Biomedical subjects

H Yasueda

Publications and source records attributed to H Yasueda.

At least 19 recordsLinked to original sources

PubMed1992

Effect of semi-random mutagenesis at the C-terminal 4 amino acids of human interleukin-6 on its biological activity.

The carboxyl(C)-terminus of human interleukin-6 (hIL-6) has a critical role in the expression of the biological activity of this cytokine. To define the structure-function relationships of this region, semi-random mutagenesis of the C-terminal Leu181-Arg182-Qln183-Met184 sequence of hIL-6 was performed. The mutants were produced in Escherichia coli, renatured, and purified. Alterations of the C-terminal 4 amino acids caused a significant reduction of the proliferative effect of the mutants on MH60.BSF2 and KT-3 cells, and also led to a drastic decrease in receptor binding affinity. These results suggest the importance of a positively charged residue at position 182 or 183 and an alpha-helix at position 181 for the biological activity of hIL-6.

Amino Acid Sequence↗

PubMed1992

Concentration of airborne mite allergens (Der I and Der II) during sleep.

Using a low-noise air sampler and a sensitive radioimmunoassay, we measured the concentration of mite allergens in the air during sleeping with Japanese bedquilts (futon). The airborne allergen levels of Der I (Der p I plus Der f I) and Der II during sleep were 223 and 87.1 pg/m3 of air, respectively. These levels were about 10-fold higher than those during usual domestic life in the living room of the same houses. When the bedquilts were changed to new ones free of mite allergens, the airborne allergen levels of Der I and Der II were decreased to 11.5 and 12.0 pg/m3, respectively. This indicated that the mite airborne allergens during sleep were generated from the used bedding, not from the floor. We believe that exposure to airborne mite allergens during sleep might be an important factor in the development of mite allergies.

Air Pollution, Indoor↗

PubMed1992

[Control of house dust mites and mite allergens by special mite-preventive futon].

We studied the effect of a recently-developed special mite-preventive futon (bedding) on mite population and mite allergen contents (Der I and Der II) in asthmatic children and their families (23 sets) for 2 years. 1. The number of mites (vacuumed samples) in the 124 sets of futon for 2 years was 6.4 (SD +/- 8.8) in average in the mite-preventive futon which was significantly smaller than the number (about 1/10) in the control futon: 70.0 (SD +/- 99) (p less than 0.001). 2. Follow-up studies were made on the mite population about 2 years (3, 6, 9 12 and 27 months afterwards). The numbers of mites in all the mite-preventive futon was always 1/10 or less than that of in the control futon (p less than 0.01). 3. The mite allergens (Der I and Der II) were measured in the cotton stuffing of the futon used for 2 years. The mite allergen counts in the mite-preventive futon averaged about 1/24 of those in the control futon. 4. Airborne mite allergens (Der I and Der II) obtained by beating the futon were measured. The ratio of the mite allergen count in the mite-preventive futon to the control futon was found to be 1:22-264. These results indicate that the special mite-preventive futon always inhibit mite infestation at least 1/10 of that in the ordinary (control) futon and that the count of mite allergens in the cotton stuffing and in the airborne particles was even lower in the mite-preventive futon.

Adult↗

PubMed1991

Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication.

The product of the rep gene of ColE2 is required for initiation of ColE2 DNA replication. The rep gene was placed under the control of the promoters, PL and PR, and the heat-labile cl857 repressor of bacteriophage lambda. The Rep protein was identified as a 35 Kd protein by the maxicell method in combination with heat-induced expression. The protein was efficiently expressed from these promoters in unirradiated cells and accumulated up to a few per cent of the total cellular proteins. It was partially purified (about 80% pure) and its properties examined. The amino acid sequence of the amino terminal portion of the partially purified protein agreed well with that predicted from the nucleotide sequence of the rep gene. One of the characteristic features of the rep gene is frequent usage of rare codons, especially those for arginine. The protein specifically stimulated replication of ColE2 DNA but not that of ColE3 DNA in crude cell extracts of Escherichia coli. Specific binding of the protein to plasmid DNA containing the origin region of ColE2 was demonstrated by the filter binding method. Neither endonuclease activity nor topoisomerase activity was detected by using ColE2 DNA.

Amino Acid Sequence↗

PubMed1991

In-vivo processing of the initiator methionine from recombinant methionyl human interleukin-6 synthesized in Escherichia coli overproducing aminopeptidase-P.

