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T Yuuki

Publications and source records attributed to T Yuuki.

34 records · Page 2Linked to original sources
PubMed1996

Direct expression of Der f2, a major house dust mite allergen, in Escherichia coli.

Der f2 protein in a highly antigenic form was directly expressed in bacteria. Plasmid pFLU11 derived from pKK233-2 was designed to express methionyl-Der f2 under the control of the trc promoter and it has the replication origin of pUC118 instead of its original to increase the copy number. This expression plasmid directed the synthesis of recombinant Der f2 (rDer f2) protein in an insoluble form of inclusion bodies in Escherichia coli cells. The high copy number plasmid pFLU11 conferred the efficient production of the Der f2 protein in E. coli, when compared to a nonchanged origin material. rDer f2 inclusion bodies were easily solubilized in urea and renatured by dialysis to assume the active form. The rDer f2 protein was purified by means of anion exchange and gel filtration chromatography. This expression system yielded about 10 mg of purified rDer f2 protein from the 1L culture. Purified rDer f2 protein reacted with IgE from patient sera almost identically to the native Der f2 in the RAST enzyme immunoassay and skin prick test.

Allergens↗
PubMed1995

Cloning and expression in Escherichia coli of cDNA encoding house dust mite allergen Der f 3, serine protease from Dermatophagoides farinae.

Der f 3 is one of the allergens produced by house dust mite Dermatophagoides farinae showing serine protease activity. Based on its amino acid sequence, a cDNA clone encoding Der f 3 was isolated from a cDNA library of D. farinae. Sequencing analysis of the clone revealed the presence of an open reading frame of 780 bp, which encodes a mature protein of 232 amino acids with 27 amino acids of pre-pro sequence at the N-terminus. When proDer f 3 was produced in Escherichia coli as a fused protein with glutathione-S-transferase, the fused protein was accumulated as inclusion bodies. The protein purified with 8 M urea and glutathione-affinity column chromatography, however, did not show protease activity. When an arginine residue was introduced at the C-terminus of the pro-region in place of threonine, removal of the pro-region to produce an active mature protease was observed. The specificity and the activity of this recombinant protease were almost the same as those of native Der f 3.

Allergens↗
PubMed1995

Analysis of the IgE-epitope of Der f 2, a major mite allergen, by in vitro mutagenesis.

Der f 2 is a major mite allergen composed of 129 amino acid residues. To determine the major epitopes on Der f 2 recognized by human IgE antibodies, artificial mutations were introduced to Der f 2 protein. The IgE-binding activity of Der f 2 was significantly decreased by deletion of 10 amino acids at the N-terminus or nine amino acids at the C-terminus. Site-directed mutagenesis with a single amino acid replacement by Ala or Leu in both N- and C-terminal regions as well as a central portion was performed to generate 42 single-site mutations. Amino acid replacement around a disulfide bond of Cys8-Cys119 caused a marked decrease in IgE-binding activity. Furthermore, a distinct decrease in IgE-binding was also caused by Ala-substitution close to a disulfide bond of Cys73-Cys78 and by mutations of a few charged residues. From these results, it was concluded that the two disulfide-forming regions of Der f 2 and several charged residues are important for forming major epitope structures recognized by human IgE antibodies.

Allergens↗
PubMed1994

Effects of amino acid variations in recombinant Der fII on its human IgE and mouse IgG recognition.

