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Biomedical subjects

E Ishikawa

Publications and source records attributed to E Ishikawa.

At least 19 recordsLinked to original sources
PubMed1992

Stimulation of cholecystokinin (CCK) release from superfused rat hypothalamo-neurohypophyseal complexes by interleukin-1 (IL-1).

This in vitro study examined the effects of interleukin-1 (IL-1) and interferon-gamma (Ifn-gamma) on the release of cholecysto-kinin (CCK) from superfused hypothalamo-neurohypophyseal complexes (HNC) of rats. An increase of CCK from HNC was elicited in a dose-dependent manner by recombinant human IL-1 alpha and -1 beta in concentrations of 0.1-10 nM. In contrast, the release of CCK from HNC was not affected by recombinant human Ifn-gamma at any dose tested (0.1, 1 and 10 nM). The increased release of CCK elicited by IL-1 was calcium-dependent, as was that induced by potassium (60 mM), but it was biphasic and had a different time course and a lower magnitude than those induced by potassium and veratridine. These results suggest that IL-1 activates pituitary-adrenal axis by stimulating CCK neurons in the hypothalamus and/or neurohypophysis to release CCK, since CCK has been implicated in the regulation of adrenocorticotropin release.

Animals↗
PubMed1992

[Ultrasensitive immunoassay of hormones].

Two methods for ultrasensitive immunoassay of peptide hormones are presented. One is to reduce the nonspecific binding of labeled reactants in two-site immunoassay by transfer of immune complexes containing labeled reactants from one solid phase to another. The other is a novel noncompetitive immunoassay method for small peptides, in which peptides are biotinylated and subsequently measured by two-site assay using anti-peptide antibody and avidin (streptavidin).

Animals↗
PubMed1992

Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys-env gp46(188-224), as antigen.

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a synthetic peptide, Cys-env gp46(188-224) of HTLV-I, is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-Cys-env gp46 (188-224) conjugate and Cys-env gp46 (188-224)-beta-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. beta-D-Galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was more sensitive and useful than the immune complex transfer enzyme immunoassay using Cys-Arg-env gp46(188-209) and other methods using HTLV-I as antigen.

Amino Acid Sequence↗
PubMed1992

Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (antihuman T-cell leukemia virus type I) immunoglobulin G in serum using a synthetic peptide, Ala-Cys-Env gp46(237-262), as antigen.

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (antihuman T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a synthetic peptide, Ala-Cys-env gp46(237-262), of HTLV-I is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-Ala-Cys-env gp46(237-262) conjugate and Ala-Cys-env gp46(237-262)-beta-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. beta-D-galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was more sensitive than other methods using HTLV-I as antigen, and most negative and positive sera were discriminated. However, some results appeared to be false positive or false negative, and the peptide, Ala-Cys-env gp46(237-262), was suggested to be useful, in combination with other peptides, for improving the reliability of serodiagnosis by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible.

Amino Acid Sequence↗
PubMed1992

Novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide.

A novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide (alpha-hANP) in plasma is described. alpha-hANP was biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-alpha-hANP [6-28] IgG-coated polystyrene ball. After washing, biotinylated alpha-hANP was eluted from the polystyrene ball with HCI and was reacted with 2,4-dinitrophenyl-fluorescein-bovine serum albumin-disulfide-rabbit anti-alpha-hANP [6-28] IgG conjugate. The complex formed was trapped onto (anti-2,4-dinitrophenyl group) IgG-coated polystyrene balls and, after washing, reacted with avidin-beta-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with epsilon N-2, 4-dinitrophenyl-L-lysine and transferred to anti-fluorescein IgG-coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2-mercaptoethylamine and transferred to (anti-rabbit IgG) IgG-coated polystyrene balls. beta-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of alpha-hANP [1-28] was 3 fg (1 amol)/tube. Interference by plasma proteins was eliminated by separation of peptides from proteins using a molecular sieve. The assay range of plasma alpha-hANP [1-28] was 0.04-120 ng/L, and plasma levels of hANP in healthy subjects (11-56 ng/L) were measured without concentration.

Atrial Natriuretic Factor↗
PubMed1992

Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG in serum using recombinant gag p24(14-214) as antigen.

