Rapid generation of stable cell lines expressing corticotropin-releasing hormone receptor for drug discovery.

Human HEK293 cells that stably express the Epstein Barr nuclear antigen 1 (EBNA1) support the episomal replication of plasmids containing the Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E) cell line expressing the human corticotropin-releasing hormone receptor subtype I (CRHR1) from an episomal plasmid was generated (293CR1s), analyzed, adapted to spinner culture, and scaled-up for production in less than 6 weeks. Forty-seven stable CHO cell lines transfected with CRHR1 were also isolated. Expression of the receptor in the best of these lines (as judged by CRH-induced cAMP production), CHO-R22, was compared to that in 293CR1s cells. Results indicate that the CRHR1 episomal expression vector in 293E cells (1) rapidly generates stable cell lines suitable for scale-up; (2) is stably maintained during 3 months in culture; (3) expresses high levels of CRHR1 mRNA; and (4) expresses significantly more CRHR1 than the CHO-R22 line. Coexpression of additional G protein alpha subunit (G alpha s) with CRHR1 in 293E cells converts a higher percentage of receptor to the agonist high-affinity G-protein-coupled state. Our data support the idea that using the EBV oriP-driven episomal system for gene expression results in greater production of protein in a relatively short period of time.

Sickness absence, respiratory impairment and smoking in industry: an australian study.

A retrospective study of sickness absence, respiratory impairment and their relation to smoking habits was conducted among a heterogeneous population of 1867 staff members of 12 Australian organizations. Data on smoking habits were used to divide the sample into current cigarette smokers (n=392), ex-cigarette smokers (n=182) and those who had never smoked (n=714). Results of the health survey by self-administered questionnaire showed that the prevalence of respiratory symptoms was higher in cigarette smokers than in non-smokers both in men and women. Smoking was particularly related to frequency of cough, and sputum production. Wheezing showed a similar, though less marked, trend. The influence of smoking on the pulmonary function value, FEV1/FVC% was assessed in a group of 972 men and 297 women. Results of analysis of variance showed that ventilatory function was significantly impaired among smokers and ex-smokers. The sickness absence records of 826 cigarette smokers, ex-smokers and non-smokers were also compared. Both frequency and duration of sickness absence were greater in cigarette smokers and ex-smokers. Significant differences between these groups were confirmed in total as well as in certified absence. Among males, the average number of days off work was 1.59 times greater in male smokers than in male non-smokers, and 1.36 times greater for female subjects who smoked. The epidemiological implications of such findings await further research. However, the consequences for the individual, industry and the general community are apparent.