Biomedical subjects

M Tsuchiya

Publications and source records attributed to M Tsuchiya.

At least 271 records · Page 15Linked to original sources

PubMed1991

Isolation and characterization of the human gene for ADP-ribosylation factor 3, a 20-kDa guanine nucleotide-binding protein activator of cholera toxin.

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro. Five different human ARFs have been identified by cDNA cloning. Northern analysis using ARF 3-specific oligonucleotides identified two mRNAs of 3.7 and 1.2 kilobases (kb). We report here the complete nucleotide sequence of the 3.7-kb ARF 3 mRNA derived from three overlapping cDNAs isolated from human hippocampus and fetal brain cDNA libraries, as well as the structure of human ARF 3 gene. Sequences of two overlapping genomic clones indicated that the ARF 3 gene spans approximately 18.3 kb and contains five exons and four introns. The conserved amino acid sequences involved in guanine nucleotide binding by ARF 3 are distributed among separate exons, as found in other GTP-binding protein genes. Translation initiates in exon 2 which includes the sequence GXXXXGK that probably participates in phosphate binding and GTP hydrolysis. The sequence DVGG in exon 3 coordinates binding of Mg2+ and the beta-phosphate of GDP. In the ARF 3 gene in contrast to those of other GTP-binding proteins, the sequence NKXD (which is thought to contribute to the specificity of interaction with the guanine ring) is divided between exons 4 and 5. The latter encodes the COOH-terminal 53 amino acids of ARF 3 and contains greater than 2500 base pairs of untranslated DNA. The sequence AATTAA is 19 bases 5' to the polyadenylation addition site of the 3.7-kb mRNA. Multiple transcription start sites were identified by primer extension and S1 and mung bean nuclease analyses. The 5'-flanking region of exon 1 contains neither a TATA nor a CAAT box, but is high in GC content (greater than 70%) and includes three potential Sp1-binding sites (GC box), consistent with the promoters described for several housekeeping genes. The 1.2-kb ARF 3 mRNA is shown to arise by use of an alternative polyadenylation signal (AACAAA) at nucleotide 1091 within the ARF 3 cDNA.

ADP-Ribosylation Factors↗

PubMed1991

Localization of an endogenous ADP-ribose acceptor, p33, in polymorphonuclear cell granules in chicken liver interlobular connective tissue.

We investigated immunohistochemically the localization of p33, an endogenous substrate protein for an arginine-specific ADP-ribosyltransferase in chicken liver. Polymorphonuclear-pseudo-eosinophilic granulocytes (heterophils) in interlobular connective tissues of the liver were exclusively and strongly stained with the antibody against p33. Strong reactivity was associated with granules in cytoplasm of the heterophils. When the chicken liver nuclear fraction was washed, the transferase activity was released into the 600 x g supernatant fraction while a nuclear enzyme poly(ADP-ribose) synthetase was retained in the pellet fraction. These results indicate that p33 and probably also ADP-ribosyltransferase, found in the liver nuclear fraction [Tanigawa et al. (1984) J. Biol. Chem. 259, 2022-2029, Mishima et al. (1988) Eur. J. Biochem. 179, 267-273], originate from interlobular heterophils of the chicken liver.

Animals↗

PubMed1991

Involvement of superoxide anion and platelet-activating factor in increased tissue-type plasminogen activator during rat gastric microvascular damages.

The role of tissue-type plasminogen activator (t-PA) was investigated in the gastric ulcer formation induced by microvascular derangement. The rat stomach was exposed and repeated electrical stimuli (irritation) were applied on the small arterial wall close to the lesser curvature to induce mucosal ischemia followed by hyperemia. The t-PA activity in the regional blood of the stomach was significantly elevated as early as 5 min after the irritation. Immunohistochemical study using anti-t-PA monoclonal antibody revealed that t-PA was detectable in the endothelial cells of capillaries and collecting venules, suggesting the involvement of endothelium-mediated fibrinolytic activity in the irritation-induced ulcer formation. Pretreatment of SOD or allopurinol significantly attenuated the irritation-induced t-PA activation, suggesting that the t-PA activity was modulated by xanthine oxidase-associated superoxide anions. CV-6209, a selective antagonist of platelet-activating factor (PAF), also prevented the activation of t-PA as well as ulcer formation, providing a concept that PAF may be associated with the local fibrinolytic activation which may cause hemorrhagic changes in the gastric mucosal microvasculature. The present study supports the hypothesis that increased t-PA activity may reflect the microvascular endothelial damages caused by vasomotor derangement and suggests that oxygen-derived free radicals may participate in the regulation of endothelium-derived fibrinolytic activities in the mucosal microvasculature.

