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M Sakai

Publications and source records attributed to M Sakai.

At least 343 records · Page 19Linked to original sources
PubMed1995

NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, increases extracellular GABA in the striatum of the freely moving rat.

We investigated the effect of an NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), on the levels of endogenous GABA in the rat striatum using in vivo microdialysis. Rats were perfused with the artificial CSF containing L-NMMA (0.1, 0.3 and 1.0 mM) or its inactive isomer D-NMMA (1.0 mM) for 1 h. Infusion of L-NMMA, but not its D-isomer, dose-dependently increased GABA concentration. Co-infusion with tetrodotoxin (1 microM) did not antagonize the increase of GABA induced by L-NMMA. These results show that decreased NO activity enhances GABA release even in the absence of depolarization of GABA neurones. We conclude that NO may be directly acting on GABA nerve terminals and tonically inhibiting GABA release or synthesis under basal conditions.

Animals↗
PubMed1995

Differential effects of an acyl-coenzyme A:cholesterol acyltransferase inhibitor on HDL-induced cholesterol efflux from rat macrophage foam cells.

When rat macrophages were converted to foam cells with acetylated low density lipoprotein (acetyl-LDL) and then reacted with high density lipoprotein (HDL) and an inhibitor of acyl-coenzyme A:cholesterol acyltransferase (58-035) (sequential incubation system), 58-035 did not enhance HDL-induced cholesterol efflux. In contrast, when macrophages were exposed to acetyl-LDL in the presence of both HDL and 58-035 (simultaneous incubation system), HDL-induced cholesterol efflux was enhanced 1.6-fold by 58-035. Cholesterol efflux with HDL alone was 2-fold greater in simultaneous incubation than in sequential incubation. These results suggest the presence of an efficient cholesterol efflux pathway in simultaneous incubation which is not available in sequential incubation. This pathway, which we refer to as the neutral cholesterol ester hydrolase-independent pathway, is characterized by the efflux of lysosome-derived cholesterol without re-esterification.

Animals↗
PubMed1995

The structure and physicochemical properties of rat liver macrophage migration inhibitory factor.

We expressed rat macrophage migration inhibitory factor (rMIF) in E. coli using the cDNA isolated from a rat liver cDNA library. rMIF specifically bound glutathione (dissociation constant = 500 microM). We purified rMIF homogeneously on SDS-PAGE by S-hexylglutathione Sepharose affinity column chromatography and Sephadex G-100 column chromatography. The amino-acid sequence of rMIF was highly homologous to that of human MIF from a T-cell line; only a single amino-acid residue was substituted if conservative amino-acid substitutions were involved. The molecular weight of rMIF was calculated to be 12.4 kDa and 23.6 kDa by SDS-PAGE and analytical ultracentrifugation, respectively. Thus, it was concluded that the native rMIF formed a homodimeric structure. Proton nuclear magnetic resonance (1H-NMR) study revealed that rMIF was less thermostable (the denaturing temperature was from 50-60 degrees C) than human MIF (the denaturing temperature is about 80 degrees C (Nishihira et al. (1993) Biochem. Mol. Biol. Int. 31, 841-850). The secondary structure of rMIF evaluated by 1H-NMR experiments revealed that the contents of alpha-helix, beta-strand, and coil were 13.8%, 55.6%, and 30.6%, respectively.

Animals↗
PubMed1995

A synthetic peptide corresponding to a critical intracellular signaling region of the human IL-4 receptor inhibits IL-4-induced proliferation.

