of nicardipine (2 micrograms/kg/min) inhibited the late pressor response but not the early one. These data suggest that bolus infusion of norepinephrine evoked the biphasic pressor response and that the late response depends on vasoconstriction by calcium influx through nicardipine-sensitive calcium channels.

Adult↗

PubMed1995

Stimulation of progesterone secretion in dispersed cells of rat corpora lutea by antioxidants.

Antioxidants were used to investigate the role of free radicals in control of luteal steroidogenesis. Corpora lutea from pseudopregnant rats were enzymatically dispersed, the cells were incubated with antioxidants, and progesterone production was measured. Addition of the antioxidants nordihydroguaiaretic acid (NDGA), butylated hydroxytoluene (BHT), and the gonadotropin luteinizing hormone (LH) resulted in a dose-dependent increase in progesterone secretion. However, the response pattern to these treatments differed with the age of the corpora lutea, and unlike LH neither NDGA nor BHT treatment resulted in an increase in the intracellular second messenger cAMP. Nevertheless, LH and antioxidant-induced progesterone stimulation could be blocked by the addition of either aminoglutethimide or ketoconazole, cytochrome P450 side-chain cleavage (cytochrome P450 SCC) enzyme inhibitors, which prevent the conversion of cholesterol to pregnenolone and thus block steroid hormone synthesis. Also, unlike exposure to LH, exposure to antioxidants resulted in an additional increase in progesterone production in luteal tissue saturated with 25 hydroxycholesterol, a soluble cholesterol analog which serves as a substrate for cytochrome P450 SCC. This study suggests that the site of antioxidant action in affecting progesterone secretion may be at the cytochrome P450 SCC enzyme. Based on these results and on studies in other steroid hormone-producing cells, it appears that free radicals may be involved in regulating synthesis by modulating activity of cytochrome P450 SCC enzyme in rat luteal tissue.

Animals↗

PubMed1995

In vivo setting behaviour of fast-setting calcium phosphate cement.

The in vivo setting behaviour of fast-setting calcium phosphate cement (FSCPC) between femoral muscles of the rat was investigated to evaluate the possible value of FSCPC for medical and dental application. Conventional CPC (c-CPC) and FSCPC were implanted between femoral muscles, and various aspects of the setting behaviour such as setting time, mechanical strength and conversion ratio of cement into hydroxyapatite (HAP: Ca10(PO4)6(OH)2) were measured by the Vicat needle method, diametral tensile strength (DTS) measurement, and quantitative powder X-ray diffraction (XRD) analysis, respectively. The setting time of FSCPC in vivo was 5-7 min, in contrast to 48 min for c-CPC. As a result of its fast setting, set specimens of FSCPC showed higher mechanical strength from the initial stage than c-CPC. Higher DTS values were observed in FSCPC than c-CPC implanted after 24 h. Powder XRD analysis revealed faster conversion of FSCPC than c-CPC into HAP, which was responsible both for the faster setting and higher mechanical strength from the initial stage. We concluded, therefore, that FSCPC may be used for a wide range of clinical applications, i.e. fields where fast setting is required such as orthopaedic, plastic and reconstructive, and oral and maxillofacial surgery.

Animals↗

PubMed1995

Enhancement of phagocytosis in mouse macrophages by pituitary adenylate cyclase activating polypeptide (PACAP) and related peptides.

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) and related peptides on phagocytosis of fluorescent latex beads by mouse peritoneal macrophages were examined using flow cytometry (FCM). PACAP38, PACAP27 and vasoactive intestinal peptide (VIP) enhanced phagocytosis in a dose-dependent manner. Relative potencies of related peptides at a concentration of 10(-6) M were PACAP38 > PACAP27 > VIP > secretin > glucagon > (peptide with NH2-terminal histidine and COOH-terminal methionine amide, in short PHM). PACAP(6-38) was as effective as PACAP38. PACAP(6-27) enhanced phagocytosis more effectively than did PACAP27. PACAP(28-38) slightly enhanced phagocytosis. The present result suggest that PACAP38 is one of the mediators that the nervous system uses to modulate the immune system.

Animals↗

PubMed1995

Enhancement of phagocytosis by dynorphin A in mouse peritoneal macrophages.