Animals↗

PubMed1991

Differentiating effect of sodium butyrate on human hepatoma cell lines PLC/PRF/5, HCC-M and HCC-T.

The in vitro effect of sodium butyrate (SB) on human hepatoma cell lines PLC/PRF/5, HCC-M and HCC-T was investigated. SB was added at the non-toxic but cytostatic concentration of 1 mM. In all these cell lines, SB reduced cell proliferation and changed the morphology of the cells into a fibroblast-like shape. In PLC/PRF/5, alpha-fetoprotein production and c-myc expression were inhibited. In contrast, gene expression of albumin, one of the normal liver-cell products, and that of integrated hepatitis B virus genome, was increased. In HCC-M and HCC-T, c-myc expression, which was enhanced in the naive state, was reduced. In HCC-M, fos expression was inhibited but the expression of N- and K-ras genes did not change. SB seemed to induce normal or mature properties of hepatocytes in human hepatoma cell lines.

Blotting, Northern↗

PubMed1991

Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin.

Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.

ADP-Ribosylation Factors↗

PubMed1991

Simultaneous determination of urinary creatinine and aromatic amino acids by cation-exchange chromatography with ultraviolet detection.

A cation-exchange chromatographic procedure for the simultaneous determination of urinary creatinine and aromatic amino acids is described. Creatinine and amino acids were separated from organic acids and/or neutral species in urine by using a preparative cation-exchange resin column. A column packed with a cation-exchange resin of low capacity was used for the analytical separations. The elution of creatinine and aromatic amino acids was monitored at 210 nm by means of an ultraviolet detector. The relationships between concentration and peak heights were reproducible with a coefficient of variation of less than 2%, and were linear from 5 to 200 microM for each compound. Overall recoveries of the analytes were more than 95%. The method was applied to the analysis of urine of patients with disorders of amino acid metabolism, such as phenylketonuria. The concentration ratios of phenylalanine to creatinine in the patients' urine were accurately and easily determined, and were quite different from those in the urine of healthy newborns.

Amino Acid Metabolism, Inborn Errors↗

PubMed1991

Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells.

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that serve as GTP-dependent allosteric activators of cholera toxin ADP-ribosyltransferase activity. Four species of mammalian ARF, termed ARF 1-4, have been identified by cloning. Hybridization of a bovine ARF 2 cDNA under low stringency with mammalian poly(A)+ RNA resulted in multiple bands that were subsequently assigned to the known ARF genes using ARF-specific oligonucleotide probes. The relative signal intensities of some bands (e.g. the 3.8- and 1.3-kilobase (kb) mRNAs) that hybridized with the cDNA were not, however, consistent with the intensities observed with the individual ARF-specific oligonucleotide probes. These inconsistencies suggested that other ARF-like mRNAs were comigrating with known ARF mRNAs. To explore this possibility, a cyclic AMP-differentiated HL-60 Lambda ZAP library was screened using the bovine ARF 2 cDNA. Clones corresponding to known ARF genes (1, 3, and 4) were identified by hybridization of positive clones with oligonucleotide probes specific for each ARF species; ARF 2 cDNA-positive, oligonucleotide-negative clones were sequenced. Two new ARF-like genes, ARF 5 and 6, encoding proteins of 180 and 175 amino acids, respectively, were identified. Both proteins contain consensus sequences believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF 5 was most similar in deduced amino acid sequence to ARF 4, which also has 180 amino acids. ARF 6, whose deduced amino acid sequence is identical with that of a putative chicken pseudogene (CPS1) except for a serine/threonine substitution, was different from other ARF species in size and deduced amino acid sequence. With mammalian poly(A)+ RNA from a variety of tissues and cultured cells, ARF 5 preferentially hybridized with a 1.3-kb mRNA, whereas ARF 6 hybridized with 1.8- and 4.2-kb mRNAs. The fact that the sizes of these mRNAs are similar to those of other ARFs (ARF 1, 1.9 kb; ARF 2, 2.6 kb; ARF 3, approximately 3.8 and 1.3 kb; ARF 4, 1.8 kb) explain the previously observed inconsistencies between the cDNA and ARF-specific oligonucleotide hybridization patterns. All six ARF cDNAs are more similar to each other than to other approximately 20-kDa guanine nucleotide-binding proteins.

