The transmembrane helix of the Escherichia coli division protein FtsI localizes to the septal ring.

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 unusual [corrected] 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.

Development of cystic fibrosis and noncystic fibrosis airway cell lines.

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.

Developmental differences in the shear stress-induced expression of endothelial NO synthase: changing role of AP-1.

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.

Telomerase activation in cervical keratinocytes containing stably replicating human papillomavirus type 16 episomes.

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.

Effects of telomerase and viral oncogene expression on the in vitro growth of human chondrocytes.

Senescent chondrocytes accumulate with aging in articular cartilage, a process that interferes with cartilage homeostasis and increases the risk of cartilage degeneration. We showed previously that chondrocyte telomere length declines with donor age, which suggests that the aging process is telomere dependent. From these results we hypothesized that telomerase should delay the onset of senescence in cultured chondrocytes. Population doubling limits (PDL) were determined for chondrocytes expressing telomerase. We found that telomerase alone did not extend PDL beyond controls that senesced after 25 population doublings. The human papillomavirus 16 oncogenes E6 and E7 were transduced into the same cell population to investigate this telomere-independent form of senescence further. Chondrocytes expressing E6 and E7 grew longer than the telomerase cDNA (hTERT) cells but still senesced at 55 population doublings. In contrast, chondrocytes expressing telomerase with E6 and E7 grew vigorously past 100 population doublings. We conclude that although telomerase is necessary for the indefinite extension of chondrocyte life span, telomere-independent senescence limits PDL in vitro and may play a role in the age-related accumulation of senescent chondrocytes in vivo.