Publications

We used an enhancerless U3 mutant retroviral vector to deliver chimeras of the phosphoenolpyruvate carboxykinase (PEPCK) promoter region to a renal epithelial cell line capable of expressing PEPCK mRNA. Chimeras consisting of the PEPCK promoter and chloramphenicol acetyltransferase, neomycin phosphotransferase or human growth hormone genes were expressed after viral infection of the NRK52E renal epithelial cell line. Virus-delivered sequences in which the direction of PEPCK promoter transcription was antegrade to the normal direction of the long terminal repeat (LTR)-initiated transcription correctly upon stimulation with dexamethasone or 8-bromo cyclic AMP and upon lowering of the extracellular pH. Fluorescent primer extension in situ using primers specific for virus-delivered sequences of antegrade constructs indicated that a large fraction of NRK52E cells could be infected by co-cultivation with virus-producing psi-2 cells without G418 selection. Virus-delivered constructs whose orientation was opposite to that of the LTRs were expressed at very low levels, with transcripts detectable by PCR only in RNA from cyclic AMP-treated cells. Using reverse transcription/PCR, we demonstrated that the chimeric transcripts were from the internal PEPCK promoter rather than a functional or reconstituted Moloney LTR. PEPCK-reporter chimeras delivered by retroviral vectors demonstrated a level of expression more consistent with the level of expression of the native PEPCK gene than did transfected chimeras. This expression system should prove useful for studies of the physiological modulation of gene expression in renal tissues.

We examined 142 biopsy specimens of smokeless tobacco-associated oral mucosal lesions from 133 professional baseball players. Four types of epithelial change were observed in the specimens: hyperparakeratosis, hyperorthokeratosis, pale surface staining, and basal cell hyperplasia. These types of epithelial change were associated with the type of smokeless tobacco used (snuff or chewing tobacco) but not with the duration (years) or amount (hours per day) of use. The thickness of hyperkeratosis in a specimen correlated directly with the amount of smokeless tobacco use. The use of snuff was more frequently associated with development of oral mucosal lesions than was the use of chewing tobacco, and snuff appeared to cause a greater variety and severity of epithelial change than did chewing tobacco.