ADP-Ribosylation Factor 6↗

PubMed1991

Morphological alteration of gut-associated lymphoid tissue after long-term total parenteral nutrition in rats.

The morphological alteration of gut-associated lymphoid tissue (GALT) induced by long-term absence of dietary stimulation was investigated. Male Wistar rats weighing approximately 230 g were maintained with total parenteral nutrition (TPN). Control rats were allowed to have the same amount of the solution orally. After two weeks, the morphological alteration of GALT was examined. Although no significant difference in weight gain was noted between the two groups, the area comprised by Peyer's patches was decreased in TPN rats. The number of transported lymphocytes and the ratio of helper T (Th) cells to suppressor/cytotoxic T (Ts/c) cells in intestinal lymph were lowered after TPN treatment. In an immunohistochemical study of the rat ileum, the number of T cells and the Th/Ts/c ratio were decreased both in the intraepithelial spaces and in the lamina propria of TPN rats. The percentage of interleukin-2 receptor-positive cells and the number of IgA-containing cells in the lamina propria were significantly reduced in TPN rats. These results suggest that dietary stimulation might play a role in the maintenance of GALT function and morphology.

Animals↗

PubMed1991

Embryolethality of new herbicides is not detected by the micromass teratogen tests.

New herbicidal compounds (11 pyrimidine-diones, 3 benzoates and 1 sulfonamide) were found to be embryolethal but not teratogenic in rats. The range of the embryolethal dose varied from 0.2 to greater than 200 mg/kg. This broad range enabled us to validate whether proposed in vitro teratogen tests can detect the embryolethality of these herbicides. The IC50 values (inhibition concentration 50%) for both differentiation and proliferation of midbrain and limb bud cells of rat embryos were determined and found to be above 50 micrograms/ml in all cases, confirming that the herbicides were not teratogenic. No correlation, however, was observed between the embryolethality in vivo and the activities in these cells. In order to test whether the potential to cause embryolethality could be predicted and detected as a general cytotoxic effect, the inhibition of colony forming ability in V79 cells was determined. The results indicated that cytotoxicity in V79 cells may be useful for preliminary testing of the embryolethal effect of herbicides.

Abnormalities, Drug-Induced↗

PubMed1991

Platelet-activating factor and granulocyte-mediated oxidative stress. Strategy for in vivo oxyradical visualization.

Platelet-activating factor (PAF) and leukotriene B4 are potent lipid mediators of endothelium-granulocyte interaction which results in granulocyte-dependent increased microvascular permeability. The specific mechanism by which PAF induces granulocyte-mediated endothelial injury has not been fully investigated. Digital imaging photonic intensified microscopy has revealed that PAF effectively induces granulocyte-mediated oxidative stress on microvascular beds. The method has made it possible to visualize luminol-dependent photonic burst released from PAF-treated microvascular beds in the rat mesentery. The photonic activities clearly corresponded to the localization of sticking granulocytes in post-capillary venules. By contrast, no significant chemilumigenic response could be detected in the leukotriene B4-induced activation of endothelium-granulocyte interaction in vivo. The present findings suggest that oxygen radicals are not prerequisites for granulocyte adhesion in the microcirculation and provide evidence for a dissociation of in vivo granulocyte function between leukotactic and oxidative activation in leukotriene B4-induced microvascular changes.

Animals↗

PubMed1991

Treatment of chronic hepatitis C with weekly administration of interferon-alpha.

To evaluate the efficacy of weekly administration of interferon (IFN)-alpha, we studied 23 anti-HCV positive patients with chronic hepatitis diagnosed by liver needle biopsy. Thirteen patients received weekly intramuscular injections of 6 MU human lymphoblastoid interferon (HLBI) for 24 weeks, and the other 10 patients were given no treatment. We examined liver-specific idiotype-bearing antibody (LSIA) in the patients' sera. This HLBI treatment was easily tolerated by all the treated patients. In the treated group serum alanine aminotransferase (ALT) level significantly decreased during HLBI treatment. Normalization of serum ALT level by the end of treatment was observed in 7/13 (54%) of the treated patients but in 0% of the non-treated patients. Anti-HCV was detectable in all the patients during the treatment. Those who had high LSIA levels did not respond to HLBI treatment. These results demonstrate that weekly administration of IFN is sufficient to suppress disease activity in patients with chronic hepatitis C and that patients with high LSIA levels are unlikely to respond to IFN therapy.