Human interleukin 6 (hIL-6) overproduced in Escherichia coli HB101 was found to partially retain the initiator methionine (Met) residue (Met-hIL-6). In order to remove the residual N-terminal Met in vivo, an attempt was made to express hIL-6 in aminopeptidase-P (Ap-P)-hyperproducing strains, since the N-terminus Met-Pro- structure of nascent recombinant hIL-6 has been shown to be a favoured substrate of the enzyme in vitro. Using a mutant with duplicated Ap-P genes (pepP) on a chromosome or some recombinant strains overproducing Ap-P, we have succeeded in removing the initiator Met from Met-hIL-6 in vivo. The content of the mature product without the initiator Met in the pepP recombinant strains could be increased to approximately 99% from 85%.

Amino Acid Sequence↗

PubMed1991

Enzymatic dispersion of mast cells from human sinus mucosa: characterization of histamine release and its comparison with chopped fragments of the tissue.

Functional characteristics of mast cells in chopped fragments from sinus mucosa, which was dissected from patients with chronic sinusitis, were compared with those from dispersed cells prepared by enzymatic treatment. The results obtained in this study were the following. (1) Both chopped fragments and dispersed cells released histamine in a dose-dependent manner when incubated with anti-IgE. However, higher histamine release was always observed in dispersed cells. (2) Although no differences in the ability to reduce histamine release with salbutamol or forskolin could be observed between chopped fragments and dispersed cells, staurosporin and p-bromophenacyl bromide were more active on dispersed mast cells than chopped fragments. (3) Passive sensitization of dispersed cells with an allergic serum containing IgE to mite could be achieved only after elution of IgE on the cells with lactic acid.

Acetophenones↗

PubMed1991

[The diagnostic value of a new whole blood histamine release test using paper disc-coupled antigens--results compared with intracutaneous test, RAST, and eye test].

We have developed a novel histamine release test (HR) using whole blood and antigen-coupled RAST paper discs, for the screening of allergens rather in a short time with a small amount of blood. Histamine was determined by a RIA kit. In order to evaluate the diagnostic value, the results of the test were compared with those of RAST, intracutaneous tests, and eye tests in 45 allergic patients. HR correlates better with RAST than intracutaneous test in almost all antigens. The closest positive correlation with the other tests was seen in mite allergen, followed by pollen, foods and mould spores in that order. When the HR was positive for house dust, 100% of the patients were also positive for the eye test, which is reported to closely correlate with bronchial provocation tests. HR seemed to be a useful test not only for the screening but also for the determination of pathogenic allergens.

Adolescent↗

PubMed1991

[Kinetics of the falling of airborne mite allergens (Der I and Der II)].

A futon (Japanese quilt) was beaten to disperse mite allergens into the air in a closed room, and the airborne allergens were collected both by Andersen air sampler for particle size analysis and by slit air sampler for kinetic analysis of the clearing of the allergens from the air. After extraction of the allergens from the agar plates in the samplers, two kinds of major mite allergens (Der I and Der II) were immunochemically quantitated. We found that the aerodynamic diameters of both allergens were mainly above 5.5 microns, and that airborne allergen levels decreased to about 10% of the starting level in 30 minutes, indicating the rapidity of the falling of both allergens.

Air Pollutants↗

PubMed1991

[Evaluation of different treatments of Japanese bed quilts for reducing mite allergens].

We evaluated the effectiveness of different treatments of Japanese bedquilt (futon) in reducing mite allergens: vacuum-cleaning, beating plus vacuum-cleaning and washing of the whole futon in water. Before and after these treatments, small amounts of cotton were taken out of the futon and mite allergens were extracted from the cotton into water. The absolute contents of two kinds of major allergens of two Dermatophagoides species were immunochemically quantitated. We found that beating and vacuum-cleaning reduced the allergen contents by only about 40%, whereas washing reduced the allergens by more than 90%. Therefore, for reducing airborne mite allergens generated from futon, we think that washing the whole futon in water is the most effective method.

Allergens↗

PubMed1991

[An enzyme-linked immunosorbent assay (ELISA) for the quantitation of sugi pollen and Dermatophagoides mite allergens and its application for standardization of allergen extracts].

A simple enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of the major allergens of sugi pollen, Cry j I and of Dermatophagoides mites, Der I (Der p I/Der f I) and Der II (Der p II/Der f II) for use in the in vitro standardization of allergen extracts. Polystyrene microplates coated with a IgG fraction of rabbit antiserum were incubated first with allergen extracts and then with biotinylated antiserum IgG. The bound allergen-biotinylated antibody complex was detected with commercially available streptavidin-enzyme conjugate followed by the addition of colorimetric substrate. The assay was very sensitive (-0.2 ng/ml) and reproducible (CV% = 1.9-13.8%). The ELISA was compared with the radioimmunoassay previously described, and the results showed a very good correlation between the assays (r = 0.967-0.990). The allergen content in three sugi pollen and three house dust extracts measured by the ELISA also demonstrated a good agreement with the relative potency of these extracts as determined by the intradermal skin test. These results indicate that the ELISA could be useful in the standardization of allergen extracts.