The effects of the opioid peptide dynorphin A (DynA) on phagocytosis in peritoneal macrophages was examined by flow cytometry (FCM). DynA enhanced phagocytosis in a dose-dependent manner. Leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), beta-neo-endorphin (beta Neo-End), DynA(9-17) and DynA(13-17) had no such activity. Alpha-Neo-endorphin (alpha Neo-End), dynorphin B (DynB), DynA(1-13) and DynA(6-17) enhanced phagocytosis less effectively than DynA. Naloxone did not inhibit the enhancement of phagocytosis induced by DynA. Unstimulated control phagocytosis was partially suppressed in Ca2+-free EGTA-containing solution and even in this solution DynA enhanced phagocytosis. However, the enhancement by DynA was suppressed in EGTA- and BAPTA-AM-containing Ca2+-free solution. The present study showed that enhancement of phagocytosis by DynA was independent of extracellular Ca2+ ([Ca2+]o) and dependent on intracellular Ca2+ ([Ca2+]i). The present results support DynA being one of the mediators from the nervous system that modulates the immune system.

Amino Acid Sequence↗

PubMed1995

Induction of functional interleukin-2 receptor in mouse microglia.

Interleukin (IL)-2, initially discovered for its mitogenic activity on T cells, also acts on monocytes, resulting in the activation of cytokine production, superoxide production, and tumoricidal activity. Because severe brain damage was observed in IL-2-transgenic mice, this cytokine may have some influence(s) on the cells of the CNS. We investigated IL-2 receptor-bearing cells in the CNS and found that activated microglia expressed alpha-chain mRNA and immunoreactive IL-2 receptor beta-chain protein in culture. Although microglia did not express IL-2 receptors under normal culture conditions, they were induced to express these receptors by lipopolysaccharide (LPS) in a time-dependent manner. The IL-2 receptors were found to be functional because the viability and growth activity of LPS-treated microglia, but not untreated controls, increased in response to recombinant mouse IL-2 as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay and bromodeoxyuridine uptake experiment, respectively. These effects of recombinant IL-2 were blocked by pretreatment with anti-mouse IL-2 receptor beta-chain antibody. Our findings suggest that activated microglia in the CNS can respond to this T cell-derived factor regulating their growth, which may be an important mechanism of communication between nervous and immune systems in physiological and pathological conditions.

Animals↗

PubMed1995

Polymorphic drug metabolism: studies with recombinant Chinese hamster cells and analyses in human populations.

Most promutagens and procarcinogens exert their genotoxicity after undergoing metabolic activation. Metabolism of chemicals is an important factor in limiting the extent of the action of a chemical. In this study, we established cell lines which carried cDNAs coding for human CYP1A2 and N-acetyltransferase (NAT); the latter functions as O-acetyltransferase for N-hydroxyarylamines formed by CYP1A2. A cell line which expresses CYP1A2 together with P450 reductase activated aflatoxin B1, but not heterocyclic amines. A cell line which carries CYP1A2 and polymorphic NAT (NAT2) in addition to P450 reductase efficiently activated IQ and some other heterocyclic amines. However, a cell line which carries CYP1A2 and monomorphic NAT (NAT1) had only low activity toward the same heterocyclic amines. In order to determine the presence of and frequency of genetic polymorphisms of CYP1A2 and NAT2 in humans, we performed in caffeine phenotyping test on 205 Japanese volunteers. Analyses of metabolic ratios of urinary metabolites showed a bimodal distribution, indicating that about 86% and 91% of Japanese were extensive metabolizers (EM) of CYP1A2 and NAT2, respectively. The genotype NAT2 determined by the PCR-RFLP method agreed completely with the phenotype. To determine the mechanism of the differences in CYP1A2 activity, genomic DNA from peripheral lymphocytes of poor metabolizers (PM) and EM was subjected to DNA sequencing. No differences in nucleotide sequence were observed between PMs and EMs in the exons, exon-intron junctions and 5'-flanking region of the CYP1A2 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

A flow cytometric assay reveals a suppression of phagocytosis by rabbit defensin NP-3A in mouse peritoneal macrophages.

Phagocytosis of fluorescein isothiocyanate (FITC)-labeled polystyrene microparticles by peritoneal macrophages from thioglycollate-elicited mice was examined by means of flow cytometry (FCM). This assay revealed that rabbit defensin NP-3A suppressed the phagocytosis in a dose-dependent manner. The present results suggest that NP-3 released from neutrophils is one of the mediators which modulates the activity of macrophages in response to infection.

Animals↗