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Advances in bioscience and biotechnology [journal]
- Increased survivin expression contributes to apoptosis-resistance in IPF fibroblasts. [JOURNAL ARTICLE]
- Adv Biosci Biotechnol 2012 Oct; 3(6A):657-664.
Fibroblasts perform critical functions during the normal host response to tissue injury, but the inappropriate accumulation and persistent activation of these cells results in the development of tissue fibrosis. The mechanisms accounting for the aberrant accumulation of fibroblasts during fibrotic repair are poorly understood, although evidence supports a role for fibroblast resistance to apoptosis as a contributing factor. We have shown that TGF-β1 and endothelin-1 (ET-1), soluble mediators implicated in fibrogenesis, promote fibroblast resistance to apoptosis. Moreover, we recently found that ET-1 induced apoptosis resistance in normal lung fibroblasts through the upregulation of survivin, a member of the Inhibitor of Apoptosis (IAP) protein family. In the current study, we sought to determine the role of survivin in the apoptosis resistance of primary fibroblasts isolated from the lungs of patients with Idiopathic Pulmonary Fibrosis (IPF), a fibrotic lung disease of unclear etiology for which there is no definitive therapy. First, we examined survivin expression in lung tissue from patients with IPF and found that there is robust expression in the fibroblasts residing within fibroblastic foci (the "active" lesions in IPF which correlate with mortality). Next, we show that survivin expression is increased in fibroblasts isolated from IPF lung tissue compared to cells from normal lung tissue. Consistent with a role in fibrogenesis, we demonstrate that TGF-β1 increases survivin expression in normal lung fibroblasts. Finally, we show that inhibition of survivin enhances susceptibility of a subset of IPF fibroblasts to apoptosis. Collectively, these findings suggest that increased survivin expression represents one mechanism contributing an apoptosis-resistant phenotype in IPF fibroblasts.
- Degradation of naturally occurring and engineered antimicrobial peptides by proteases. [JOURNAL ARTICLE]
- Adv Biosci Biotechnol 2011 Dec; 2(6):404-408.
We hypothesized that current antimicrobial peptides should be susceptible to proteolytic digestion. The antimicrobial peptides: Griffithinsin, RC-101, LL-37, LSA-5, PSC-RANTES and DJ007 were degraded by commercially available proteases. Two different species of anaerobic vaginal flora, Prevotella bivia and Porphyromonas asaccharolytica also degraded the materials. Griffithsin was resistant to digestion by 8 of the 9 proteases and the bacteria while LL-37 was the most sensitive to protease digestion. These data suggests most of the molecules may not survive for very long in the proteolytic rich environments in which they are intended to function.
- pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein. [JOURNAL ARTICLE]
- Adv Biosci Biotechnol 2011 Jun; 2(3):132-137.
Green florescent protein (GFP) variants that are sensitive to changes in pH are invaluable reagents for the analysis of protein dynamics associated with both endo- and exocytotic vesicular trafficking. Ratiometric pHluorin is a GFP variant that displays a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidification, pHluorin excitation at 395 nm decreases with a corresponding increase in the excitation at 475 nm. GFP2, a GFP variant that contains mammalianized codons and the folding enhancing mutation F64L, displays ~8-fold higher florescence compared to pHluorin upon excitation at 395 nm. Using GFP2 as a template, an enhanced ratiometric pHluorin (pHluorin2) construct was developed to contain fully mammalianized codons, the F64L mutation and ten of the thirteen pHluorin-specific mutations. As a result, pHluorin2 displays markedly higher florescence when compared to pHluorin while maintaining the ratiometric pH-sensitivity. Unlike native pHluorin, pHluorin2 expressed in the ligand-binding domain of the parathyroid hormone 1 receptor is readily detectable by confocal microscopy and displays a marked increase in florescence upon ligand-induced endocytosis to intracellular vesicles. Thus, pHluorin2's enhanced florescence while sustaining ratiometric pH-sensitivity represents a significant improvement for this methodological approach.
- Direct measurement of oxygen consumption rates from attached and unattached cells in a reversibly sealed, diffusionally isolated sample chamber. [JOURNAL ARTICLE]
- Adv Biosci Biotechnol 2010 Dec 1; 5(5):398-408.
Oxygen consumption is a fundamental component of metabolic networks, mitochondrial function, and global carbon cycling. To date there is no method available that allows for replicate measurements on attached and unattached biological samples without compensation for extraneous oxygen leaking into the system. Here we present the Respiratory Detection System, which is compatible with virtually any biological sample. The RDS can be used to measure oxygen uptake in microliter-scale volumes with a reversibly sealed sample chamber, which contains a porphyrin-based oxygen sensor. With the RDS, one can maintain a diffusional seal for up to three hours, allowing for the direct measurement of respiratory function of samples with fast or slow metabolic rates. The ability to easily measure oxygen uptake in small volumes with small populations or dilute samples has implications in cell biology, environmental biology, and clinical diagnostics.