Biomedical subjects

C Nishiyama

Publications and source records attributed to C Nishiyama.

At least 19 recordsLinked to original sources

PubMed1999

The transcription factors Elf-1 and GATA-1 bind to cell-specific enhancer elements of human high-affinity IgE receptor alpha-chain gene.

Key regulatory regions necessary for the expression of the gene encoding FcepsilonRI alpha-chain, a component of the high-affinity IgE receptor primarily responsible for IgE-dependent allergic response, were investigated. Two regions, -74/-69 and -55/-47, which contained binding motifs for proteins belonging to the Ets family and the GATA family, respectively, were shown to be necessary for the activation of the alpha-chain promoter. Both the regulatory elements enhanced the promoter activity only in alpha-chain-producing cells PT18 and RBL-2H3 (mast cell lines), indicating that the elements required specific trans-acting proteins present in the alpha-chain-producing cells. EMSA using nuclear extracts and in vitro-translated proteins revealed that Elf-1 and GATA-1 bound to the enhancer elements. This is the first report describing the regulation in the expression of the FcepsilonRI alpha-chain.

Animals↗

PubMed1999

Analysis of human IgE epitope of Der f 2 with anti-Der f 2 mouse monoclonal antibodies.

Der f 2 is one of the major mite allergens recognized by human IgE antibodies of allergic patients. Using five anti-Der f 2 mouse monoclonal antibodies, human IgE epitopes of Der f 2 were analyzed. Among them, two monoclonal antibodies 15E11 and 13A4 inhibited the binding between Der f 2 and human IgE antibodies. To determine major IgE epitopes of Der f 2, epitopes for the monoclonal IgG antibodies were analyzed using 43 single site Der f 2 mutants constructed by site-directed mutagenesis. Binding ability of 13A4 and 15E11 was decreased by the amino acid replacement around the C-terminus, and around 73rd, respectively. These results suggest that the C-terminal portion and the central portion around 73rd of Der f 2 were recognized by human IgE antibodies as major epitopes. The location of the putative IgE epitopes on 3-D structure of Der f 2 is also discussed.

Allergens↗

PubMed1998

Solution structure of Der f 2, the major mite allergen for atopic diseases.

House dust mites cause heavy atopic diseases such as asthma and dermatitis. Among allergens from Dermatophagoides farinae, Der f 2 shows the highest positive rate for atopic patients, but its biological function in mites has been perfectly unknown, as well as the functions of its homologs in human and other animals. We have determined the tertiary structure of Der f 2 by multidimensional nuclear magnetic resonance spectroscopy. Der f 2 was found to be a single-domain protein of immunoglobulin fold, and its structure was the most similar to those of the two regulatory domains of transglutaminase. This fact, binding to the bacterial surface, and other small pieces of information hinted that Der f 2 is related to the innate antibacterial defense system in mites. The immunoglobulin E epitopes are also discussed on the basis of the tertiary structure.

Allergens↗

PubMed1998

Functional interactions between nuclear receptors recognizing a common sequence element, the direct repeat motif spaced by one nucleotide (DR-1).

Direct repeat motifs composed of two hexamer half-sites spaced by a single nucleotide (DR-1) are recognized by several members of the nuclear hormone receptor superfamily. We examined, by means of gene transfection assays, the interplay between the DR-1-binding nuclear receptors commonly expressed in liver, peroxisome proliferator-activated receptor alpha (PPARalpha), hepatocyte nuclear factor-4 (HNF-4), and chicken ovalbumin upstream transcription factor I (COUP-TFI). Both PPARalpha and HNF-4 efficiently bound to the acyl-CoA oxidase gene enhancer element, but PPARalpha exhibited much stronger transactivation than HNF-4. As a result, HNF-4 suppressed the gene-activating function of PPARalpha, when they were expressed together, due to competition for a common binding site. On the other hand, HNF-4, but not PPARalpha, effectively bound to the apolipoprotein CIII gene element, and activated gene transcription. PPARalpha had no effect even when co-expressed with HNF-4. COUP-TFI bound to both elements, and suppressed the gene activation by PPARalpha and HNF-4. Thus, these nuclear receptors have individual functions in gene regulation, and exhibit complex compound effects when they co-exist.

Animals↗

PubMed1997

Engineering of the major house dust mite allergen Der f 2 for allergen-specific immunotherapy.

A major problem with allergen-specific immunotherapy involving repeated injection of allergens is the risk of an anaphylactic reaction. We engineered the major house dust mite allergen, Der f 2, to reduce its capacity to induce skin test reactivity and histamine release from peripheral blood basophils in allergic patients. The engineered allergen, in which the disulfide bond that linked the N- and C-terminal sequences of Der f 2 was disrupted, retained T-cell epitopes essential for immunotherapy and ability to stimulate T-cell proliferation. Such engineered allergens are potentially useful for safer and more effective immunotherapy for allergies.

