Biomedical subjects

Shannon M Conley

Publications and source records attributed to Shannon M Conley.

4 recordsLinked to original sources

PubMed2005

Alterations in human trabecular meshwork cell homeostasis by selenium.

Epidemiological evidence indicates that selenium supplementation may increase risk for glaucoma and ocular hypertension. The purpose of this study was to determine the effects of selenium on trabecular meshwork cells, a likely site of pathology for glaucoma. Human trabecular meshwork (HTM) cells and human umbilical vein endothelial cells (HUVECs) were treated with selenium (MSeA) at or near physiologically relevant concentrations. Selenium uptake by cells was monitored using mass spectrometry. Alterations in protein secretion, intracellular signaling, and cell morphology were monitored; and the role of integrin signaling in MSeA-induced morphological alterations was investigated using divalent cation treatments. Radiolabeling was used to assess protein synthesis and secretion, while luciferase and MTT assays monitored total cellular ATP and cell viability, respectively. Whereas detectible changes in intracellular selenium were observed after exposure to 1-10 microM MSeA for 24hr, the majority remained in the conditioned medium. Selenium-induced morphological changes (< or =3 hr) occurred before alterations in protein secretion and intracellular signaling (3-6 hr). Zinc treatment prevented selenium-mediated alterations in protein secretion and changes in cell-matrix adhesion. MSeA treatment (5 microM) led to a 60% decrease in protein synthesis after 3 hr and a 30% reduction in secretion, although significant alterations in cell viability and total ATP were not observed after MSeA treatment. Selenium altered several indicators of HTM cell homeostasis, but did not affect viability at physiologically relevant doses. Similar results with HUVECs have implications for understanding selenium's mechanisms of action as an anti-angiogenic agent.

Adult↗

PubMed2005

Barriers to productive transfection of trabecular meshwork cells.

PURPOSE: A critical function of trabecular meshwork cells is to degrade cellular debris, including DNA. We hypothesize that low transfection efficiencies of primary human trabecular meshwork (HTM) cell cultures with plasmid DNA are a function of retained capacity to efficiently degrade exogenous DNA in vitro. METHODS: To determine mechanisms responsible for low transfection efficiencies of cultured HTM cells, steps of DNA entry into cytoplasm and nucleus were characterized. Following synchronization with sequential serum starvation and serum reintroduction, the HTM cell cycle was characterized using 5-bromo-2-deoxyuridine incorporation into replicating DNA. HTM cells were transfected during S-phase with plasmid DNA encoding green fluorescence protein (GFP) or plasmid DNA conjugated with Cy3. In some experiments, cells were treated with a DNase I inhibitor, 100 nM aurintricarboxylic acid. Uptake of plasmid DNA was measured by intracellular fluorescence of Cy3 and productive transfection efficiency was measured by intracellular fluorescence of GFP. RESULTS: HTM cells enter S-phase between 18 and 20 h after synchronization. Plasmid DNA reached the cytosolic compartment in 95% of transfected cells, regardless of synchronization. Synchronization dramatically increased productive transfection efficiency in HTM cells, from 3.0 to 9.0%. DNase I inhibition increased productive transfection efficiency of HTM cells two fold. CONCLUSIONS: Cultured HTM cells have a lower transfection efficiency than other primary ocular cell cultures, likely due partially to cytoplasmic digestion of DNA. We suggest that the difficulties in transfecting cultured HTM cells may be related to the filter function of the cells in vivo where the cells must degrade exogenous DNA.

Adult↗

PubMed2004

Longitudinal decline in lung function: evaluation of interleukin-10 genetic polymorphisms in firefighters.

During annual medical monitoring, some firefighters are found to have rates of decline in forced expiratory volume in one second (FEV1) far exceeding their peers. Interleukin-10 (IL-10) suppresses inflammation, and single nucleotide polymorphisms (SNPs) in the IL-10 gene may confer variable susceptibility to more rapid decline in lung function. In 1204 firefighters with at least six annual FEV1 measurements, increased age and greater initial FEV1 were associated with more rapid decline in lung function. DNA collected from 379 of these firefighters was screened for IL-10 SNPs at -1117, -854, 919, 1668, and 1812. A statistically significant difference in decline in lung function was found based on genotyping at the 1668 SNP. Evaluation of gene polymorphisms regulating lung inflammation may help to explain some of the variation in rate of decline in lung function in firefighters.

Adult↗

PubMed2004

Selenium's effects on MMP-2 and TIMP-1 secretion by human trabecular meshwork cells.

PURPOSE: Because of the observed increase in incidence of glaucoma among some individuals taking selenium as a dietary supplement, the present study was undertaken to investigate mechanisms of selenium-induced changes in homeostasis of human trabecular meshwork (HTM) cells. Specifically, the impact of selenium on matrix metalloproteinases (MMPs), their inhibitors (tissue inhibitors of metalloproteinases; TIMPs), and the second messengers that regulate MMP expression was investigated in an HTM cell culture model. METHODS: HTM cell cultures were treated with an organic selenium compound (methyl seleninic acid), and changes in secretion and activity of MMPs and TIMPs were analyzed by Western blot and zymography. Changes in extracellular-signal-related kinases 1 and 2 (ERK1/2) and phospho-ERK1/2 levels were monitored by Western blot analysis of whole-cell lysates prepared from selenium-treated cells. Photographs of cultures over time were used to document selenium-induced changes in cell morphology. RESULTS: Treatment of HTM cells with selenium for 24 hours at doses ranging from 1 to 10 micro M caused a dose-dependent decrease in the secretion of MMP-2 and TIMP-1. Treatment for 6 hours revealed a significant decrease in MMP-2 and TIMP-1 at the highest dose. MMP-1, -3, and -9 and TIMP-2 were either not detected or their secretion was not consistently influenced by selenium treatment. Selenium treatment caused a significant decrease in ERK1/2 phosphorylation, but no change in overall ERK protein levels. Selenium treatment resulted in dose-dependent, reversible changes in HTM cell-matrix associations. CONCLUSIONS: Selenium-induced changes in MMP-2/TIMP-1 secretion may alter the balance of extracellular matrix turnover in the conventional outflow pathway and cause an increase in intraocular pressure that eventually leads to glaucoma.

Blotting, Western↗