In this study, we utilized the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell line, designated NuLi-1 (normal lung, University of Iowa), was derived from HAE of normal genotype; three cell lines, designated CuFi (cystic fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the ion channel physiology expected for the genotypes. The CF transmembrane conductance regulator defect in the CuFi cell lines could be corrected by infecting from the basolateral surface using adenoviral vectors. Using nuclear factor-kappaB promoter reporter constructs, we also demonstrated that the NuLi and CuFi cell lines retained nuclear factor-kappaB responses to lipopolysaccharide. These cell lines should therefore be useful as models for studying ion physiology, therapeutic intervention for CF, and innate immunity.

Endothelial nitric oxide synthase (eNOS) mRNA and protein levels increase during late gestation and then decrease postnatally in sheep lung parenchyma. The increase in fluid shear stress at birth, resulting from increased pulmonary blood flow, is an important mediator of postnatal eNOS gene expression. Our objective was to identify factors stimulating eNOS expression in pulmonary arterial endothelial cells (PAEC) in response to shear stress and to determine if these factors are developmentally regulated. PAEC were isolated from fetal lambs and adult sheep. Transcriptional activity from a 1,600-bp eNOS promoter fragment increased in both fetal and adult PAEC exposed to 8 h of shear stress. Conversely, activity driven from an 840-bp promoter fragment containing a putative activator protein (AP)-1 binding site was increased only in fetal PAEC. This increase was completely abolished in an identical construct containing a mutant AP-1 sequence. The AP-1 protein c-Jun was localized to the cytosol in static adult PAEC and to the nucleus in static fetal PAEC. After shear, c-Jun was nuclear localized in both cell types. However, transcriptionally active phosphorylated c-Jun was elevated only in the nuclei of sheared fetal PAEC. Resting levels of eNOS and NO were 2- and 20-fold higher, respectively, in fetal cells. Shear increased eNOS and NO in both cell types: levels were approximately 2.5-fold higher in fetal PAEC. Phosphorylation of Akt and eNOS was evident in sheared fetal but not adult PAEC. We have therefore identified mechanisms of eNOS regulation at the transcriptional level and to be enzyme activation specific to the fetal pulmonary arterial circulation.

Retroviral transduction and expression of the human papillomavirus type 16 (HPV-16) E6 gene has been shown to activate telomerase in human cervical and foreskin keratinocytes. There still remains some controversy, however, as to whether expression of E6 in the context of the whole HPV-16 genome can activate telomerase. In this study, we have generated human cervical keratinocyte clones that contain stably replicating HPV-16 episomes. Interestingly, the majority of the clones exhibited low or no telomerase activity at early passage and this was associated with low transcript levels of the reverse transcriptase component of telomerase, hTERT. The HPV-16-containing clones became immortal without a crisis and, at later passage, exhibited elevated levels of telomerase and higher levels of hTERT without any apparent increase in HPV-16 copy number, E6 transcript levels, or ability to degrade p53. These results indicate that HPV-16 by itself does not necessarily cause telomerase activation in cervical keratinocytes, but rather, supports a model in which HPV-16 facilitates telomerase activation in conjunction with other viral or cellular changes over time.

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FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of about a dozen division proteins that localize to the septal ring. FtsI comprises a short amino-terminal cytoplasmic domain, a single transmembrane helix (TMH), and a large periplasmic domain that encodes the catalytic (transpeptidase) activity. We show here that a 26-amino-acid fragment of FtsI is sufficient to direct green fluorescent protein to the septal ring in cells depleted of wild-type FtsI. This fragment extends from W22 to V47 and corresponds to the TMH. This is a remarkable finding because it is usual for a TMH to target a protein to a site more specific than the membrane. Alanine-scanning mutagenesis of the TMH identified several residues important for septal localization. These residues cluster on one side of an alpha-helix, which we propose interacts directly with another division protein to recruit FtsI to the septal ring.The use of fluorescence microscopy to visualize proteins in bacteria has revealed that many proteins are not distributed randomly but instead localize to specific subcellular sites, such as the midcell or pole(s) (22, 37). Moreover, proteins that are targeted to specific sites often fail to function properly if they are mislocalized. Despite the importance of proper localization, little is known about how targeting information is encoded in the amino acid sequences of bacterial proteins. In this report, we describe a small peptide from a bacterial cell division protein, FtsI, that is sufficient to target green fluorescent protein (GFP) to the division site in Escherichia coli. Interestingly, this peptide is a transmembrane helix (TMH). These findings help to clarify how targeting information is encoded in FtsI's primary sequence and demonstrate that a bacterial TMH can serve as a targeting signal.FtsI, also known as penicillin-binding protein 3 (PBP3), is a transpeptidase needed for cross-linking septal peptidoglycan (1,3,38). Previous studies from a number of laboratories have shown that FtsI is one of over a dozen proteins that localize to the division site, where they form a structure called the septal ring (for recent reviews, see references 12 and 43). As division proceeds, the ring constricts so as to remain at the leading edge of the developing septum. The septal ring is thought to be a multiprotein complex that mediates cell division. Studies of septal ring assembly in various mutant backgrounds have revealed that, at least in E. coli, the division proteins are recruited to the ring in a sequential fashion. In this hierarchy, FtsI is one of the last proteins to join the ring; localization of FtsI appears to depend upon the prior localization of FtsZ, FtsA, ZipA, FtsEX (though this is a leaky requirement), FtsK, FtsQ, FtsBL, and FtsW. This scheme suggests that FtsI is recruited to the septal ring by a cascade of protein-protein interactions involved in the assembly of a multiprotein complex. Moreover, FtsI might localize by binding to ...