Alanine Transaminase↗

PubMed1991

Lack of alcohol dehydrogenase isoenzyme activities in the stomach of Japanese subjects.

Alcohol dehydrogenase (ADH) isoenzymes different from those of the liver were shown to be present in the human gastric mucosa. Two ADH activity bands present in the gastric mucosa of surgical specimens from all 7 black and 11 white Americans studied were absent in 14 and barely detectable in 3 of 21 Japanese subjects evaluated. Similar ethnic differences pertained to both genders and were independent of the gastric pathology. The mobility of these bands on starch gel electrophoresis corresponded to those recently reported and named mu-ADH or sigma-ADH. The absence of these bands was associated with a 70% decreased capacity to reduce m-nitrobenzaldehyde, a preferred substrate for sigma-ADH, suggesting that the bands missing from the Japanese stomachs comprise this isoenzyme.

Alcohol Dehydrogenase↗

PubMed1991

Kupffer cell-mediated oxidative stress on colon cancer cell line visualized by digital imaging fluorescence microscopy.

Temporal and spatial changes of lipid peroxides in a cultured colon cancer cell line, Colo-205 cells, were investigated after culturing with Kupffer cells by using 2',7'-dichlorofluorescein diacetate and a digital imaging processor equipped with an inverted microscope. By this method, we successfully visualized the alteration of lipid peroxides in the individual cancer cell. Without any prior activation, Kupffer cells isolated from an intact rat liver caused rapid increase in the intensity of dichlorofluorescein in tumor cells in a time-dependent manner. The increase of the fluorescent intensity was significantly attenuated by pretreatment with superoxide dismutase. In ex vivo study using isolated perfused rat liver, dichlorofluorescein-preloaded cancer cells, which were transportally injected, were found to adhere to hepatic sinusoids and then to enhance their fluorescence. The present study suggested that the resident Kupffer cell-derived oxidative stress participates in the cytotoxic process against cancer cells by inducing intracellular lipid peroxidation. It may be sustained that Kupffer cells play a role in the host defence mechanisms against the liver metastasis of colon cancer cells.

Animals↗

PubMed1991

Analgesia produced by pituitary ACTH and dopaminergic transmission in the arcuate.

Analgesia (NAA) caused by nonacupuncture point (abdominal muscle) stimulation after lesioning the analgesia inhibitory system (AIS) or treating the subject with proglumide was abolished by hypophysectomy or adrenalectomy. The final sector of the NAA afferent pathway from the nonacupuncture point to the pituitary gland and the initial sector of the descending pain inhibitory system were found in the anterior and posterior arcuate nucleus (A-HARN and P-HARN), respectively. Analgesia caused by ACTH microinjected into the P-HARN disappeared after denervation of the A-HARN, but that caused by dopamine did not. Firing rates of P-HARN neurons were increased by nonacupuncture point simulation (NAPS) after lesion of the AIS or treatment with proglumide. The NAPS responsive neurons also responded to ultramicroinjected dopamine, but not to ultramicroinjected ACTH. Both NAA and NAPS responsive neuron activity that were abolished by hypophysectomy were restored by concurrent application of NAPS and intraperitoneal ACTH. Reduction of sodium ions due to adrenalectomy was found to abolish NAA. It was concluded that NAA production involves dopaminergic transmission in the HARN and ACTH acting presynaptically on this transmission.

Adrenal Glands↗

PubMed1991

Dopaminergic transmission in the hypothalamic arcuate nucleus to produce acupuncture analgesia in correlation with the pituitary gland.