We have previously identified a critical region for growth signal transduction in the cytoplasmic domain of the human IL-4 receptor (hIL-4R). Since the entire cytoplasmic domain of this receptor lacks known catalytic activities such as the tyrosine kinase domain, it is likely that the IL-4R associates with other signal-transducing molecules through this critical cytoplasmic region. We test here whether a synthetic peptide corresponding to this critical cytoplasmic region, designated SP-1, interferes with IL-4-induced proliferation by competing with the IL-4R for binding to intracellular signal-transducing molecules. Our data indicated that 100 micrograms/ml SP-1 peptide completely inhibits human IL-4 (hIL-4)-induced proliferation of Ba/F3 transfectants expressing the full-length hIL-4R (hIL-4R-Ba/F3 transfectants). In contrast, a wide concentration range of an unrelated synthetic peptide, designated SP-2, did not affect hIL-4-induced proliferation of hIL-4R-Ba/F3 transfectants. This difference between SP-1 and SP-2 peptides was not due to their differential uptake by cell, since approximately 100 times more SP-2 peptide could be found in cytoplasmic extracts than SP-1 peptide in experiments using radiolabeled peptides. The specificity of SP-1-mediated inhibition of IL-4-induced proliferation was supported by the fact that the SP-1 peptide had no effect on IL-3-induced proliferation of the same hIL-4-Ba/F3 transfectants. In addition, the SP-1 peptide did not affect either IL-2-induced proliferation of Ba/F3 transfectants expressing the human IL-2 receptor beta chain (hIL-2R beta) or hIL-4-induced proliferation of Ba/F3 transfectants expressing a chimeric receptor consisting of the hIL-4R extracellular domain and the hIL-2R beta cytoplasmic domain. SP-1 was unable to inhibit IL-4-induced proliferation of other IL-4-responsive cell lines such as human erythroleukemic cell line TF-1 and mouse T cell lines HT2 and CTLL-2. In addition, SP-1 caused only a 50% inhibition of Ba/F3 cell proliferation induced by mouse IL-4. The failure of SP-1 to inhibit IL-4-induced proliferation in these various cell lines while producing excellent inhibition of hIL-4-induced proliferation of hIL-4R-Ba/F3 transfectants appeared to be related to the number of IL-4Rs expressed on each cell type.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Immunocytochemical localization of GTP cyclohydrolase I in the brain, adrenal gland, and liver of mice.

GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), the cofactor of phenylalanine, tyrosine, and tryptophan hydroxylases, the enzymes that synthesize tyrosine, catecholamines (dopamine, noradrenaline, and adrenaline), and serotonin, respectively. We produced for the first time polyclonal antibody with highly sensitive immunoreactivity against an oligopeptide of rat enzyme, GEPERELPRPGA, by immunization of rabbits with the peptide conjugated to hemocyanin by glutaraldehyde. The specificity of the antibody was confirmed by Western blot analysis. Using this antibody specific for GCH, we observed strong GCH immunostaining in the liver cells, in the dopamine-, noradrenaline-, adrenaline-, or serotonin-containing cells of the brain, and in the adrenal gland of mice. Immunocytochemical studies revealed GCH to be localized in monoamine-containing perikarya in the periglomerular cells of the olfactory bulb, zona incerta, arcuate nucleus, ventral tegmental area, substantia nigra pars compacta, locus ceruleus, nucleus tractus solitarius, area postrema, and ventrolateral area of the medulla oblongata. GCH immunostaining was particularly strong in serotoninergic nuclei, such as dorsal and median raphe nuclei, nucleus raphe pallidus, and nucleus raphe magnus. By immunoelectron microscopy, GCH-labeled cytoplasm and microtubules in the processes were observed ultrastructurally, but no staining was found in the mitochondria, and Golgi apparatus. Immunostaining was observed neither in the group D neurons that contain only aromatic amino acid decarboxylase without tyrosine hydroxylase, nor in glial cells and endothelial cells. These results indicate the abundant presence of GCH in catecholaminergic and serotoninergic neurons as well as in the adrenal medulla and liver, where BH4 is synthesized as the cofactor of tyrosine, tryptophan, and phenylalanine hydroxylases.

Adrenal Glands↗
PubMed1995

Intrauterine ultrasonography with a high-frequency probe: preliminary report.

Intrauterine ultrasonography was performed using miniature probes and high-frequency ultrasound to examine the possible clinical applications of this technique to gynecology. There were 44 women in the study population (age 19-76; ten with a normal uterus, nine fibromyoma, 15 endometrial cancer, ten cervical cancer). The probe was easily introduced into the endometrial cavity in 36 subjects (81.8%). No notable complications were encountered. In subjects with a normal uterus, the endometrium in the secretory phase showed a clearly demarcated hyperechoic area, and higher resolution was obtained than with transvaginal scanning. In patients with fibromyomas, myoma nodules were not clearly visualized because of the attenuation of ultrasound. Myometrial invasion of endometrial cancer was estimated correctly in ten of the 11 patients in which the lesion was visualized. In the subjects with cervical cancer, intracervical scanning was completed in only five cases, and an endocervical lesion was identified in three cases. Intrauterine ultrasonography with a high-frequency miniature probe may be a useful tool, especially in the preoperative evaluation of endometrial cancer and possibly in infertility practice. However, this modality does not appear to be satisfactory for cervical lesions.

