Biomedical subjects

C Nishiyama

Publications and source records attributed to C Nishiyama.

At least 19 recordsLinked to original sources

PubMed2001

Cloning of full-length genomic DNA encoding human FcepsilonRI alpha-chain and its transcriptional regulation.

Two novel exons, named exon 1A and exon 2A, were found at 18.4 and 12.6 kb upstream from the exon known as the first exon of human FcepsilonRI alpha-chain gene. Transcription from the promoter present in the upstream of exon 1A was decreased by mutations introduced into the "first intron" between exon 1A and exon 2A, suggesting the presence of an intronic regulatory element in the intron. Consistent with this, electrophoretic mobility shift assay revealed the presence of a nuclear factor which bound the region in FcepsilonRI alpha-chain positive cells.

Base Sequence↗

PubMed2001

Molecular cloning of rat USF2 cDNA and characterization of splicing variants.

The complete nucleotide sequence of rat USF2 cDNA was determined. In addition to the full length clone (USF2FL), four isoforms (delta1, delta2, delta3, and delta4) suggested to be generated by alternative splicing were isolated. USF2delta1 and delta2 lacked 27 and 67 internal amino acid residues, respectively. USF2delta3 and delta4 lacked most of the entire sequence but encoded short peptides of an N-terminal portion of USF2FL. Overexpression of USF2FL increased the transcription of the human high affinity IgE receptor (FcepsilonRI) alpha chain gene through specific binding to the CAGCTG motif in the first intron. On the other hand, overexpression of USF2delta1 or delta2 reduced the transcription of the human FcepsilonRI alpha chain gene. Both USF2FL and USF2delta1 bound to CACGTG as well as CAGCTG, while USF2delta2 bound to CACGTG but not to CAGCTG. These results suggested the presence of a different and definitive role of each variant in the expression of the alpha chain gene.

Alternative Splicing↗

PubMed2001

[A case of Folliculitis barbae Candidomycetica].

A 71-year-old man was referred to our department on January 30, 1998 with hard red papules that had developed on the philtrum in mid-January. On January 2, the patient had received high-dose steroid therapy (pulse therapy) for cluster asthma attacks and antibiotics at the Department of Internal Medicine of our hospital. Infiltrative, protruding reddish plaques were observed on the philtrum, which contained a number of small pustules at sites corresponding to hair follicles. There was partial opacity and slight irregularity of the nail plates on the first and second toes of the right foot. Fungal elements were detected from a lesion on the mustache and the nail. Histological examination of the lesion on the philtrum revealed infiltration of inflammatory cells comprising neutrophils, lymphocytes, and macrophages around the hair follicles. Beard hair and nail cultures revealed Candida albicans A, indicating that the patient had candidal sycosis and candidal onychia. He was treated with oral fluconazole (100 mg/day). The lesion was clinically improved within 50-days. Recently, extensive use of steroids and antibiotics has produced an increase in reports of patients with Folliculitis barbae Candidomycetica. We believe that the present case was also induced by high-dose steroid therapy and antibiotics.

Administration, Oral↗

PubMed2001

A complex composed of USF1 and USF2 activates the human FcepsilonRI alpha chain expression via a CAGCTG element in the first intron.

The high-affinity IgE receptor, FcepsilonRI, is a key regulatory molecule in the allergic reaction. During the course of studies to find cis-acting elements for FcepsilonRI alpha chain gene expression, a CAGCTG sequence located in the first intron was revealed to serve as a crucial enhancer element. Electromobility shift assays using antibodies and in vitro translation products showed that the CAGCTG element was recognized by the USF1/USF2 complex. As was the case for other intronic cis-elements, the CAGCTG element regulated the promoter in an orientation- and position-dependent manner. Overexpression of USF2 antisense repressed the FcepsilonRI alpha chain gene promoter and decreased the amount of alpha chain mRNA in mast cell lines. All these results indicated that the USF1/USF2 complex activates the human FcepsilonRI alpha chain gene expression via the CAGCTG element in the first intron.

Animals↗

PubMed2000

Splice isoforms of transcription factor Elf-1 affecting its regulatory function in transcription-molecular cloning of rat Elf-1.

To elucidate the role of Elf-1 in Fc epsilonRI alpha chain expression, rat Elf-1 cDNAs were isolated and characterized. The rat Elf-1 cDNA of 2744 bp contained an open reading frame of 1848 bp. In addition to the full length rat Elf-1 cDNA (named type 1), two splice isoforms were isolated. One of the two isoforms lacked the amino acid residues from 85th to 120th (type 2), and the other from 85th to 175th (type 3). Similar isoforms were also observed in human tissue. Overexpression of rat Elf-1 (type 1) using a transient coexpression system inhibited of the alpha chain promoter activity. The inhibition activity was different between the isoforms; the inhibition activity of type 2 was lower than that of type 1, and type 3 did not have an inhibitory effect. This observation suggested that each Elf-1 isoform played a different role in the gene expression under its control.

Amino Acid Sequence↗

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↗