Allergens↗

PubMed1990

Histamine release with a solid-phase-coupled allergen.

Mite allergen was coupled to Sepharose, paper disk or microcrystalline cellulose, and the immobilized allergen was used for histamine release assay in human leukocytes. In terms of the spontaneous release and the reproducibility, the histamine release assay using immobilized allergen was comparable to that using soluble allergen. Histamine release increased progressively with the concentration of the immobilized allergen up to maximal release of histamine which persisted with further increase in immobilized allergen. Histamine release with soluble allergen decreased at higher concentrations of the allergen. The concentration of immobilized allergen required for maximal histamine release was the same as that with soluble allergen, but the maximal release with immobilized allergen was always about 20% lower than that with soluble allergen. Histamine release was not dependent on the density of allergen molecules on Sepharose beads. Although there was a significant correlation between histamine release obtained with a commercial disk, with a disk prepared in our laboratory, and with soluble allergen, the magnitude of the release by both assays using a paper disk was reduced significantly.

Allergens↗

PubMed1990

High-level direct expression of semi-synthetic human interleukin-6 in Escherichia coli and production of N-terminus met-free product.

We have developed a direct expression system for high-level production of recombinant human interleukin-6 (rhIL-6) in Escherichia coli. In this system, (i) the natural N-terminal coding region of the hIL-6 gene was replaced by a synthetic sequence containing A-T rich codons, (ii) dual Shine-Dalgarno (SD) sequences were employed, (iii) an A-T rich segment was inserted in front of the initiation codon to avoid putative mRNA secondary structure in the region and (iv) the natural amber termination codon of the hIL-6 gene was changed to an ocher stop codon. The hIL-6 polypeptide, synthesized at a high level, formed cytoplasmic inclusion bodies. After refolding, the N-terminal methionine was removed by aminopeptidase-P in vitro. The purified recombinant hIL-6 had B-cell differentiation activity equivalent to natural IL-6 from a human T-cell culture.

B-Lymphocytes↗

PubMed1989

Structural and functional organization of ColE2 and ColE3 replicons.

The complete nucleotide sequences of the 1.5 kb regions of ColE2 and ColE3 plasmids containing the segments sufficient for autonomous replication have been determined. They are quite homologous (greater than 90%), indicating that these two plasmids share common mechanisms of initiation of replication and its regulation. An open reading frame with a coding capacity for a protein of about 300 amino acids is present in both ColE2 and ColE3 and it actually specifies the Rep (for replication) protein, which is the plasmid specific trans-acting factor required for autonomous replication. The amino acid sequences of the Rep proteins of ColE2 and ColE3 are quite homologous (greater than 90%). The cis-acting sites (origins) where replication initiates in the presence of the trans-acting factors consist of 32 bp for ColE2 and 33bp for ColE3. They are the smallest of all the prokaryotic replication origins so far reported. They are nonhomologous only at two positions, one of which, a deletion of a single nucleotide in ColE2 (or an insertion in ColE3), determines the plasmid specificity in interaction of the origins with the Rep proteins. Both plasmids carry a region with an identical nucleotide sequence and the one in ColE2, the IncA region, has been shown to express incompatibility against both ColE2 and ColE3. These results indicate that these plasmids share a common IncA determinant. A possibility that a small anti-sense RNA is involved in copy number control and incompatibility (IncA function) was suggested.

Bacteriocin Plasmids↗

PubMed1989

Quantitation of platelet-activating factor by high-performance liquid chromatography with fluorescent detection.

Platelet-activating factors, 1-O-hexadecyl- and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-AGEPC and C18AGEPC), were measured by reverse-phase high-performance liquid chromatography with fluorescent detection. C16AGEPC, C18AGEPC, and 1-O-hexadecyl-2-propionyl-sn-glycero-3-phosphocholine, which was suitable for use as an internal standard, were hydrolyzed with phospholipase C, and then the resulting hydrolyzed products were derivatized with 7-methoxycoumarin-3-carbonyl chloride or 7-methoxy-coumarin-4-acetic acid to form 7-methoxycoumarin ester derivatives which permit a fluorometric detection. The lower limit of detection of the derivatives was about 100 pg at a signal-to-noise ratio of 5:1. A commercial platelet-activating factor was demonstrated to contain C16AGEPC (70%) and C18AGEPC (12.8%) by the present method. The present method was also applicable to the measurement of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activity in a lysate of human polymorphonuclear leukocytes.

Acetyltransferases↗

PubMed1989

Comparative analysis of physicochemical and immunochemical properties of the two major allergens from Dermatophagoides pteronyssinus and the corresponding allergens from Dermatophagoides farinae.

