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Biomedical subjects

K Uchida

Publications and source records attributed to K Uchida.

At least 1,135 records · Page 63Linked to original sources
PubMed1989

Production and characterization of monoclonal antibodies to rat liver arginase.

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.

Animals↗
PubMed1989

Immunochemical and biochemical distinction of subtypes of atrial natriuretic peptide receptor.

The presence of subtypes of atrial natriuretic peptide (ANP) receptor has been demonstrated by examining the immunological features and ligand specificities of the receptors in various bovine tissues. The antibody probe used was the antiserum raised against the bovine lung ANP receptor, and the tissues examined for the possible presence of different types of ANP receptor were the lung, kidney, adrenal cortex, ovary, choroid plexus, and vascular tissues. When incubated with Triton extracts of these tissues, the antiserum strongly cross-reacted with the ovary, kidney, and choroid plexus receptors as well as the homologous lung receptor (type I). The adrenal and vascular receptors were recognized only weakly, however, suggesting the presence of distinct ANP receptors (type II). In support of this immunochemical subtyping, type I and type II receptors showed a marked difference in their ability to bind the ANP analog atriopeptin I (ANP5-25): type I receptors in the lung exhibited a moderate affinity for atriopeptin I with a KD of 10(-9) M; however, type II receptors in adrenal and artery showed only a weak affinity for the analog with a KD of 10(-6) M. Structural analysis of affinity-labeled ANP receptors by SDS-PAGE indicated that type I and type II receptors have similar disulfide-linked dimeric structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1989

Purification and characterization of a new hydrolase for conjugated bile acids, chenodeoxycholyltaurine hydrolase, from Bacteroides vulgatus.

A new hydrolase for conjugated bile acids, tentatively named chenodeoxycholyltaurine hydrolase, was purified to homogeneity from Bacteroides vulgatus. This enzyme hydrolyzed taurine-conjugated bile acids but showed no activity toward glycine conjugates. Among the taurine conjugates, taurochenodeoxycholic acid was most effectively hydrolyzed, tauro-beta-muricholic and ursodeoxycholic acids were moderately well hydrolyzed, and cholic and 7 beta-cholic acids were hardly hydrolyzed, suggesting that this enzyme has a specificity for not only the amino acid moiety but also the steroidal moiety. The molecular weight of the enzyme was estimated to be approximately 140,000 by Sephacryl S-300 gel filtration and the subunit molecular weight of the enzyme was 36,000 by SDS-polyacrylamide gel electrophoresis. The optimum pH was in the range of 5.6 to 6.4. The NH2-terminal amino acid sequence of the enzyme was Met-Glu-Arg-Thr-Ile-Thr-Ile-Gln-Gln-Ile-Lys-Asp-Ala-Ala-Gln. The enzyme was activated by dithiothreitol, but inhibited by sulfhydryl inhibitors, p-hydroxymercuribenzoate, N-ethylmaleimide, and dithiodipyridine.

Amidohydrolases↗
PubMed1989

Subcellular distribution of the transmissible agent in Creutzfeldt-Jakob disease mouse brain.

To determine the intracellular localization of the Creutzfeldt-Jakob disease (CJD) agent in mouse brain, cerebrum tissue of the mouse brain affected with the Fukuoka-1 strain was separated into six subcellular fractions (microsome, nerve ending, myelin, mitochondria, nucleus, and soluble fractions) by differential sucrose density gradient, and then the CJD infectivity of these fractions was examined. Serially diluted samples of each subfraction were inoculated intracerebrally into groups of BALB/c mice, and the infectivity was determined as to end point titration value, incubation period, and number of affected mice. On the basis of the protein content, the highest CJD infectivity was observed in the microsomal fraction. The nerve ending (synaptic plasma membrane) and myelin fractions were also infective. The mitochondria and nucleus fractions showed the lower infectivity. The infectivity of the soluble fraction was the lowest among the six subcellular fractions. From the findings obtained in this study two possibilities as to the intracellular localization of CJD agent were suggested: 1) the transmissible agent of CJD is closely associated with surface membranes of neuronal and/or glial cells, including their processes; 2) the CJD agent is diffusely present intracellularly, including in the surface membranes, but for manifestation of infectivity the agent needs membrane components as prerequisite factors.

Animals↗
PubMed1989

Synthesis of L-3,4-dihydroxyphenylalanine by tyrosine hydroxylase cDNA-transfected C6 cells: application for intracerebral grafting.

