Molecular cloning and characterization of mouse cardiac junctate isoforms.

Junctate is a newly identified integral ER/SR membrane calcium binding protein, which is an alternative splicing form of the same gene generating aspartyl beta-hydroxylase and junctin. Screening a mouse heart cDNA library using canine junctin cDNA as a probe yielded three complete mouse heart cDNAs. One of the cDNAs is homologous to the previously reported human junctate. The three mouse junctate proteins are composed of 270, 259, and 215 amino acids (we named them junctate-1, -2, and -3). The apparent molecular masses of the mouse junctates in SDS-PAGE were in the range between 40 and 53 kDa. Northern and Western blot analyses indicate that mouse junctates are expressed in heart, brain, spleen, lung, liver, kidney, and stomach, but not in skeletal muscle. The apparent molecular weights of junctates from heart and brain were somewhat different from those from the other tissues tested, suggesting that there are tissue-specific expression patterns of the different junctate isoforms. Immunohistochemical studies showed that junctates were expressed both in ventricular and atrial tissues. This is the first study that shows the presence of 3 distinct cardiac junctate isoforms expressed in various mammalian tissues.

IgE immune response to Ginkgo biloba pollen.

BACKGROUND: The ginkgo (Ginkgo biloba L.) continues to be planted as a shade tree in preference to other species in Seoul, Korea. The proportion of ginkgo to total shade trees was 43.2% in 1998, but the allergenic characteristics of ginkgo pollen has not been elucidated. OBJECTIVES: This study was undertaken to obtain information regarding the skin reactivity rate to ginkgo pollen in a population of Korean subjects with respiratory allergy. Possible ginkgo pollen allergens and the cross-reactivity of ginkgo pollen with other prevalent pollens were also examined. METHODS: Four hundred and forty-seven patients with asthma and/or allergic rhinitis were skin prick tested with extract of ginkgo pollen (1:20 wt/vol). Of these patients, positive skin responders (A/H ratio > or =2+) were selected for ELISA and immunoblot experiments. RESULTS: A total of 21 patients (4.7%) showed skin reactivity (A/H ratio > or =2+) to ginkgo pollen in the skin prick test. They were also cosensitized to many other tree, grass, and weed pollens. Sixteen (76%) of the 21 positive skin responders showed specific IgE responses to ginkgo pollen in ELISA. In inhibitory ELISA, IgE binding to ginkgo pollen was inhibited by more than 80% by oak, ryegrass, mugwort, and ragweed; and 34% by hop Japanese; and 10% by rBet v 2 at 10 microg/mL. In immunoblot, 10 out of 21 sera (48%) reacted to the 15-kD protein of ginkgo pollen, 9 (43%) to 33-35 kD, and 8 (38%) to 36-38 kD. In inhibitory immunoblot, IgE binding to ginkgo pollen proteins was almost completely inhibited by oak, ryegrass, mugwort and ragweed, but only partially by hop Japanese and rBet v 2. CONCLUSION: The skin reactivity rate to ginkgo pollen is approximately 4.7% in a population of Korean subjects with respiratory allergy. Since ginkgo pollen has a high cross-reactivity with other prevalent pollens, it could cause clinical symptoms during its pollen season by cross-reacting with the IgE produced in response to other pollens in patients sensitized to multiple pollens.

cDNA cloning and characterization of human cardiac junctin.

Junctin is a calsequestrin binding protein detected in junctional sarcoplasmic reticulum of striated muscles. In the present study, the human cardiac junctin cDNA has been cloned by human heart cDNA library screening and RT-PCR, and the cDNA sequence has been determined. The deduced amino acid sequence of human junctin (210 aa) has 84% sequence identity to that of canine junctin identified previously. A human junctin isoform (isoform 1, 225 aa) was also identified and characterized. The isoform 1 has a 15 aa insertion at the amino acid residue 55 of the human junctin. Northern blot analysis revealed that the human junctin was present both in cardiac and skeletal muscles, and the sizes of the transcripts were approximately 3.0 and 4.2kb. Amino acid residues 6-78 of human junctin and 35-107 of human aspartyl beta-hydroxylase (hAspH) overlapped perfectly. The gene copy number of human junctin and hASPH was investigated by genomic Southern blot analysis using various restriction enzymes and a common DNA probe. The result showing a single hybridized DNA band at each restriction enzyme suggests that the same genomic region codes both junctin and hASPH.

Identification and characterization of the major allergen of the Humulus japonicus pollen.