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using recombinant gag p24(14-214) of HTLV-I is described. The recombinant gag p24(14-214) is soluble in the absence of detergents and allows the use of enzymes other than horseradish peroxidase as a label in the assays. The usefulness of recombinant gag p24(14-214) was examined with 305 sera characterized by other methods including gelatin particle agglutination, enzyme-linked immunosorbent assay (ELISA) using HTLV-I, and Western blotting. This assay was more sensitive than other methods using HTLV-I as antigen. The specificity could be tested by preincubation of test serum with excess of the recombinant protein. Most of negative and positive sera were discriminated. However, some results appeared to be false-positive or false-negative, and recombinant gag p24(14-214) was suggested to be useful, when used with other recombinant proteins and/or peptides, for improving the reliability of serodiagnosis by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant gag p24(14-214) conjugate and recombinant gag p24(14-214)-beta-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation Studies as Topic↗
PubMed1992

A sensitive sandwich enzyme immunoassay for gamma-seminoprotein and its application to sex discrimination of blood and bloodstains.

A sensitive sandwich enzyme immunoassay for gamma-seminoprotein (p30, prostate-specific antigen) is described for sex discrimination of blood and bloodstains. A polystyrene ball coated with rabbit anti-gamma-seminoprotein IgG was incubated with gamma-seminoprotein and, after washing, with affinity-purified rabbit anti-gamma-seminoprotein Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as hydrogen donor. The detection limit of gamma-seminoprotein was 0.15 pg per assay. Blood levels of gamma-seminoprotein, measured using 1-10 microliters of blood, were at least 3.3-fold higher in male adults than in female adults. The ratio of gamma-seminoprotein in terms of pg to hemoglobin in terms of mg was significantly higher in male adults than in female adults. Thus, the measurement of gamma-seminoprotein or both gamma-seminoprotein and hemoglobin was useful for the discrimination of blood and bloodstains of male and female adults, although with some limitations.

Adolescent↗
PubMed1992

Usefulness of thyroglobulin antibody detected by ultrasensitive enzyme immunoassay: a good parameter for immune surveillance in healthy subjects and for prediction of post-partum thyroid dysfunction.

OBJECTIVE: Using newly developed ultrasensitive enzyme immunoassay (EIA) for thyroglobulin antibody (TgAb), we have evaluated physiological and pathological implications of the antibody in healthy subjects as well as in autoimmune thyroid diseases. MEASUREMENTS: This EIA was based on the immune complex transfer method, and was 10(4)-fold more sensitive compared with the conventional haemagglutination assay (HA); the detection limit was 0.1 micrograms IgG/I, and the specificity of the assay was confirmed from the unequivocal decrease in the fluorescence intensity by the preincubation of test serum with Tg and/or inactive beta-D-galactosidase which blocks antibodies to the enzyme. RESULTS: TgAb was detectable in 159 (91%) of 175 healthy subjects aged 3rd to 7th decade (96 men and 79 women), and did not exhibit age or sex-associated change. In nine healthy women, the TgAb level significantly decreased as pregnancy progressed but increased transiently after delivery. TgAb was detectable in 52 (98%) of 53 patients with Graves' disease and all (100%) of 107 patients with chronic thyroiditis. Abnormal high TgAb values (> 40 micrograms/I), determined from the 95th percentile in healthy subjects, were shown in 40 (75%) with the former disease and 94 (88%) with the latter disease. Moreover, in 14 goitrous patients with biopsy-proved chronic thyroiditis with negative HA results, 12 (86%) showed abnormal high TgAb levels. In 69 patients with post-partum thyrotoxicosis in Graves' disease, 15 (79%) of 19 patients with the TgAb level of more than 2 x 10(3) micrograms/I in early pregnancy showed destructive thyrotoxicosis and 46 (92%) of 50 with less than this level showed stimulative thyrotoxicosis. This TgAb test could discriminate the two types of thyrotoxicosis more clearly than could the conventional TGHA test. In chronic thyroiditis, the mean TgAb value in early pregnancy was significantly higher in patients with postpartum hypothyroidism than in those without thyroid dysfunction. Hypothyroidism developed in 80% of the patients with a TgAb value of more than 10(3) micrograms/I. CONCLUSIONS: The ultrasensitive TgAb EIA was useful for detecting the physiological changes in autoantibody formation in healthy subjects and the TgAb value was useful for predicting post-partum thyroid dysfunction in autoimmune thyroid diseases. This EIA is useful for the evaluation of the immune surveillance in patients with autoimmune thyroid diseases as well as in healthy subjects.

Adult↗
PubMed1992

Characterization of parathyroid hormone-related protein in the human term placenta.

