Effects of Smoking Cessation on Gene Expression in Human Leukocytes of Chronic Smoker

OBJECTIVE: The risks of cigarette smoking concerning higher systemic disease mortality are lessened by smoking cessation. METHODS: Microarray analysis compared the expression profiles of smokers who were successful and not successful at smoking cessation, with the goal of identifying genes that might serve as potential biomarkers or that might be valuable in elucidating distinct biological mechanisms. The mRNAs were isolated and compared from peripheral leukocytes of six smokers who were successful in cessation and six smokers who failed in smoking cessation. RESULTS: Two hundred ninety nine genes displayed significantly different expression; 196 genes were up-regulated and 103 genes were down-regulated in the success group compared to the failure group. Twenty four of these genes were identified with biological processes including immunity, cytoskeleton and cell growth/cycle. Real-time PCR confirmed the differential gene expression. The mRNA levels of HEPACAM family member 2 (HEPACAM2) and tropomodulin 1 (TMOD1) were significantly more expressed in the success group, while the mRNA ubiquitin specific peptides 18 (USP18) were significantly less expressed in the success group compared to the failure group. CONCLUSION: The results suggest that smoking cessation can modulate cell adhesion and immune response by regulating expression levels of genes, especially HEPACAM2, TMOD1 and USP18, which have an important relationship with smoking cessation.

Identification of Oocyte-Specific Diva-Associated Proteins using Mass Spectrometry / 대한불임학회지

OBJECTIVE: We previously described that Diva is highly expressed in matured metaphase II (MII) oocytes compared to immature germinal vesicle (GV) oocytes in mouse.1 We report here that the expression of Diva transcript as well as protein is oocyte-specific. To elucidate its physiological role in oocyte, the binding partner(s) of Diva has been identified by using immunoprecipitation (IP) followed by Mass Spectrometry. METHODS: NIH/3T3 cells were transiently transfected for 24 h with either empty vector for control or FLAG-tagged mouse Diva construct, and IP was performed with anti-FLAG antibody. The immuno-isolated complexes were resolved by SDS-PAGE on a 12% gel followed by Coomassie Blue staining. For in-gel digestion, 15 bands of interest were excised manually and digested with trypsin. All mass spectra were acquired at a positive reflector mode by a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Proteins were identified by searching the NCBI nonredundant database using MASCOT Peptide Mass Fingerprint software (Matrixscience, London). RESULTS: Diva-associated complexes were formed in FLAG-tagged mouse Diva-overexpressed NIH/3T3 cells via IP using anti-FLAG-conjugated beads. Among the excised 15 bands, actin and actin-binding proteins such as tropomyosin, tropomodulin 3, and alpha-actinin were identified. Binding between Diva and actin or tropomyosin was confirmed by IP followed by Western blot analysis. Both bindings were also detected endogenously in mouse ovaries, indicating that Diva works with actin and tropomyosin. CONCLUSIONS: This is the first report that immuno-isolated Diva-associated complexes are related to actin filament of the cytoskeletal system. When we consider the association of Diva with actin and tropomyosin, oocyte-specific Diva may play a role in modulating the cytoskeletal system during oocyte maturation.