Bronchi ; cytology ; metabolism ; Cell Line ; Epithelial Cells ; cytology ; metabolism ; Erythromycin ; pharmacology ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Glutathione ; genetics ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; Up-Regulation ; drug effects

AIM:To investigate the transcriptional regulating function of the catalytic subunit(GCLC)gene of ?-glutamylcysteine synthase(?-GCS),the rate-limiting enzyme of glutathione(GSH)synthetic enzymes.METHODS:The regulating region of the human GCLC gene was cloned by PCR method to construct the wild type plasmid,which expressed luciferase reporting gene.Many mutated plasmids expressing luciferase reporting gene were also constructed by the method of exonuclease Ⅲ with S1 nuclease.The transcription regulation of GCLC gene was investigated by transient transfection of the wild type and the mutated plasmids through the observation of relative activities of luciferase.RESULTS:We found that the-2 515～-2 236 bp,-864～-838 bp,-769～-538 bp,-421～-341 bp regions upside from the transcriptional start site up-regulated the gene transcription are the new positive transcription-regulating regions and the-810～-769 bp(especial the-782～-769 bp)region is the negative transcription-regulating region,which is more precise than ever.CONCLUSION:The results of this experiment provide one good foundation to study the transcription management of the GCLC gene and GSH synthesis.

Objective To find whether heNOS could be expressed in mouse lung after intratracheal administration of recombinant adenovirus vector.Methods heNOS cDNA was obtained by RT-PCR from total RNA which extracted from human HUVEC.The replication-deficient heNOS recombinant adenovirus vector was constructed with a ligation method.High titer of recombinant adenovirus was obtained by chromatographic methods.The expression of heNOS protein was determined by immunohistochemistry staining after intratracheal administration of recombinant adenovirus.Results Sequence confirmed the cloned cDNA containing the whole ORF and the heNOS cDNA was about 3731 bp.It showed 99.93% identity with that of heNOS cDNA in GenBank(163729).The biological activity of heNOS protein was examined in transfected cos7 cell line with heNOS cDNA in eukaryotic expression vector of pcDNA3.0.The replication-deficient recombinant adenovirus vector containing heNOS cDNA was constructed successfully and the purified viral titer was 2.0?1010pfu/mL.After intratracheal administration of the recombinant adenovirus,heNOS expression was found in the majority of bronchial epithelium,alveolar lining cells,endothelial cellsand smooth muscle cells of pulmonary vessels.In control group,little endogenous eNOS immunoreactivity was detected in pulmonary vessels and no eNOS immunoreactivity was shown in bronchial and alveolar epithelial cells.Conclusion The replication-deficient recombinant human endothelial nitric oxide synthase mediated by adenovirus vector constructed could be delivered into the lung tissue of mouse by intratracheal administration of recombinant adenovirus and can be expressed in lung tissue with high-efficiency.

Objective To analyze the influence of adenovirus latent infection on gamma-glutamylcysteine systhetase(γ-GCS) in rat alveolar epithelial cells. Methods The rat alveolar epithelial cells were stably transfected with the plasmid pE1Aneo and control plasmid. Glutathione(GSH) contents, the activity of γ-GCS were detected in oxidant stress. Then the leuel of protein expression, mRNA expression, and promoter transcriptional activity of glutamate-cysteine ligase catalytic subunit(GCLC) were further detected. Results GSH contents decreased because of adenovirus E1A expression in oxidant stress. E1A repressed the expression and activity of γ-GCS, messenger RNA expression, and promoter transcriptional activity of GCLC. Conclusion Adenovirus E1A decreased the activity of γ-GCS probably by repressed promoter transcriptional activity of GCLC. As a result, GSH contents were downregulated in oxidant stress. Thus Adenovirus latent infection amplified the oxidant/antioxidant imbalance in rat alveolar epithelial cells in oxidants stress, which may be an important mechanism of COPD.

Objective To research the effect of PEGylation on rBla g 2 from Blattella germanica.Methods rBla g 2 allergen expressed in E.coli was purified by Ni+affinity chromatographY,then was PEGylated by mPEG2-NHS(Mr,10×103).The PEG-rBla g 2 was purified by CM-Sepharose,a cation exchange chromatography. SDS-PAGE,Western blot and ELISA were used to characterize its biologicat actovoty.Results The relative molecular mass of the purified rBla g 2 was about 39×1023.PEG-rBla g 2 was analyzed by SDS-PAGE.Five bands were visualized by staining with Coomassie Brilliant Blue R-250,while seven bands by staining with I2-KI.Cation exchange chromatography could separate rBla g 2 and PEG-rBla g 2.The 100×103(Mr)and 130×103(Mr)of PEG-rBla g 2 could combined with the special IgE from sera of one cockroach-allergic patient by Western blot.The immunological activities of PEG-rBla g 2 in vitro decreased remarkablv bv ELISA,which was only 42%of rBla g 2.Conclusion PEGylated allergen can retain the ability of combining with special IgE from sera,while its immunological activities reduce enormously,which establishes the basic work of researching recombinant low-sensitive allergens.