Allergens↗

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↗

PubMed1996

IgE responsiveness to Dermatophagoides farinae in young asthmatic children: IgE binding study using recombinant allergens of Der f1, Der f2 and mutant proteins of Der f2.

IgE reactivities to two recombinant (r) allergens, rDer f1 and rDer f2, synthetized by cDNA clones from Dermatophagoides farinae (Df) were analyzed using sera of Df-sensitive asthmatic patients of varying ages. Positive RAST responses to rDer f1 and rDer f2 were found in 88 and 80%, respectively, with sera from young asthmatic children aged 0-1, positive RAST rates to these antigens increased to 100% up to 5 years of age. There was a significant correlation between RAST levels of rDer f1 and rDer f2 in these young children aged 0-1. IgE reactivities to four mutant proteins of rDer f2, which have at least one amino acid substitution, were similarly examined in the asthmatic sera. IgE reactivity to D7A, in which Asp7 was replaced by Ala, was reduced to 30-60% compared to the rDer f2. IgE binding to C8/119S, in which both Cys8 and Cys119 were replaced by Ser, lacking a disulfide bond between Cys8 and Cys119, was reduced almost to a background level at all ages. In contrast, A72L and A120L, in which Ala72 and Ala120 were substituted for Leu, respectively, almost retained the same IgE binding activity as the rDer f2 at all ages. These results suggest that Der f1 and Der f2 are important antigens associated with early sensitization to house dust mite in young asthmatic children. In addition, the disulfide bond between Cys8 and Cys119 and the N-terminal region including the 7th amino acid residue are considered to maintain IgE epitopes of the Der f2 allergen.

Adolescent↗

PubMed1996

Active leprosy treated effectively with ofloxacin.

The patient is a 25-year-old Filipino who showed polymorphous eruptions over the whole body, right ulnar nerve paresis, polyneuropathy and hypalgesia in the area of eruptions. Because the biopsy specimen showed foam cells, histiocytes, epithelioid cells, many Mycobacterium leprae and no giant cells, the diagnosis of borderline-lepromatous (BL) type was made. The symptoms were improved by the administration of 300 mg/day ofloxacin. Because the monotherapy using ofloxacin has been reported to be effective in all 5 previously reported cases of BL type leprosy, it may be recommended for a larger number of leprosy cases.

Adult↗

PubMed1996

[A case showing recurrence of myelopathy and optic nerve disturbance accompanied by pusturosis palmaris et plantaris and positive anticardiolipin antibody].

The patient is a 62-year-old woman who was admitted to our hospital with chief complaints of paresthesia in the four extremities and the trunk and gait disturbance in February 1993. Habitual abortions and left retrobulbar optic neuritis were present in the past history. She had been suffering from pusturosis palmaris et plantaris for 10 years. On admission, weakness of the distal muscles in the right upper extremity and severe disturbance of the deep sensations in the bilateral lower extremities and the right upper extremity were present. With the progress of the neurological symptoms, pusturosis palmaris et plantaris progressed. After the admission, the neurological symptoms became better by the administration of corticosteroid hormone medicine. After that, myelopathy and left optic nerve disturbance relapsed and IgG anti-cardiolipin antibody in the serum was present. The neurological symptom, pusturosis palmaris et plantaris and positive IgG anti-cardiolipin antibody in the serum may be caused by immunological mechanism. Anti-phospholipid antibodies such as IgG anti-cardiolipin antibody in the serum should be examined in patients with clinical multiple sclerosis like our case.

Abortion, Habitual↗

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

Presence of role of the 5SrRNA-L5 protein complex (5SRNP) in the threonyl- and histidyl-tRNA synthetase complex in rat liver cytosol.

