Orai1 is the key subunit of the Ca2+-release-activated Ca2+ channel. Our previous report has demonstrated that Orai1 expression in the airway was upregulated in the ovalbumin (OVA)-induced allergic rhinitis (AR) mouse models. To observe whether inhibition of Orai1 expression in the airway could suppress symptoms in a murine model of AR and to assess the impacts of this inhibition on the responses of local and systemic immunocytes, we administered recombinant lentivirus vectors that encoded shRNA against ORAI1 (lenti-ORAI1) into the nostrils of OVA-sensitized mice before the challenges, and analyzed its effect on allergic responses, as compared with the unsensitized mice and untreated AR mice. Administration of lenti-ORAI1 into the nasal cavity successfully infected cells in the epithelial layer of the nasal mucosa, and significantly decreased the frequencies of sneezing and nasal rubbing of the mice. Protein levels of leukotriene C4, OVA-specific IgE, and IL-4 in the nasal lavage fluid and serum and eosinophil cation protein in the serum were also significantly reduced by lenti-ORAI1, as were the mRNA levels of these factors in the nasal mucosa and spleen. These data suggested that administration of lenti-ORAI1 into the nasal cavity effectively decreased Orai1 expression in the nasal mucosa, alleviated AR symptoms, and partially inhibited the hyperresponsiveness of the local and systemic immune cells including T cells, B cells, mast cells and eosinophils that are involved in the pathogenesis of AR.
Animals ; Calcium Channels/analysis/*genetics/immunology ; *Down-Regulation ; Eosinophil Cationic Protein/blood/genetics ; Glutathione Transferase/blood/genetics/immunology ; Immunoglobulin E/blood/genetics/immunology ; Interleukin-4/blood/genetics/immunology ; Lentivirus/genetics ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa/immunology/metabolism ; Ovalbumin/immunology ; RNA, Messenger/genetics ; RNA, Small Interfering/*administration & dosage/genetics ; Rhinitis, Allergic, Perennial/*genetics/immunology ; Spleen/immunology/metabolism ; *Transfection

Inflammation has been known to be an important underlying condition for development of various diseases including cancer. The aims of this study were to investigate whether tobacco smoke exposure increases the level of inflammation biomarkers and the GSTM1 and GSTP1 gene polymorphisms are associated with inflam matory response due to tobacco smoke exposure. We measured urinary cotinine level in 300 healthy university students. Total serum TNF-alpha levels and blood WBC counts were determined to evaluate inflammatory response. Allelic loss of the GSTM1 and the GSTP1 (Ile105Val) polymorphism were determined by PCR and RFLP. Tobacco smoke exposure was found to be associated with increase of both TNF-alpha level and WBC count. Particularly, smokers with combination of GSTM1 null and GSTP1 AG or GG genotypes showed higher TNF-alpha level than those with the other genotype combinations (p=0.07). This result suggests that smoking may induce inflammation measured as TNF-alpha level or WBC count and combinations of the GSTM1 and GSTP1 polymorphisms may modify the effect of smoking on serum TNF-alpha level.
Tumor Necrosis Factor-alpha/blood ; Students ; Smoking/*epidemiology/*genetics/immunology ; Risk Factors ; Risk Assessment/*methods ; Prevalence ; Polymorphism, Single Nucleotide/genetics ; Male ; Korea/epidemiology ; Inflammation/*epidemiology/*genetics/immunology ; Humans ; Glutathione Transferase/*genetics ; Glutathione S-Transferase pi/*genetics ; Genetic Predisposition to Disease/epidemiology/genetics ; Female ; Adult