Amino acid sequencing of the major mite allergen Der fII purified from mite body extract revealed that it is a mixture of at least two variants. Substitutions were found only at positions in which amino acid variations were predicted from the nucleotide sequences of three cloned cDNAs. When cDNAs corresponding to the three variants were expressed in Escherichia coli, Der fII proteins were produced as inclusion bodies. Denaturation and renaturation with urea converted recombinant Der fII into a protein with three intramolecular disulfide bonds (Cys8-Cys119, Cys21-Cys27, and Cys73-Cys78), which were identical to those previously identified in native Der fII. All the variants, including native Der fII, were equally recognized by human IgE antibodies from 14 different sera of mite-allergic patients. These results suggested that recombinant Der fII proteins assumed a conformation identical to that of the native Der fII and that all the Der fII variants acted as allergens. This result also suggested that IgE antibody binds to the region where there were no amino acid variations. The binding ability of the monoclonal antibody (mAb) 18G8 for the variant clones 1, 2, and 11 Der fII was almost the same. However, mAb 15E11 bound to clone 1 Der fII more efficiently than to clones 2 and 11, whereas mAb 13A4 recognized clone 2 Der fII as the most preferable antigen. This suggested that these antibodies recognized the region of amino acid variations. The stability of Der fII variants was analyzed by measuring their IgE antibody binding activity after heating, freezing, or acid treatment, and was analyzed by resistance to trypsin digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗
PubMed1993

Determination of three disulfide bonds in a major house dust mite allergen, Der f II.

Der f II is a major mite allergen consisting of 129 amino acid residues. Der f II contains six cysteine residues, suggesting the existence of three disulfide bonds which would stabilize this small protein. As the first step in revealing the relationship between the structure and the allergenic property of Der f II, the formation of disulfide bonds was examined. Der f II purified from Dermatophagoides farinae was treated with lysyl-endopeptidase or proline-specific endopeptidase, and the peptide fragments thus generated were separated by reverse phase high performance liquid chromatography. Determination of the amino acid sequence of each peptide collected in this way proved the existence of three disulfide bonds between Cys8 and Cys119, Cys21 and Cys27, and Cys73 and Cys78.

Allergens↗
PubMed1991

Synthesis of biologically active recombinant Der f II.

A cDNA library corresponding to mite protein was screened employing anti-Der f II antibody. Two possible clones containing plasmids pFL1 and pFL11 were obtained. The two plasmids had insertions of about 500 basepairs. The DNA sequences of the two insertions were determined, from which the amino acid sequences were deduced. The amino acid sequence of the purified native Der f II protein could be determined to 45 residues from the N terminus. As a result of comparison, we concluded that the cDNAs prepared from live Dermatophagoides farinae mite corresponded to the mite allergen Der f II. The recombinant Der f II was biologically active.

Allergens↗
PubMed1991

Cloning and expression of cDNA coding for the major house dust mite allergen Der f II in Escherichia coli.

A cDNA library corresponding to mite protein was screened using anti-Der f II, a major allergen from the house dust mite Dermatophagoides farinae, antibody. Three possible clones were obtained that contained cDNA fragments coding for Der f II, and the nucleotide sequences of the fragments were determined. There were minor differences observed affecting the deduced amino acid sequence among the three cDNA fragments. The amino acid sequence of the purified native Der f II protein could be analyzed to 45 residues from the N-terminus. As a result of comparison, all the three cDNA fragments code for a mature protein with a derived molecular weight of about 14,000. The amino acid sequence was not homologous to any known protein sequences and it contained six cysteine residues and no N-glycosylation sites.

Allergens↗
PubMed1990

Cloning and sequencing of cDNAs corresponding to mite major allergen Der f II.

A cDNA library corresponding to mite protein was screened employing anti-Der f II antibody. Two possible clones were obtained, which contained plasmids, pFL1 and pFL11, respectively. Both plasmids had insertions of about 500 base pairs. The DNA sequences of the two insertions were determined, from which the amino acid sequences were deduced. The amino acid sequence of the purified native Der f II protein could be determined to 45 residues from the N-terminus. As a result of comparison, we concluded that the cDNAs prepared from live mite Dermatophagoides farinae corresponded to the mite allergen, Der f II.

Allergens↗
PubMed1989

Amino acid residues stabilizing a Bacillus alpha-amylase against irreversible thermoinactivation.