In the present study, we obtained genetically manipulated nonneuronal cells which synthesize a catecholamine precursor for future use in intracerebral grafting. Human type 1 tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was inserted into eukaryotic expression vector pKCRH2 and was co-transfected into C6 cells with plasmid pSV2neo. Expression of the TH minigene was screened by immunohistochemical staining with TH antibody and immunoblotting analysis. Several clones of the C6 transfectants that produce TH molecules were obtained. These cells showed TH activity, and the product, L-3,4-dihydroxyphenylalanine (L-DOPA), was detected intracellularly due to the absence of L-amino acid decarboxylase (EC 4.1.1.28) activity. It was found that a large amount of L-DOPA was released from the cells into the culture medium. These transfectants were transplanted into rat brain, and the expression of TH was examined immunohistochemically. On the 10th day following transplantation, a mass of C6 cells which was heavily stained with TH antibody was observed in the brain. These findings may provide us with an opportunity to investigate the effects of intracerebral transplantation of nonneuronal cells that produce catecholamine or its precursor.

Animals↗
PubMed1989

Effect of antibiotics on colony formation from mouse granulocyte-macrophage progenitors (CFU-GM), megakaryocyte progenitors (CFU-M) and erythrocyte progenitors (CFU-E, BFU-E) in vitro.

Mouse bone marrow cells were cultured in vitro in semisolid medium containing various concentrations of beta-lactam antibiotics, moxalactam (LMOX), cefotetan (CTT), flomoxef (FMOX), ceftizoxime (CTZ), cefmenoxime (CMX), cefotasime (CTX), cefamandole (CMD), cefotiam (CTM), carbenicillin (CBPC) or sulbenicillin (SBPC). All of these antibiotics dose-dependently suppressed colony formation from granulocyte or macrophage progenitors (CFU-GM) and from megakaryocyte progenitors (CFU-M). The suppressive potencies of these antibiotics for both progenitors, as evaluated from the drug concentrations that induced 50% suppression of colony formation (IC50), were ranked as the weakest for CBPC and SBPC, moderate for LMOX, CTT, CMX, FMOX and CTZ, and strongest for CMD, CTX and CTM. LMOX, FMOX, CTX, CBPC and SBPC also showed dose-dependent inhibition of colony formation from erythroid progenitors (CFU-E and BFU-E). In the ranking of suppression of colony formation from CFU-E, CMD and CTX showed comparatively strong inhibition but others showed a weak effect, and in that from BFU-E, the effect was weakest for CBPC and SBPC, moderate for LMOX and FMOX, and strongest for CMD and CTX. The suppressive effect of LMOX and CMD on colony formation from each progenitor could almost be prevented by washing the cells after incubation with these antibiotics for 24 hr.

Animals↗
PubMed1989

Correlation between depression of catabolite control of xylose metabolism and a defect in the phosphoenolpyruvate:mannose phosphotransferase system in Pediococcus halophilus.

Pediococcus halophilus X-160 which lacks catabolite control by glucose was isolated from nature (soy moromi mash). Wild-type strains, in xylose-glucose medium, utilized glucose preferentially over xylose and showed diauxic growth. With wild-type strain I-13, xylose isomerase activity was not induced until glucose was consumed from the medium. Strain X-160, however, utilized xylose concurrently with glucose and did not show diauxic growth. In this strain, xylose isomerase was induced even in the presence of glucose. Glucose transport activity in intact cells of strain X-160 was less than 10% of that assayed in strain I-13. Determinations of glycolytic enzymes did not show any difference responsible for the unique behavior of strain X-160, but the rate of glucose-6-phosphate formation with phosphoenolpyruvate (PEP) as a phosphoryl donor in permeabilized cells was less than 10% of that observed in the wild type. Starved P. halophilus I-13 cells contained the glycolytic intermediates 3-phosphoglycerate, 2-phosphoglycerate, and PEP (PEP pool). These were consumed concomitantly with glucose or 2-deoxyglucose uptake but were not consumed with xylose uptake. The glucose transport system in P. halophilus was identified as a PEP:mannose phosphotransferase system on the basis of the substrate specificity of PEP pool-starved cells. It is concluded that, in P. halophilus, this system is functional as a main glucose transport system and that defects in this system may be responsible for the depression of glucose-mediated catabolite control.

Aldose-Ketose Isomerases↗
PubMed1989

Subtype switching of ANP receptors during in vitro culture of vascular cells.