Two major allergens, DP1 (Der p I) and DP2 (Der p II), were isolated from the whole culture extract of Dermatophagoides pteronyssinus, and the physicochemical and immunochemical properties of these allergens were compared with those of the corresponding allergens from Dermatophagoides farinae, DF1 (Der fI) and DF2 (Der fII). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polyacrylamide gel isoelectric focusing and amino acid analysis, both DP1 and DP2 were demonstrated to have close physicochemical similarity with DF1 and DF2, respectively. On immunodiffusion with the use of rabbit antisera, the two Der I allergens showed the reaction of typical partial identity, while the two Der II allergens showed the reaction of almost complete identity. Radioallergosorbent test (RAST) and RAST absorption experiments with the use of sera from mite-allergic patients showed that human IgE antibody response to the Der I allergens was directed against both cross-reactive and species-specific determinants. In contrast, IgE antibodies to the Der II allergens were demonstrated to react almost completely to cross-reactive determinants.

Allergens↗

PubMed1989

Measurement of allergens associated with dust mite allergy. I. Development of sensitive radioimmunoassays for the two groups of Dermatophagoides mite allergens, Der I and Der II.

Sensitive radioimmunoassays were developed to measure the two groups of major allergens (Der I and Der II) of Dermatophagoides pteronyssinus and Dermatophagoides farinae, in which radiolabeled protein A served as a general tracer. Glass rods covalently coupled with F(ab')2 fragments of rabbit antiserum IgG were incubated first with mite or house dust extracts, and then with affinity-purified rabbit antibodies. The bound allergen-antibody complex was detected with a 125I-labeled protein A. The two Der I allergens, Der p I and Der f I, were measured separately with the use of immunoabsorbed rabbit antibodies directed against species-specific determinants, while the two Der II allergens, Der p II and Der f II, were measured as 'Der II' with the use of antibodies directed against common determinants on both allergens. Each assay demonstrated consistently parallel dilution curves with mite and house dust extracts. The mean intraassay and interassay coefficient of variation for each assay ranged from 4.3 to 7.7% and from 3.6 to 9.0%, respectively. The Der p I and Der f I assays were shown to be highly species-specific so that the ratio of Der p I: Der f I would provide a good index of the distribution of the two mite species in a dust sample. The concentrations of the Der I and Der II allergens in different types of mite extract and dust samples from houses were compared using these assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗

PubMed1989

Measurement of allergens associated with dust mite allergy. II. Concentrations of airborne mite allergens (Der I and Der II) in the house.

Assays of mite allergens (Der p I, Der f I and Der II) in the air of houses became feasible with the use of a low-noise air sampler and a sensitive radioimmunoassay described previously. The levels of the airborne allergens Der I (Der p I + Der f I) and Der II in the living room of 10 houses during usual domestic life were very low, 29.5 and 6.3 pg/m3, respectively, with a Der I: Der II ratio of 4.7:1. At the time of bedmaking, they greatly increased, about 1,000-fold, to 30,900 and 12,600 pg/m3, respectively, with a Der I: Der II ratio of 2.5:1. The amounts of Der I and Der II in the floor dust of the living room were 2,040 and 2,690 ng/g of fine dust, respectively, with a Der I: Der II ratio of 0.8:1. Der I seemed more prone to become airborne than Der II.

Air↗

PubMed1989

[Measurement of IgE, IgG1 and IgG4 antibodies against mite Sephadex fractions and purified allergens by means of enzyme-linked immunosorbent assay].

Mite antigens (Dermatophagoides farinae) were fractionated by a Sephadex G-200 column and their reactivities with IgE, IgG1 and IgG4 antibodies were investigated with enzyme-linked immunosorbent assay (ELISA). High IgE antibody values were observed in fractions with low molecular weight (allergenic part), while high IgG1 and IgG4 antibody values were observed in fractions with high molecular weight. High IgG4 antibody values to crude mite extract and fractions with high molecular weight were detected in individuals who had received immunotherapy. However, IgG4 antibodies directed to allergenic part were found in only one out of 12 sera tested. IgG4-ELISA using DF1 (major allergen of Dermatophagoides farinae) as antigen was also performed. In the group treated with mite, significant IgG4 antibody levels were detected in only one out of 13 sera tested. In the group treated with house dust, significant IgG4 antibodies were detected in only one out of 12 sera tested. Patients who showed high IgG4 antibody responses to crude mite extract and to high molecular weight did not show responses to allergenic part and DF1. The only case who showed positive IgG4 responses to allergenic part also reacted with DF1. Those results suggest that IgG1 and IgG4 antibody values in ELISA using crude mite extract as antigen do not reflect major allergen-specific antibody values. The importance of the use of partially purified antigens in measuring major allergen-specific IgG4 antibodies was also suggested.

Allergens↗