The alpha-amylase of Bacillus licheniformis (BLA) is stable and active at high temperature. More than 80% of its activity is retained after heat treatment at 90 degrees C for 30 min, and the optimum temperature for its activity is 80-85 degrees C. In contrast, the alpha-amylase of Bacillus amyloliquefaciens (BAA), the amino acid sequence of which shows 80% homology with that of BLA, is rapidly inactivated at 90 degrees C. Various chimeric genes were constructed from the structural genes for the two enzymes, and their products were analyzed for stability as to irreversible thermoinactivation. Two regions in the amino acid sequence of BLA comprising Gln178 (region I) and the 255th-270th residues (region II), respectively, were shown to determine the thermostability of BLA. Region I plays a major role in determining the thermostability. By means of site-directed mutagenesis of the BAA gene, deletion of Arg176 and Gly177 in region I and substitutions of alanine for Lys269 and aspartic acid for Asn266 in region II were shown to be responsible for the enhancement of the thermostability. Mutant BAAs containing the above deletion and substitutions showed almost the same thermostability as BLA as to irreversible thermoinactivation. Nevertheless, the mutant BAAs showed a temperature optimum as low as that of BAA (65 degrees C), indicating that they are still susceptible to reversible inactivation at temperatures higher than 65 degrees C.

Amino Acid Sequence↗
PubMed1985

Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences.

The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.

Amino Acid Sequence↗
PubMedDate not supplied

Non-anaphylactic combination of partially deleted fragments of the major house dust mite allergen Der f 2 for allergen-specific immunotherapy.

Allergen-specific immunotherapy, in which repeated injections of allergens over prolonged periods are used to induce tolerance, has proven an effective treatment of allergy. A major side effect of allergen-specific immunotherapy is anaphylactic reaction. House dust mite allergens are major causative factors associated with various allergic diseases. Der f 2 is the major house dust mite allergen composed of 129 amino acid residues. Analysis using deletion mutants of Der f 2 suggested that T-cell epitopes of Der f 2 were multiple in mite-allergic patients. We found that some IgE epitopes were renatured by dialysis of a mixture of two denatured C- and N-terminal deletion mutants, 1-112 and 85-129 in 13 patients out of 14. On the other hand, IgE binding activity was negative in the separately dialyzed fragments and their mixture in each patient tested. Furthermore, we demonstrated that neither of the two separately prepared polypeptides induced in vivo skin prick test reactivity. These findings are important for improvement of T-cell targeting allergen-specific immunotherapy and development of monovalent IgE haptens. The use of combinations of overlapping non-anaphylactic fragments of allergen covering all of the T-cell epitopes achieves the removal of IgE reactivity, the cause of harmful anaphylactic reactions, without affecting the T-cell reactivity essential for immunotherapy, offering potentially safer and more effective treatment for allergic disease.

Allergens↗
PubMedDate not supplied

Inflammatory cytokines in vitreous fluid and serum of patients with diabetic vitreoretinopathy.

To determine whether inflammatory cytokines are increased in proliferative diabetic retinopathy. We measured concentrations of interleukin-6, 8 (IL-6, 8) and tumor necrosis factor (TNF)-alpha by enzyme-linked immunosorbent assay (ELISA) in vitreous and serum from 47 patients with proliferative diabetic retinopathy and 21 patients with vitreous noninflammatory retinopathies. Vitreous concentration of IL-6 were 64.7+/-12.8 pg/ml in proliferative diabetic retinopathy, much greater (P<.005) than in noninflammatory retinopathy (2.8+/-4.5 pg/ml). Amounts of IL-8 in vitreous fluid also were greater in proliferative retinopathy than in noninflammatory retinopathy (34.0+/-11.5 vs. 6.1+/-2.0 pg/ml, P<.005). Concentrations of TNF-alpha in vitreous fluid were not statistically different in proliferative retinopathy from those in noninflammatory retinopathy. In sera, concentrations of IL-6 and IL-8 were not different between proliferative and noninflammatory retinopathy. However, serum TNF-alpha was much greater in proliferative retinopathy than in noninflammatory retinopathy (0.81+/-0.72 vs. 0.09+/-0.00 pg/ml, P<.001). Elevated TNF-alpha in serum then may be diagnostically useful in proliferative diabetic retinopathy. And inflammatory cytokines in vitreous may be pathogenically important in this concentration.