Inflammation has been known to be an important underlying condition for development of various diseases including cancer. The aims of this study were to investigate whether tobacco smoke exposure increases the level of inflammation biomarkers and the GSTM1 and GSTP1 gene polymorphisms are associated with inflam matory response due to tobacco smoke exposure. We measured urinary cotinine level in 300 healthy university students. Total serum TNF-alpha levels and blood WBC counts were determined to evaluate inflammatory response. Allelic loss of the GSTM1 and the GSTP1 (Ile105Val) polymorphism were determined by PCR and RFLP. Tobacco smoke exposure was found to be associated with increase of both TNF-alpha level and WBC count. Particularly, smokers with combination of GSTM1 null and GSTP1 AG or GG genotypes showed higher TNF-alpha level than those with the other genotype combinations (p=0.07). This result suggests that smoking may induce inflammation measured as TNF-alpha level or WBC count and combinations of the GSTM1 and GSTP1 polymorphisms may modify the effect of smoking on serum TNF-alpha level.
Tumor Necrosis Factor-alpha/blood ; Students ; Smoking/*epidemiology/*genetics/immunology ; Risk Factors ; Risk Assessment/*methods ; Prevalence ; Polymorphism, Single Nucleotide/genetics ; Male ; Korea/epidemiology ; Inflammation/*epidemiology/*genetics/immunology ; Humans ; Glutathione Transferase/*genetics ; Glutathione S-Transferase pi/*genetics ; Genetic Predisposition to Disease/epidemiology/genetics ; Female ; Adult

GABARAP, a microtuble-associated protein, is identified to interact with GABAA receptor. Anchoring of the GABAA receptor to GABARAP helps to cluster the receptor at the synaptic termini and to mediate fast synaptic transmission. GABARAP may mediate interaction of gephyrin with the GABAA receptor to stabilize clusters by forming multimeric structures. Furthermore, GABARAP and gephyrin may play more of a role in receptor sorting and transport to the cell surface than in anchoring to the cytoplasm, because at inhibitory synapses GABARAP appears to associate with transport vesicles rather than the cell surface. The association of GABARAP with NSF (N-ethylmaleimide sensitive factor), a protein involved in intracellular vesicle transport, supports this hypothesis. We cloned cDNA encoding full-length human GABARAP by nested PCR and inserted it into eukaryon expression vector pcDNA6HA and GST fusion protein expression vector pGEX4T2. The recombinant plasmid pGEX4T2-hGABARAP was transformed into E. coli BL21, from which GST-hGABARAP fusion protein was purified after IPTG induction by affinity chromatography with glutathione Sepharose-4B column. The antiserum against GABARAP was generated by immunizing rabbits with the purified GST-hGABARAP and was purified with GST-hGABARAP coupled NHS-activated Sepherose 4 column. The purified polyclonal antibody was effective for Western blotting and immunostaining. The hGABARAP was located both in the cytoplasm and nucleus with an abundant distribution around the peripheral nucleus.
Adaptor Proteins, Signal Transducing ; biosynthesis ; genetics ; immunology ; Animals ; Antibodies ; blood ; immunology ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Immunization ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

A cDNA fragment locating at the putative envelop protein 2(E2) region of GBV-C/HGV fused with Schistosoma japonicum, glutathione S-transferase(GST) was amplified with PCR from plasmid pGEX-E2. The amplified DNA fragment was inserted into plasmid pGEX-5X-1, at the downstream of the coding sequences of GST, in the same reading frame with the gene of GST. The fusion gene fragment of GST-E2 was amplified with PCR, using the recombinant plasmid pGEX-5X-1-E2 as the template. The amplified 1324 bp DNA fragment of GST-E2 was inserted into Pichia pastoris expression vector pPIC9K in reading frame with alpha-factor secreting signal peptide. The plasmid pPIC9K-GST-E2 was transformed into Pichia pastoris GS115 with electroporation. The transformants (His+ Muts) were selected and induced to express the 54kD GST-E2 fusion protein, which could be specially recognized by both the antisera directed against E2 and against GST. The GST-E2 fusion protein was purified with Sepharose 4B glutathione affinity chromatography to a purity of 95%. The expression was optimized to achieve the highest expression level of GST-E2 fusion protein which was accumulated up to 50% of total proteins in the culture supernatant. The GST-E2 protein derived from the recombinant Pichia pastoris was proved possessing antigenicity and high specificity by ELISA, probed with sera from the patients infected by GBV-C/HGV.
Animals ; Antigens, Viral ; genetics ; immunology ; isolation & purification ; GB virus C ; genetics ; immunology ; Gene Expression ; Genetic Engineering ; Glutathione Transferase ; genetics ; Hepatitis Antibodies ; blood ; immunology ; Humans ; Pichia ; Recombinant Fusion Proteins ; genetics ; immunology ; isolation & purification ; Schistosoma japonicum ; enzymology ; Viral Envelope Proteins ; genetics ; immunology ; isolation & purification