To characterize parathyroid hormone-related protein (PTHrP) in the human placenta, we measured PTHrP-like immunoreactivity (PRP-LI) in the term placenta and studied the elution profiles of placental tissue extracts on Sephadex G-75 chromatography with a specific RIA. We also examined the gene expression of PTHrP mRNA by Northern blot analysis and the localization of PRP-LI in the placenta by immunohistochemistry. The amount of PRP-LI in placental extracts (n = 7) was 20.9 +/- 2.2 pg/g wet tissue (mean +/- SE). Dilution curves of placental tissue ran parallel to those of synthetic PTHrP (1-34) standards. Sephadex G-75 gel chromatography demonstrated two major PRP-LI peaks; the first peak was eluted around the molecular size between 10 kilodaltons (Kda) and 20 Kda and the other around 5 Kda. Northern blot analysis of PTHrP mRNA extracted from placental tissues showed a major hybridization signal around 18S. PTHrP immunohistochemistry showed PRP-LI staining in the cytoplasm of syncytiotrophoblasts and stroma cells (Hofbauer cells) in the term placenta. These results suggest that syncytiotrophoblasts and stroma cells in the term placenta synthesize PTHrP in two major molecular forms, 10 Kda-20 Kda and around 5 Kda.

Adult↗
PubMed1991

Interleukin-1 (IL-1) stimulates the release of corticotropin-releasing factor (CRF) from superfused rat hypothalamo-neurohypophyseal complexes (HNC) independently of the histaminergic mechanism.

We demonstrated previously that interleukin-1 (IL-1) (recombinant human IL-1 alpha and -1 beta) stimulated the release of corticotropin-releasing factor (CRF) from the superfused rat hypothalamo-neurohypophyseal complex (HNC), independently of the cholinergic system. In the present study we studied the effects of IL-1 on the release of CRF not only from the HNC but also from the isolated hypothalamus of rats in a superfusion system to define the origin of measured CRF and the site of IL-1 action. We also studied the possible involvement of the histaminergic system in the mediation of the stimulation by IL-1. An increase in CRF was elicited from the HNC and the isolated hypothalamus in a dose-dependent manner by human recombinant IL-1 beta in concentrations of 0.1-10 nM with similar time courses. Histamine in concentrations of 1-100 nM also elicited qualitatively similar increases of CRF from these two types of explants. The increases in CRF release from the HNC induced by 10 nM of histamine were completely suppressed in the combined presence of pyrilamine (10 microM) and cimetidine (10 microM), an H1 and an H2 receptor antagonist, respectively. On the other hand, the increase in CRF release induced by 10 nM IL-1 beta was not affected by the combination of these two antagonists. These results indicate that IL-1 stimulates CRF release from the median eminence through an action on the hypothalamus, and that the stimulatory effect of IL-1 is probably independent of the histaminergic system.

Animals↗
PubMed1991

Stimulation by interleukin-1 (IL-1) of the release of rat corticotropin-releasing factor (CRF), which is independent of the cholinergic mechanism, from superfused rat hypothalamo-neurohypophysial complexes.

The effects of interleukin-1 (IL-1) and interferon-gamma (Ifn-gamma) on the release of corticotropin-releasing factor (CRF) from superfused hypothalamo-neurohypophysial complexes (HNC) of rats were examined in the present study. In this in vitro system, the release of CRF from HNC was not affected by any dose of human recombinant Ifn-gamma tested (0.1, 1 and 10 nM). In contrast, a rapid increase of CRF from HNC was elicited in a dose-dependent manner by human recombinant IL-1 alpha and -1 beta in concentrations of 0.1-10 nM. The involvement of the cholinergic system in the mediation of the stimulatory effect of IL-1 on CRF release was evaluated. Acetylcholine in concentrations of 1-100 nM also elicited a rapid increase of CRF. The increase in CRF release induced by 10 nM of acetylcholine was completely suppressed in the presence of both hexamethonium (10 microM) and atropine (50 microM), a nicotinic and a muscarinic receptor antagonist, respectively. On the other hand, the increase in CRF release induced by 10 nM IL-1 alpha or -1 beta was not affected by these two antagonists. These results indicate that IL-1 stimulates CRF release through an action on the hypothalamo-neurohypophysial system, most likely on the hypothalamus, and that the stimulatory effect of IL-1 is probably independent of the cholinergic system.

Acetylcholine↗
PubMed1991

Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Env gp46(188-209), as antigen.

An immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a chemically and safely synthesized peptide, env gp46(188-209), is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with dinitrophenyl bovine serum albumin-env gp46(188-209) conjugate and env gp46(188-209)-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. beta-D-Galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was sensitive and detected anti-HTLV-I IgG in serum samples which were negative by the conventional enzyme immunoassay and Western blotting. And the specificity of this assay was confirmed by preincubation of test serum with excess of env gp46(188-209). However, some disadvantages were also noted.

Antibodies, Viral↗
PubMed1991

Time-resolved fluorometric sandwich immunoassay for human growth hormone in serum and urine.