BACKGROUND: Pollen of Humulus japonicus has been known as one of the important causes of pollinosis in Korea and China. To date, the major allergen of H. japonicus has not been determined. OBJECTIVE: To identify the major allergen of H. japonicus pollen and characterize its biochemical properties. METHODS: With the sera of 29 patients reactive to H. japonicus, the major allergen of H. japonicus was determined from the results of IgE immunoblotting and ELISA inhibition. The biochemical properties of the major allergen of H. japonicus were evaluated by lectin blotting assay and 2-dimensional PAGE blot. N-terminal amino acid sequences were determined by the Edman degradation method. The suggested major allergen was purified by DEAE anion exchange and gel filtration chromatography. RESULTS: Twenty-nine sera contained IgE bound to the 10, 16, 20, 29 and 42 kDa proteins of H. japonicus in immunoblot analysis. A protein of 10 kDa was the most prevalent allergen in the sera of H. japonicus-reactive patients (72%). The ELISA optical density of H. japonicus-specific IgE was not inhibited by pollen extracts of birch, oak, rye grass and mugwort. The 10-kDa allergen was neither stained with PAS nor bound with ConA and five other lectins. The isoelectric point of the 10-kDa allergen was approximately pH 5.1. We sequenced the N-terminal amino acids of the 10-kDa allergen, which was not homologous with any previously characterized allergen. The 10-kDa allergen could be purified with DEAE anion exchange and gel filtration chromatography. Maximum inhibitions of H. japonicus-specific IgE ELISA by whole extract of H. japonicus and purified 10-kDa allergen were more than 97 and 88%, respectively, while the 50% inhibitory concentration of the whole extract of H. japonicus and purified 10 kDa were 38 and 20 ng/mL, respectively. CONCLUSION: The 10-kDa peptide could be a major allergen of H. japonicus. Its isoelectric point was 5.1 and it did not bind with lectins. The N-terminal amino acid sequence of the 10-kDa major allergen was also determined.

Cross-reactivity between pollen extracts from six artemisia species.

Pollen extracts of six different ARTEMISIA species, A. VULGARIS, A. SCOPARIA, A. PRINCEPS, A. TRIDENTATA, A. ANNUA, and A. CAMPESTRIS were compared using SDS-PAGE, IEF, immunoblotting, and immunoelectrophoretic methods. The band patterns obtained after SDS-PAGE and IEF showed a large degree of similarity between the extracts. Immunoblotting of these gels using a pool of sera from patients allergic to A. VULGARIS gave essentially the same IgE-binding band pattern with all the extracts, demonstrating an extensive degree of cross-reactivity between A. VULGARIS and the other ARTEMISIA species. FRIE using a polyspecific antiserum against A. VULGARIS showed that all the extracts contained several antigens that were immunologically identical to antigens in A. VULGARIS extract. Antigens showing immunological identity to the important A. VULGARIS allergens Ag 12 and ART V II were present in all the extracts. The cross-reactivity between A. VULGARIS and A. PRINCEPS was further verified by screening of ten Korean and nine Norwegian individual patient sera against extracts of both species in SDS-PAGE or IEF immunoblotting. Both groups of patients had essentially the same pattern of reactivity towards both pollen extracts.

Immunoelectron-microscopic localization of IgE binding site of mugwort pollen.

To elucidate the IgE binding site of mugwort (Artemisia vulgaris r.) pollen, pollen grains were frozen and fixed using a cryocut. They were incubated with antibodies according to the following sequence: Sera pool of individuals who showed mugwort-RAST class 3 or 4, biotin-labeled goat anti-human IgE antibody, streptavidin-peroxidase and diaminobenzidine. Then, they were observed under electron microscopy. The control section was incubated with the sera pool from individuals who showed a negative result on a skin prick test to mugwort pollen. Antigenic activity (electrondense line) was noted on the surface of the exine. There was no activity in cytoplasm or the intine layer. The control section was completely free of activity. It was suggested that the IgE binding site of mugwort pollen was present on the surface of the exine.

Identification and partial purification of pollen allergens from Artemisia princeps.

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.

Bronchial challenge responses in asthmatic patients sensitized to Artemisia spp. pollen.

To characterize the patients whose asthma may be caused by Artemisia pollen extracts, we studied the bronchoprovocation test with Korean Artemisia pollen extracts (1:20 w/v), methacholine bronchial challenge test and wormwood-RAST in 32 asthmatic patients sensitized to Artemisia pollen. Twenty-six(81%) developed a 15% or greater decrease in FEVI after the inhalation of Artemisia pollen extracts and 13 patients showed early responses, 8 dual, and 5 late only. Thirteen(50%) out of 26 positive responders complained of seasonal aggravation of their asthmatic symptoms. Seven(53.8%) of the 13 seasonal type patients, 10(76.9%) of the 13 perennial type and 5(100%) of the 5 negative responders showed concurrent positive responses in the house dust bronchoprovocation test. The bronchial responsiveness to allergen(PD15) was more dependent upon the specific IgE level(bound radioactivity on wormwood-RAST) and multiple regression analysis revealed that the specific IgE level and methacholine PC20 may be contributory to allergen PD15. These results suggested that specific IgE to Artemisia pollen appears to be the major contributor to susceptibility to Artemisia bronchial challenges and this pollen may be considered as one of the important allergenic etiologies of atopic asthma in this country.