Vascular receptors for atrial natriuretic peptide (ANP) have been characterized immunochemically and found to be classified into two subtypes that show differential localization and expression. An antiserum, recognizing the protein core of the receptor, was raised against the purified bovine lung ANP receptor and used for the immunochemical subtyping. ANP receptors solubilized from the bovine jugular vein reacted strongly with the antiserum as well as the homologous lung receptor; however, the receptors from the aorta and carotid artery were recognized only weakly, indicating the presence of two types of ANP receptor in the vascular system. The one that reacts strongly with the antiserum and is distributed mainly in the venous side is termed type I and the other reacting weakly and predominating in the arteries, type II. The two subtypes were also distinguishable in their ligand specificities; the type I receptor showed a remarkably higher affinity for the ANP analogue atriopeptin I than the type II receptor. Surprisingly, similar immunochemical and biochemical analysis of the receptors on cultured vascular smooth muscle and endothelial cells revealed that the arterial cells originally expressing the type II receptor begin to express the type I receptor when cultured in vitro.

Animals↗
PubMed1989

Immunohistochemical localization of atrial natriuretic peptide receptor in bovine kidney and lung.

Receptors for atrial natriuretic peptide (ANP) were localized in the alveoli and bronchiolar smooth muscle cells of bovine lung and in podocytes of the kidney by immunofluorescence and immunoperoxidase methods. Two specific antisera were raised against the ANP receptor purified from bovine lung plasma membranes: anti-Rc 140 and anti-Rc 70. Anti-Rc 140 was raised against the 140 KD native receptor having a homodimeric structure, and anti-Rc 70 was elicited by immunizing a rabbit with the 70 KD reduced subunits. Essentially identical staining patterns were obtained with both antisera. Identification of ANP receptor sites would provide useful information in understanding the pulmonary and renal actions of ANP.

Animals↗
PubMed1989

Production and some properties of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa.

A set of 16 serotype specific monoclonal antibodies (MoAbs) against Pseudomonas aeruginosa classified by Homma were obtained by the mouse hybridoma technique. This set of MoAbs was useful for classification of their serotypes especially O-antigen groups G and M. MoAb obtained from serotype 15 of Homma's classification reacted with serotype 17 of the same classification. We also obtained two MoAbs which reacted with 7 Homma's serotypes of 6 different O-antigen groups and one MoAb which recognized all 4 Homma's serotypes of O-antigen group B. These MoAbs were also useful in mice for the preventive effect against the infections of P. aeruginosa.

Animals↗
PubMed1989

[Effect of a new thromboxane A2 antagonist, S-145, on platelet aggregation].

The newly synthesized compound S-145, (+/-)-5(Z)-7-(3-endo-phenylsulfonylamino [2.2.1] bicyclohept-2-exo-yl)heptenoic acid, inhibited arachidonic acid (AA)-, 9,11-methanoepoxy-PGH2 (U46619)-, collagen- and ADP-induced human platelet aggregation in vitro with IC50 values of 0.25, 0.34, 0.22, and 0.08 microM, respectively. The inhibiting potency of this compound to AA- or U46619-induced platelet aggregation was about twice that of ONO-3708 and 1/7-1/14 that of SQ29,548 in human platelets, about 7 times that of ONO-3708 and 1/3-1/7 that of SQ29,548 in guinea pig platelets, and 250-800 times that of ONO-3708 and 1-7 times that of SQ29,548 in rabbit platelets. When S-145 was administered orally to guinea pigs at the dose of 0.1 mg/kg, AA-induced platelet aggregation was completely inhibited at 30 and 60 min after the administration, but not at 3 and 6 hr. The minimum effective doses of S-145 (p.o.) to AA- and collagen-induced platelet aggregation at 60 min after the administration were 0.01 mg/kg and 0.03 mg/kg, respectively. The potency of S-145 (p.o.) to inhibit AA- and collagen-induced guinea pig platelet aggregation was 30-300 times that of ONO-3708 or SQ29,548 and 300-1000 times that of aspirin. These results suggest that S-145 is a thromboxane A2 antagonist showing a potent inhibiting effect on platelet aggregation by oral administration.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1989

Effect of N-methyltetrazolethiol on liver microsomal vitamin K reductase.