A specific and sensitive time-resolved fluorometric sandwich immunoassay for human growth hormone (hGH) in serum and urine is described. A monoclonal anti-hGH IgG1 (5802)-coated polystyrene ball was incubated with serum or dialyzed urine and subsequently with europium ion-labeled monoclonal anti-hGH IgG1 (5801). Europium ion bound to the polystyrene ball was measured by time-resolved fluorometry. The detection limit of hGH was 0.3 pg/tube, which was 15-fold higher than that by sandwich enzyme immunoassay using horseradish peroxidase as label. The assay range of serum hGH was 15-15,000 ng/liter using 20 microliters of serum. The assay range of urinary hGH was 2-2,000 ng/liter using 150 microliters of dialyzed urine. The detection limits of hGH in serum and urine by this immunoassay were satisfactory for measuring hGH levels in serum and urine of healthy children and in serum of healthy adults and higher levels but not in urine of healthy adults and in serum and urine of patients with hGH deficiency.

Adolescent↗
PubMed1991

Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys-Gag p19(100-130), as antigen.

An immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a synthetic peptide, cys-gag p19(100-130), is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl bovine serum albumin-cys-gag p19(100-130) conjugate and cys-gag p19(100-130)-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Beta-D-Galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was 100-fold more sensitive than the conventional enzyme immunoassay, in which a cys-gag p19(100-130)-bovine serum albumin-coated polystyrene ball was incubated with test serum and, after washing, with (anti-human IgG gamma-chain) Fab'-peroxidase conjugate. The degree of inhibition by preincubation of test sera with excess of cys-gag p19(100-130) in combination with an appropriate cut-off value for the fluorescence intensity of bound beta-D-galactosidase activity discriminated almost all seropositive samples from seronegative ones.

Adolescent↗
PubMed1991

Novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for alpha-human atrial natriuretic peptide in plasma.

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for alpha-human atrial natriuretic peptide (alpha-hANP) in plasma, which uses only one monoclonal IgG for the ring structure of alpha-hANP, is described. Plasma was filtered through polysaccharide membrane to separate peptides from proteins. The plasma filtrate was incubated with N-hydroxysuccinimidobiotin to biotinylate alpha-hANP and subsequently with a polystyrene ball coated with monoclonal IgG for the ring structure of alpha-hANP to trap biotinylated alpha-hANP. The polystyrene ball was washed to eliminate unreacted N-hydroxysuccinimidobiotin and other biotinylated substances, and biotinylated alpha-hANP was eluted from the polystyrene ball with HCl. The eluate was neutralized and incubated with horseradish peroxidase-labeled antibody IgG for the ring structure of alpha-hANP and subsequently with two streptavidin-coated polystyrene balls. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. The detection limit of alpha-hANP was 20 amol, and the assay range of plasma alpha-hANP was 0.8-1,200 ng/L using 100 microliters of plasma filtrates corresponding to 75 microliters of plasma. Plasma levels of hANP in healthy subjects were 9.8-21.5 ng/L. These values were significantly lower than those measured by a two-site enzyme immunoassay probably due to the presence of alpha-hANPs lacking some N-terminal amino acids, which were as reactive as alpha-hANP in the two-site enzyme immunoassay but less reactive in the hetero-two-site enzyme immunoassay.

Adult↗
PubMed1991

Sensitive detection of anti-human T-cell leukemia virus type I IgG in human serum by a novel enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant gag-env hybrid protein as antigen.

Anti-human T-cell leukemia virus type I IgG (anti-HTLV-I IgG) in human serum was detected with high sensitivity by a novel enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant gag(14-139)-env(197-295) hybrid protein. Anti-HTLV-I IgG in test serum was reacted simultaneously with dinitrophenyl bovine serum albumin-recombinant gag-env hybrid protein conjugate and recombinant gag-env hybrid protein-horseradish peroxidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified anti-dinitrophenyl group IgG. After washing the polystyrene balls to eliminate nonspecific IgG in the test serum and excess of the peroxidase conjugate, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified anti-human IgG gamma-chain IgG. Peroxidase activity bound to the polystyrene balls was remarkably reduced by transfer of the complex and the detection limit of anti-HTLV-I IgG in serum was lowered 300 to 3000-fold compared with that by Western blotting and the conventional enzyme immunoassay, in which a recombinant gag-env hybrid protein-coated polystyrene ball was incubated with the test serum and, after washing, with anti-human IgG gamma-chain Fab'-peroxidase conjugate. The immune complex transfer enzyme immunoassay may overcome some difficulties with currently used methods.

Gene Products, env↗