Blood↗
PubMedDate not supplied

Effect of SG-210, a novel aldose reductase inhibitor, on impaired polyol pathway in rats received diabetic manipulations.

To investigate the effect of SG-210, a potent inhibitor selective to aldose reductase (ARI), on the impaired polyol pathway, we examined biochemically and histologically the potencies of this compound in streptozotocin-induced diabetic or galactosemic rats. The study with diabetic rats showed that SG-210 (1-10 mg x kg(-1)) dose-dependently inhibited sorbitol accumulations in erythrocytes, sciatic nerves, lens, and retina with ED50 values of 1.4, 1.3, 3.5, and 4.6 mg x kg(-1), respectively. Zenarestat, currently under clinical trials both in Japan and the United States, was about two or over five times less potent than SG-210 in suppressing sorbitol contents of erythrocytes or other tissues, respectively. Epalrestat, commercially available, was much less potent in reducing the contents with ED50 values of more than 30 mg x kg(-1) in all of the cells and the tissues examined. An extensive study using galactosemic rats indicated that SG-210 (3-30 mg x kg(-1)) inhibited galactitol accumulations in lens and retina as well as in erythrocytes, preventing the progression of histological abnormalities in lens accompanied by the reduction in galactitol contents. Epalrestat (3-30 mg x kg(-1)) failed to show any significant effects. Pharmacokinetic studies suggested that SG-210 has a high bioavailability and possesses a long half-life in rats (ca. 10 h). Taken together with its excellent pharmacokinetic profiles, the potent suppressive effects of SG-210 observed in this study may be available as a new treatment of diabetic complications.

Aldehyde Reductase↗
PubMedDate not supplied

Expression of ephrin in retinal neovascularization and iris rubeosis.

We investigated expression of ephrin-B2 and Eph-B4 in the retinal tissues of six primate eyes with neovascularization and iris rubeosis secondary to laser-induced central retinal vein occlusion and in tissue from 10 human eyes with proliferative diabetic retinopathy. Two primate eyes with rubeosis and retinal neovascularization were enucleated 1, 2 and 4 weeks after the creation of central retinal vein occlusion. Antibodies were localized using the avidin-biotin reaction. In the primate eyes, ephrin-B2 was negative at I week and positive at 2 and 4 weeks in the rubeotic tissue, but was positive only at 2 weeks in the retinal neovascular membrane. Eph-B4 was negative in all the primate eye specimens. In the human tissue, ephrin-B2 was detected in two of the five eyes with rubeosis and three of the five eyes with retinal neovascularization. These data suggest that ephrin-B2 is a key regulator of neovascularization.

Angiogenic Proteins↗
PubMedDate not supplied

Immunohistochemical detection of endotoxin in endotoxemic rats.

BACKGROUND/AIMS: Although there are various methods to detect endotoxin mainly after the intravenous injection of purified endotoxin in a host, its uptake and distribution among the various organs is not well understood. In the present study, the time course of the distribution and disappearance of endotoxin in various rat organs following injection via two different routes was evaluated by an immunohistochemical staining method using a newly developed monoclonal antibody against Factor C. MATERIALS AND METHODS: The time course of the distribution of lipopolysaccharide (LPS) into the liver, spleen, lung, and kidney after intravenous (i.v.) or intraperitoneal (i.p.) injection of LPS was studied by immunohistochemical staining using a newly developed monoclonal antibody against Factor C in rats. Moreover, plasma endotoxin levels were measured by a modification of a chromogenic endotoxin-specific assay. RESULTS: At 30 minutes after injection in the i.v. group and at 12 and 24 hours in the i.p. group, endotoxin was present on Kupffer cells by staining and on some sinusoidal endothelial cells in the liver as well as on macrophages in the marginal zone of the spleen. The plasma endotoxin levels in the i.v. group decreased gradually after injection. However, levels in the i.p. group gradually increased, reaching a maximum level at 6 hours after injection, and then gradually decreasing. CONCLUSION: These results suggest that, regardless of the route of injection, endotoxin can be detected by an immunohistochemical staining method using a monoclonal antibody against Factor C.

Animals↗