Acupuncture analgesia (AA) caused by low frequency stimulation of the acupuncture point (AP) was abolished by hypophysectomy and adrenalectomy. Termination of the AA producing pathway from the AP to the pituitary gland was in the medial hypothalamic arcuate nucleus (M-HARN). The origin of the descending pain inhibitory system associated with AA was in the posterior HARN (P-HARN). AA in the hypophysectomized rats, and enhanced neuronal activity in the P-HARN that were abolished during acupuncture stimulation, were both restored by intraperitoneal microinjection of 0.5 mg/kg morphine or 0.1 micrograms beta-endorphin into the P-HARN during acupuncture stimulation. Of the analgesia produced by dopamine or beta-endorphin injected into the P-HARN, that caused by beta-endorphin disappeared after denervation of the M-HARN. The P-HARN neurons that responded to acupuncture stimulation also responded to iontophoretic dopamine, but not to iontophoretic morphine nor ultramicroinjected beta-endorphin. The transmission between the M-HARN and P-HARN may be dopaminergic, and beta-endorphin might presynaptically modulate this transmission. Reduction of sodium ions may have been the reason for abolition of AA after adrenalectomy.

Acupuncture Analgesia↗

PubMed1991

Involvement of platelet-activating factor in endothelin-induced vascular smooth muscle cell contraction.

This study was designed to elucidate the participation of platelet-activating factor (PAF) in endothelin-induced vascular constriction in vivo and in vitro. The microvascular hemodynamic changes in rat mesentery induced by the superfusion of endothelin-1 (ET-1) were visualized through an intravital microscopic system. It was revealed in vivo that ET-1 in a range of 100 fM-10 pM caused a sustained arteriolar constriction in a dose-dependent manner. Pretreatment with CV-6209, a selective PAF antagonist, significantly attenuated the constrictive change in arterioles. Changes of intracellular Ca2+ mobilization after treatment with ET-1 were investigated in vitro using a cell line (A-10 cell) derived from rat arterial smooth muscle cells. ET-1 caused a prompt rise in the fura-2-associated fluorescence intensity in the individual A-10 cell and it fell to a lower plateau level that was still higher than the baseline value. CV-6209-pretreated cells did not show the rapid-phase mobilization of Ca2+, but showed the slow late phase of Ca2+ activation. The present study demonstrates that PAF may be involved in endothelin-induced microvascular constriction by mediating the mobilization of Ca2+ in vascular smooth muscle cells.

Animals↗

PubMed1991

cDNA clones of Japanese hepatitis C virus genomes derived from a single patient show sequence heterogeneity.

Twelve cDNA clones of Japanese hepatitis C virus (HCV) have been isolated from liver tissue of a single non-A, non-B hepatitis patient. These clones represented the non-structural domains of HCV. The degree of substitution in the nucleotide sequences and deduced amino acid sequences between these clones was 9.5 and 7.7%, respectively. This high level of substitution suggested that repeated infections of different HCVs may have occurred in the patient.

Amino Acid Sequence↗

PubMed1991

Impaired autonomic nervous system in alcoholics assessed by heart rate variation.

The suppression of heart rate variation reflects cardiac autonomic nervous dysfunction and is known to be associated with a poor prognosis or sudden death in diabetic patients. We investigated consecutive changes in the heart rate variation in 51 alcoholics using the coefficient of variation of R-R interval (CVRR). To correct for age effects, a ratio of CVRR to the standard predicted value (CVP) was calculated. On the whole, CVRR/CVP was suppressed on admission and on the 7th day of abstinence and increased on the 30th day. However, alcoholics could be divided into two groups by their CVRR/CVP on the 30th day: one group with transient autonomic dysfunction whose CVRR/CVP was more than 0.8 (n = 32), and the other group with persistent autonomic dysfunction whose CVRR/CVP was less than 0.8 (n = 19). Withdrawal hypertension occurred more frequently (63% vs. 19%) and mean systolic pressure (159 +/- 24 mmHg vs. 138 +/- 17 mmHg) was higher in the latter group than in the former, suggesting that persistent autonomic damage might, at least in part, contribute to withdrawal hypertension. To investigate further the relationship between the persistent autonomic damage and other complications, the CVRR/CVP on the 30th day of abstinence was analyzed in an additional 85 alcoholics (total n = 136). Persistent suppression of the CVRR/CVP was more frequently found in alcoholics with leg paresthesia (64%, n = 22), the Wernicke-Korsakoff syndrome (73%, n = 11), or diabetes mellitus (69%, n = 68), than in alcoholics without these complications (31%, n = 35).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