AIM:To observe the influence of adenovirus latent infection on the oxidant/antioxidant balance in rat alveolar epithelial cells.METHODS:The rat alveolar epithelial cells were stably transfected with the plasmid pE1Aneo and control plasmid pneo.GSH and MDA contents,the activities of major anti-oxidative enzymes including SOD,CAT,GPx,GST and ?-GCS were detected in oxidant stress.RESULTS:Adenovirus E1A expression repressed the activity of ?-GCS,and decreased GSH contents in oxidant stress.As a result,the activity of GPx and GST was decreased.The contents of MDA maintained high in oxidant stress.CONCLUSION:Adenovirus latent infection amplifies the oxidant/antioxidant imbalance in rat alveolar epithelial cells in oxidants stress,and adenovirus E1A protein decreases the activity of ?-GCS,which plays an important role in this process.

AIM: To explore the role of basic-fibroblast growth factor (bFGF) in the development of pulmonary hypertension induced by hypoxia. METHODS: 1) The pulmonary arteries of SD rats with hypoxia for one and two weeks were isolated, from which the total RNA were extracted by acid guanidinium thiocyanate-phenol-chlorform .Then the levels of mRNA were measured by RT-PCR. 2) About 3mm-long arterial rings cut from SD rat pulmonary arterial stem were suspended between stainless steelhooks in chamber with warmed (37℃) Kreb's solution. Different concentrations of bFGF were added in a cumulative fashion into the chamber where the rings were suspended. The cumulative concentration response curve was obtained. RESULTS: 1)The levels of bFGF mRNA in pulmonary artery of rats with hypoxia were increased significantly compared with those that without hypoxia (2578?384 counts?min -1 (control) vs 5303?756 (hypoxia) for 1 week and 4054?547 (hypoxia) for 2 weeks, P all

Aim To investigate the role of physiological concentration of glucocorticoids on the inflammation mediator IL-6 expression in response to LPS in rat alveolar epithelial cells(CCL149).Methods The CCL149 were treated with LPS,H2O2 and glucocorticoid respectively.Flammtory mediator IL-6 protein expression was measured with ELISA,and the activity of histone deacetylase(HDAC) was measured using colorimetric HDAC activity assay kit.Results IL-6 protein levels were increased in cells exposed to 10 mg?L-1 LPS.Hydrocortisone decreased IL-6 protein expression induced by LPS.Such effect of hydrocortisone was blunt by HDAC inhibitor trichostatinA treatment(10 ?g?L-1).LPS decreased HDAC activity.Hydrocortisone increased HDAC activity.The expression of IL-6 protein induced by LPS was further enhanced by H2O2 treatment.Pretreatment with H2O2 resulted in the inhibition of antiflammtion effect of glucocorticoids.Conclusion Physiological concentration of glucocorticoids could suppress inflammatory response,and this effects requires recruitment of HDAC.Oxidants such as H2O2 may cause the failure of glucocorticoids to function effectively,and the reason may be related to the reduction of HDAC activity.This mechanism may contribute to the pathogenesis of pulmonary disorder.

Airway inflammation in atopic asthma is characterized by T -cell activation, leading to the release of certain cytokines. In this study the IL-2 activity and IL-2 mRNA express in the lymphocytes in guinea pigs with OVA -induced bronchial asthma was measured. It was found that eosinophils and lymphocytes were predominant cells in the peritoneal lavage fluid and bronchoalveolar lavage fluid (BALF) in asthmatic guinea pigs. Compared with contol group, the IL-2 activities in lymphocytes from the BALF (22.56? 2.44 ng/ml vs 14.23 ?4.71ng/ml, P

In order to assess whether there is any abnormality in oncogene expression in hypoxia induced pulmonary hypertension, the expression of oncogene erbB in the lung tissue of rats with and without hypoxia was detected by in situ hybridization with digoxingenin as a prob label. The results showed that there was a slight expression of erbB mRNA in control normoxic rats. After hypoxia for 7 to 21 days, its expression increased significantly as compared with that in control (P