A complex containing Thr-RS and His-RS was purified about 1000 to 2000-fold from rat liver cytosol by successive column chromatographies on Sephadex G-200, Phenyl-Sepharose CL-4B, and tRNA-Sepharose. The ratio of the specific activity of Thr-RS and His-RS was relatively constant throughout the purification steps, suggesting that the two synthetases were co-purified as a complex. Chromatographic analyses of the tRNA-Sepharose fraction by Sephadex G-150 column chromatography showed the presence of a hybrid form of the Thr-RS monomer and the His-RS monomer in addition to dimer forms of both enzymes from the pattern of activity of both enzymes. The monomer form of Thr-RS showed high activity comparable to the dimer form and the monomer form of His-RS showed definite activity. An association form of Thr-RS and His-RS dimers was detected by Sephadex G-200 chromatography of rat liver cytosol. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 55SrRNA blot analysis of the tRNA-Sepharose fraction using an antibody against ribosomal protein L5, showed the presence of ribosomal protein L5 in this fraction. These findings suggest that the presence of a 5SRNA-L5 protein complex (5SRNP) in the Thr-RS and His-RS complex. 5SRNP enhanced the activity of Thr-RS in a freshly prepared tRNA-Sepharose fraction. It also enhanced the activity of the rat liver cytosol for the attachment of [3H]threonine to endogenous tRNA. This activity was inhibited by an antibody against protein L5, and the inhibition was reversed by addition of 5SRNP. These results indicate that 5SRNP plays a role as a positive effector of Thr-RS in the complex.

Animals↗

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

Metabolic characteristics of rat liver cytosolic 5SRNA.

5SRNA were purified from the cytosol, ribosomes, nucleoli and nucleoplasm of rat liver. The 5'-terminal and 3'-terminal sequences of these 5SRNA species were shown to be the same. The time course of changes in specific activities of these 5SRNA in rat liver, 20 min to 16 h after intraperitoneal injection of [14C]orotic acid, were investigated. The specific activity of cytosolic 5SRNA increased almost linearly for 3 h, without a lag, to a high value, then decreased rapidly. The specific activity of cytosolic 5SRNA was much higher than the specific activities of 5SRNA from the other cellular components over 45 min to 6 h after intraperitoneal injection, although at 20 min the specific activity was similar to that of nucleoplasmic 5SRNA. Thus, cytosolic 5SRNA of rat liver is characterized by a high rate of turnover, which was shown to be comparable to that of heterogeneous nuclear RNA. The specific activity of 5SRNA, 45 min to 6 h after intraperitoneal injection, was as follows; cytosolic > nucleoplasmic > nucleolar > ribosomal 5SRNA. The specific activity of nucleoplasmic RNA that migrated slightly slower than nucleoplasmic 5SRNA on PAGE was much higher than that of cytosolic 5SRNA or nucleoplasmic 5SRNA, and so may be the precursor of nucleoplasmic 5SRNA. Together, these results indicate that cytosolic 5SRNA is transferred from the nuclei to the cytosol immediately after maturation of the precursor of nucleoplasmic 5SRNA without entering the nucleoplasmic pool of 5SRNA.

Animals↗

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

Prenatal and postnatal development of the CGRP-immunoreactive innervation in the rat kidney.

Calcitonin gene-related peptide (CGRP) immunoreactive afferent nerves were seen intrarenally by prenatal day 16. They grow rapidly and CGRP positive nerves are observed at birth associated with the arterial and venous vascular tree, forming a rich plexus in the pelvic region and in the connective tissue located near the corticomedullary region. The distribution and extent of CGRP immunoreactive nerves suggest that at birth the rat is able to provide information on the status of the kidney to be integrated in the mechanisms of body fluid homeostasis.

Aging↗

PubMed1990

[Relation between movement of acid or alkaline proteinase from Trichophyton rubrum and the growth of fungus, pH in medium].

We have previously reported that T. rubrum produced a new type of extracellular proteinase in liquid medium containing bovine serum albumin. This enzyme had an optimal pH 4.5 for azocoll. In this paper, we studied about the relation between movement of acid or alkaline proteinase and environmental pH. In the beginning of cultivation, acid proteolytic activity increased radically, and 3 or 4 weeks later, it was decreased. In place of acid proteinase, alkaline proteinase which had an optimal pH 8.0 was increased gradually. During incubation of T. rubrum in bovine serum albumin medium which was adjusted to pH 6.0, the pH of medium was gradually increased to about pH 7.0 toward 4 weeks. The growth of T. rubrum increased until pH in medium was fixed to pH 7.0.

Aspartic Acid Endopeptidases↗

PubMed1989

[The relation between the growth and extracellular proteolytic activity in trichophyton rubrum, comparing isolates from granuloma trichophyticum with tinea superficialis].

Trichophyton rubrum, a major pathogenic fungus in dermatophytosis, produced a new type of extracellular proteinase, in addition to those previously reported, when cultivated in liquid medium containing bovine serum albumin. This enzyme had an optimal pH 4.5 for azocoll and was found in both isolates from tinea superficialis and granuloma trichophyticum. The proteolytic activity of isolates from granuloma trichophyticum were superior to isolates from tinea superficialis in both acid and alkaline proteinase. But the growth of T. rubrum isolates from tinea superficialis were better than that from granuloma trichophyticum.

Endopeptidases↗