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.

BACKGROUND: As a method to effectively improve organic tolerance and velocity, altitude training has been gradually accepted by the coaches,and it is a problem to be solved to find the optimal altitude.OBJECTIVE: To establish rat models of stimulating altitude training, and observe the changes of myocardial cell structure in the altitude training, so as to provide scientific animal models for the physiological studies of altitude training, and provide more scientific theoretical evidence for the altitude training of athletes.DESIGN: Based on the division of altitude region in aerospace medicine,the experimental animals were divided into different altitude training groups and control groups. Morphological characters of cellular and subcellular structures were observed under electron microscope, the biochemical analysis of cardiac troponin T were performed, and then the myocardial injury was completely analyzed from two pathways of morphology and biochemistry.SETTING: Laboratory of Sports and Human Sciences, Tsinghua University.MATERIALS: The experiments were carried out in the Laboratory of Sports and Human Sciences, Tsinghua University from January 2000 to December 2002. Totally 372 male SD rats were used. The experimental materials included hypobaric oxygen chamber, Japanese H-500 transmission electron microscope and the kit for testing serum cardiac troponin T index.METHODS: After adaptive swimming training for 1 week, all the 372 rats were randomly divided into 9 groups and 54 subgroups, only 6 groups of them were analyzed in this experiment, including stimulating acute altitude hypoxia training at 4 000 m group (n=32), hypoxia without training group (n=24), stimulating acute altitude training at 4 000 m followed by reoxygen training for 1 week group (n=32), stimulating acute altitude training at 4000 m followed by reoxygen training for 2 weeks group (n=32), increasing altitude training group (n=32) and plain training control group (n=24). Rats in the plain training control group lived and trained in normal conditions,and those in the other groups all trained with swimming in the stimulating altitude environment for 4 weeks. They were trained for 7 days every week,once a day and 60 minutes for each time for 4 weeks. The serum cardic troponin T index of rats was detected and observed with enzyme-linked immunoabsorbent assay (ELISA). After stimulating acute altitude training at 4 000 m for 4 weeks followed by reoxygen training for 2 weeks, the rats were killed, and a small piece of myocardial tissue was taken from left and right ventricles, then double-stained with uranyl acetate and lead citrate,and the ultrastructure of the sections was observed with Japanese H-500 transmission electron microscope.MAIN OUTCOME MEASURES: The serum cardiac troponin T index of rats trained at different altitude, and the results of spectrum and observation of myocardial ultrastucture under electron microscope were mainly observed.RESULTS: ① Changes of body mass: The myocardial increasing rate in the hypoxia training group was significantly different from those in the hypoxia without training control group and plain training control group (P ＜ 0.01), and there was also significant difference between the latter two groups (P ＜ 0.05). The increasing rate of body mass was significantly different among the hypoxia training group, hypoxia without training control group and plain training control group (22.41％, 28.14％, 33.48％,P ＜ 0.01-0.05). ② Results of cardiac troponin T: Compared with the hypoxia without training group, the value of cardiac troponin T in the hypoxia training groups was significantly different, and significantly increased with weeks of training (P ＜ 0.01). During the hypoxia altitude training below 4 000 m for 1-4 weeks, the value of cardiac troponin T ranged in creased from 0.09 to 0.128 μg/L, and it gradually recovered to 0.09 μg/L after reoxygen training in plain for 2 weeks. ③ The results of observation under electron microscope showed that after acute hypoxia training at 4 000 m for 4 weeks, myocardial mitochondrial structure occurred mild swelling with the prolongation of training, and for the rats received reoxygen training in plain for 2 weeks, the thick and thin myofilaments of myocardial fibers formed light and dark bands were clear and distinct, and the myotomes arranged orderly, besides clustered or scattered glycogen granules, a great amount of mitochondria also existed among muscular bundles, no swelling, degeneration of vacuoles, lysis necrosis, interstitial edema and other pathological changes were observed.CONCLUSION: It is safe to train rats at any altitude below 4 000 m. The acute exercise training of great intensity directly at 4 000 m will cause the mild injury of cardiac subcellular structure, and the increase of cardiac troponin T concentration. The training by means of increasing altitude has slighter influence on the myocardial injury, and the recovery after training is also faster.

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.

Biomarkers, Tumor ; Early Detection of Cancer ; Facial Neoplasms ; diagnosis ; Head and Neck Neoplasms ; diagnosis ; Humans ; Mouth Neoplasms ; diagnosis

Objective To observe the effects of training on intrinsic hand muscles after digital flexor tendonrepair. Methods A total of 60 patients with digital flexor tendon repair were randomly assigned into an experimentgroup (n = 30) and a control group (n = 30). Patients in the experiment group received intrinsic hand muscles train-ing, in addition to the passive mobilization of the flexor tendons and active mobilization of the extensor tendons admin-istered to those in the control group. Minnesota manual dexterity test and Purdue pegboard test were adopted to as-sessment the outcome after 3 months of training. Results After 3 months of training, there were significant differ-ences between the two groups in terms of the results of Minnesota manual dexterity test and Purdue pegboard test( P < 0.01 ). Conclusion Intrinsic hand muscles training after digital flexor tendon repair can improve fine move-ment and coordination of hand.

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.

Adenoviridae ; genetics ; Cell Line ; Genetic Vectors ; Humans ; Nitric Oxide Synthase Type III ; genetics ; Virus Replication

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 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.