ABSTRACT
BACKGROUND: The level of the Dermatophagoides mite group 1 (Der 1) allergens in reservoir dust has been used as an index of exposure in most studies. However, the mite allergen level in reservoir dust cannot directly reflect the personal exposure level. OBJECTIVE: We sought to develop a new method for quantifying the Der 1 allergens on bedding and human skin surfaces as an index of exposure to mite allergens. METHODS: Samples were obtained with a small adhesive tape from the forearm skin of 30 healthy volunteers and from their regularly used mattresses. The level of Der 1 allergens collected onto the adhesive tape was measured by a newly developed sensitive fluorometric ELISA for Der p 1 and Der f 1. RESULTS: The Der 1 allergens could be detected in all the samples from bedding surfaces and in 28 of the 30 samples from skin surfaces. The Der 1 levels by adhesive tape sampling from the mattresses correlated with those by reservoir dust sampling. The sampling of the skin and bedding surface with adhesive tape correlated, but skin sampling did not correlate with reservoir sampling. CONCLUSION: The Der 1 allergens on bedding surfaces and on human skin surfaces could be quantified with a very simple sampling technique. The system developed in this study will provide a new tool for the assessment of mite allergen exposure in daily life.
Subject(s)
Allergens/analysis , Antigens, Dermatophagoides/analysis , Beds , Skin , Specimen Handling/methods , Adolescent , Adult , Aged , Arthropod Proteins , Child , Child, Preschool , Cysteine Endopeptidases , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Infant, Newborn , Male , Middle AgedABSTRACT
Mouse non-selective cation channel 1 (mNSC 1) cDNA from mouse pancreatic beta-cell line, MIN6, have recently been cloned. Since the number of non-selective cation channel in pancreatic duct cells has been reported to increase 9-fold in 5 h incubation with cAMP, the effect of cAMP on the gene expression of mNSC1 in MIN6 cells was examined. Dibutyryl cAMP (db-cAMP) was shown to increase the level of the mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The copy number of the mRNA was increased 4-fold in 6 h incubation with db-cAMP by competitive PCR. Western blot analysis also indicated a 4-fold increase in the quantity of the newly synthesized protein in 9 h incubation with db-cAMP. Experiments with 5'-flanking region and with a transcriptional inhibitor suggested that db-cAMP affected transcription, and protected the mRNA from its degradation as well. It is concluded that the expression of mNSC1 is indeed increased by cAMP in the pancreatic beta-cells.
Subject(s)
Cyclic AMP/pharmacology , Ion Channels/genetics , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Animals , Base Sequence , Cell Line , DNA Primers/genetics , Gene Expression/drug effects , Half-Life , Histone-Lysine N-Methyltransferase , Mice , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
We have previously cloned mouse non-selective cation channel 1 (mNSC1) cDNA inducing cation current, from a mouse insulin secreting beta-cell line, MIN6. The current has characteristics of the Ca2+-activated non-selective (CAN) cation channel, and the mRNA is localized in the brain, heart, and lung. To understand the molecular mechanisms of the transcriptional regulation, we have cloned and characterized the 5'-flanking region of mNSC1. By the PCR method, we obtained 987 bp of mouse genomic fragment. The computer program-based analysis revealed that it contained several consensus motifs; insulin responsive element (IRE), AP-2, PEA3, and GC box-like region. But there were neither typical TATA box nor CAAT box. Primer extension analysis and RNase protection assay were performed to identify the transcription start site. Transient transfection analyses using a series of 5'-end deletion and reporter gene constructs with CHO and LA-4 cells demonstrated some relatively active regions. The significantly active border correlated with IRE consensus with CHO cells. This observation may support that CAN current is activated by insulin.
Subject(s)
Ion Channels/genetics , Promoter Regions, Genetic , Animals , Base Sequence , Cell Line , Cloning, Molecular , DNA, Complementary/chemistry , Genes, Reporter , Genetic Techniques , Histone-Lysine N-Methyltransferase , Ion Channels/chemistry , Mice , Molecular Sequence DataABSTRACT
A homologue of the capsaicin receptor-nonselective cation channel was cloned from the rat kidney to investigate a mechanosensitive channel. We found this channel to be inactivated by membrane stretch and have designated it stretch-inactivated channel (SIC). SIC encodes a 563-amino acid protein with putative six transmembrane segments. The cDNA was expressed in mammalian cells, and electophysiological studies were performed. SIC-induced large cation currents were found to be regulated by cell volume, with currents being stimulated by cell shrinkage and inhibited by cell swelling. Single channel analysis showed a conductance of 250 pS with cation permeability (PCl/PNa < 0.1), and the channel possessed some of the characteristics of a stretch-inactivated channel in that it was permeable to calcium, sensitive to membrane stretch, and blocked by Gd3+. Therefore, we cloned one of the mechanosensitive cation channels of mammals, which is considered to regulate Ca2+ influx in response to mechanical stress on the cell membrane.