NADH-dependent vitamin K reductase activity in rat liver microsomes was measured by detecting the amount of the reduced form of vitamin K from the oxidized form of the vitamin. The enzyme activity was not detected when intact microsomes were employed as the enzyme source, but the solubilization of the microsomal enzyme with 1.5% Triton X-100 caused a development of the activity. Although the enzyme activity decreased gradually with time after the solubilization, the enzyme was stabilized by the addition of 20% glycerol and 2 mM vitamin C. Some optimal assay conditions for the vitamin K reductase were determined using the solubilized enzyme, and the standard assay method is described. Vitamin K reductase activity was not affected by warfarin and N-ethylmaleimide (NEM), but pyridoxal-5-phosphate (PAL-P) inhibited the activity, especially when microsomes were preincubated with PAL-P. The enzyme activity was not inhibited by N-methyltetrazolethiol (NMTT) and NMTT-containing antibiotics, suggesting that the hypoprothrombinemia caused by beta-lactam antibiotics was not due to the inhibition of NADH-dependent vitamin K reductase.

Animals↗
PubMed1989

Studies on oxacephem antibiotics: comparison of the effects of 1-oxa and 1-thia cephalosporins on blood coagulation activities and vitamin K metabolism in rats.

Oxacephem antibiotics have been developed to increase the antibacterial activity of cephem antibiotics, but the effect of 1-oxygen replacement of cephem antibiotics on blood coagulation activities is not yet known. Therefore, latamoxef (LMOX), flomoxef (FMOX) and their 1-S congeners were examined for their effects on prothrombin time, activated partial thromboplastin time, plasma prothrombin and Factor VII levels, plasma and liver descarboxyprothrombin (PIVKA-II) levels, and liver microsomal vitamin K epoxide reductase activities in rats kept on a vitamin K-deficient diet. Under the vitamin-deficient states, LMOX, FMOX and their 1-S congeners inhibited the vitamin K epoxide reductase, although the effect of FMOX or its congener was much less than that of LMOX, and they decreased the blood clotting activities in rats fed a vitamin K-deficient diet. However, no difference was found in these effects between LMOX and its 1-S congener or between FMOX and its 1-S congener. This result suggests that the 1-oxygen replacement of cephem antibiotics is not responsible for the hypoprothrombinemic effect of the antibiotics.

Animals↗
PubMed1989

Plasma 18-hydroxycorticosterone and aldosterone responses to angiotensin II and corticotropin in diabetic patients with hyporeninemic and normoreninemic hypoaldosteronism.

To examine the nature of adrenal abnormalities in diabetic patients with hyporeninemic and normoreninemic hypoaldosteronism, responses of plasma 18-hydroxycorticosterone and plasma aldosterone to angiotension II infusions and ACTH injection were investigated in 8 diabetic patients with hyporeninemic hypoaldosteronism and 9 diabetic patients with normoreninemic hypoaldosteronism compared to 11 control subjects. In both the patients with hyporeninemic and normoreninemic hypoaldosteronism, plasma 18-hydroxycorticosterone and plasma aldosterone were low, whereas plasma cortisol and plasma corticosterone were within normal ranges. Percent increments of plasma 18-hydroxycorticosterone and plasma aldosterone above their baseline levels after angiotensin II infusions were low or somewhat low in the patients with hyporeninemic hypoaldosteronism and low in the patients with normoreninemic hypoaldosteronism. Percent increments of plasma 18-hydroxycorticosterone and plasma aldosterone above their baseline levels after ACTH injection were similar in three groups. These results suggest that in diabetic patients with isolated hypoaldosteronism, the adrenal abnormality, regardless of whether it is primary or secondary, is mainly due to impaired adrenal responsiveness to angiotension II and atrophy and the zona glomerulosa.

18-Hydroxycorticosterone↗
PubMed1989

Vitamin K-reversible hypoprothrombinemia in rats: comparison of hypoprothrombinemic changes in various strains of rats caused by vitamin K deficiency and antibiotic treatment.

Hypoprothrombinemic changes were compared in rats fed various vitamin K-deficient diets. Changes such as prolongation of prothrombin time and activated partial thromboplastin time, decrease in plasma levels of prothrombin and clotting factor VII, and increase in plasma descarboxyprothrombin, appeared in rats maintained on vitamin K-deficient diet, but in rats on ordinary diet (vitamin K-sufficient diet). As the development of hypoprothrombinemia was not significantly different among animals fed various vitamin K-deficient diets, the blood coagulation parameters were concluded to be regulated only by the vitamin K level. Following the development of hypoprothrombinemia, hemorrhaging in various organs was detected in vitamin K-deficient rats, with strain differences in the severity of hemorrhage; Fischer and Wistar strains were more sensitive than the Sprague Dawley strain. Administration of a beta-lactam antibiotic, latamoxef (LMOX), to vitamin K-deficient rats led to enhancement of the hypoprothrombinemic conditions, but LMOX-associated changes in plasma enzyme levels were not detected.

Animals↗