Adult↗
PubMed1995

Effects of monocular enucleation at birth upon learning of a vertical-horizontal discrimination in hooded rats.

Previously we have demonstrated that adult albino rats with one eye removed at birth (OEB) relearn a black-white discrimination faster than those monocularly enucleated at maturity (OET) when relearning is conducted after lesioning of the visual cortex contralateral to the remaining eye (Type A experiment). This faster relearning phenomenon is considered to be one behavioral expression of the functioning of the expanded uncrossed visual pathways (expanded UXVPs) resulting from monocular enucleation at birth. However, neither OEBs nor OETs were able to master the discrimination when the experiment was conducted without previous learning following the same surgical treatment (Type B experiment). We hypothesized that this occurs because the cues to discriminate might be close to the threshold of discrimination for either the normal UXVPs or the expanded UXVPs. In order to gain insight into the hypothesis, the present study was undertaken using hooded rats as subjects which genetically possess larger and presumably more efficient functioning UXVPs. The questions addressed were as follows: 1) Whether or not the UXVPs can mediate a vertical-horizontal discrimination in OEBs and OETs. 2) If they can, is there any difference in the upper limit of discrimination capacity between the normal UXVPs and the expanded UXVPs? Three experiments were carried out. In the Type A experiment OEBs relearned discrimination of the 10-mm stripes [0.44 cycles/degree (c/d)] faster than OETs (Experiment 1), yet in the Type B experiment neither OEBs nor OETs were capable of acquiring that discrimination (Experiment 2). However, they could originally master the discrimination equally well when the width of stripes was broadened to 30 mm (0.15 c/d). And when the width of stripes was systematically reduced thereafter, the width of the smallest stripes for the expanded UXVPs to discriminate was found to be 6 mm (0.73 c/d) and that for the normal UXVPs 10 mm (0.44 c/d) [Experiment 3]. These findings were discussed in relation to the hypothesis advanced on our previous data in albino rats.

Animals↗
PubMed1995

A quick test for sound discrimination ability of rats in a single session after preparatory training.

We developed a sound discrimination test combined with a preparatory training procedure. After water deprivation of 48 h, rats were trained to respond to a sound with pedal-pressing to receive a reward of water. Six of the eight trained rats showed pedal-pressing responses to the sound within 4 h of training, and these six were exposed to two different sounds, response to only one of which was rewarded with water. In a single session of 10 h, the rate and latency of pedal-pressing in response to the two sound stimuli were continuously monitored. All six tested rats showed behavioral discrimination between the rewarded and unrewarded sounds within 6 h.

Acoustic Stimulation↗
PubMed1995

Analysis of the alternative promoters that regulate tissue-specific expression of human aromatic L-amino acid decarboxylase.

Previously we identified two alternative first exons (exon N1 and exon L1) coding for 5' untranslated regions of human aromatic L-amino acid decarboxylase (AADC) and found that their alternative usage produced two types of mRNAs in a tissue-specific manner. To determine the cis-acting element regulating the tissue-specific expression of human AADC, we produced three kinds of transgenic mice harboring 5' flanking regions of the human AADC gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The transgene termed ACA contained -7.0 kb to -30 bp in exon N1, including the entire exon L1; ACN contained -3.6 kb to -30 bp in exon N1; and ACL contained -2.8 kb to -42 bp in exon L1. The ACA transgenic mice expressed CAT at extremely high levels in peripheral nonneuronal tissues, such as pancreas, liver, kidney, small intestine, and colon, that contained endogenous high AADC activity, whereas CAT immunoreactivity was not detected in either catecholaminergic or serotonergic neurons in the CNS. Thus, it was suggested that the ACA transgene contained the major part of cis-regulatory elements for the expression of AADC in peripheral nonneuronal tissues. On the other hand, the ACN transgenic mice moderately expressed CAT in various tissues except for the lung and liver, and the ACL transgenic mice showed moderate CAT expression only in the kidney.

Animals↗
PubMed1995

Hepatitis C virus core region: helper T cell epitopes recognized by BALB/c and C57BL/6 mice.

In this study, we characterized the B cell and T cell responses to the hydrophilic portion of hepatitis C virus (HCV) core protein in two strains of mice and identified the respective antigen determinants. BALB/c (H-2d) and C57BL/6 (B6:H-2b) mice were immunized by a subcutaneous injection of recombinant HCV core protein together with Freund's complete adjuvant. The level of antibody production, as determined by ELISA, was consistently higher in BALB/c than in B6 mice. However, antibodies in sera from each strain bound to the N-terminal region of the core protein within amino acids 1 to 28 (MSTNPKPQRKIKRNTNRRPQDVKFPGGG), according to an experiment using non-overlapping peptides that covered the hydrophilic portion of HCV core protein. The T cell responses were also higher in BALB/c than in B6 mice with respect to the proliferative responses of the draining lymph node cells in vitro. By limiting dilution cultures of the draining lymph node cells in vitro repetitively stimulated with recombinant core protein, T cell clones were established from both strains of mice and characterized. The surface markers of these clones were Thy-1.2+, CD3+, TCR alpha beta+, CD4+ and CD8+. The proliferative responses were inhibited in the presence of anti-CD4 or anti-MHC class II monoclonal antibodies. The T cell lines in BALB/c mice recognized an epitope in HCV core at amino acids 72 to 91 (EGRAWAQPGYPWPLYGNEGL). The T cell lines in B6 mice recognized an epitope at amino acids 55 to 74 (RPQPRGRRQPIPKARQPEGR). Thus, mice with different MHC haplotypes recognized different non-overlapping T cell antigenic determinants of HCV core proteins.

Amino Acid Sequence↗
PubMed1995

Xanthine oxidase mediates paraquat-induced toxicity on cultured endothelial cell.

The role of xanthine oxidase in paraquat toxicity was investigated using cultured bovine pulmonary artery endothelial cells. Exposure to paraquat 0.1 mM was done for 24 hr with or without tungsten pretreatment and in the presence or absence of xanthine oxidase inhibitors. Exposure to paraquat significantly increased O2- production and relative xanthine oxidase activity (xanthine oxidase activity divided by total xanthine dehydrogenase plus xanthine oxidase) while depressing cell growth. In contrast, tungsten and allopurinol inhibited the increase of xanthine oxidase activity and decreased O2- release. Cell injury was assessed by leakage of lactate dehydrogenase and by fluorescein diacetate staining; it was found that oxidase inhibitors (both allopurinol and tungsten) reduced paraquat cytotoxicity. Thus the toxicity of paraquat was at least partly due to intracellular O2- production mediated by xanthine oxidase and the subsequent formation of other free radicals.

Allopurinol↗
PubMed1995

Reduced threshold for myocardial cell calcium intolerance in the rat heart with aging.

To determine whether advancing age is accompanied by a reduced Ca2+ tolerance, we measured Ca(2+)-dependent diastolic pressure, prolonged relaxation and systolic functional deterioration, spontaneous sarcoplasmic reticulum (SR)-generated Ca2+ oscillations [detected as scattered laser light intensity fluctuations (SLIF)], aftercontractions, and ventricular fibrillation in isolated, isovolumic, atrioventricular-blocked intact hearts from 24- to 26-mo (old) and 6- to 8-mo (young) male Wistar rats. In enzymatically isolated single cardiac myocytes, the likelihood of the occurrence of spontaneous contractile waves driven by spontaneous SR Ca2+ release was also determined. In response to stepwise increase in perfusate Ca2+ concentration (Cao), a reduction in the maximum developed pressure accompanied by an elevation in end-diastolic pressure and a prolonged contraction duration was observed at lower Cao in old vs. young hearts (P < 0.01 for each parameter). Furthermore, Ca(2+)-dependent ventricular fibrillation occurred during pacing in six old but in no young hearts (P < 0.01), aftercontractions were observed in seven old vs. one young heart (P < 0.01), and SLIF increased to a greater extent in old vs. young hearts. In single cardiac myocytes, spontaneous contractile waves occurred more frequently with increasing age (P < 0.01). These results indicate that aging is associated with an increased likelihood for the occurrence of SR-generated Ca2+ oscillations and functional abnormalities that result from these oscillations.

Aging↗