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Mitchell D McCartney

Publications and source records attributed to Mitchell D McCartney.

4 recordsLinked to original sources
PubMed2006

Protein expression in a transformed trabecular meshwork cell line: proteome analysis.

PURPOSE: Characterization of the human trabecular meshwork (TM) proteome is hindered by the small mass of intact tissue and the slow growth of cultured cell strains. We have previously characterized a transformed TM cell strain (GTM3) that demonstrates many of the same protein expression and cell signaling systems of nontransformed cell strains. The aim of this study was to initiate a proteomic survey of GTM3 cells as the initial step toward characterization of the complete human TM proteome. METHODS: GTM3 cells were cultured to confluence, harvested and solubilized in urea/Nonidet. The protein extract (600 mug) was focused in immobilized isoelectric focusing (IEF) strips, separated by 10% SDS PAGE, and visualized with colloidal Coomassie Blue. Spots of interest were excised, destained, and the contained proteins subjected to in-gel reduction, derivatization, and tryptic digestion. Tryptic peptides were extracted and analyzed by electrospray LC/MS/MS. Protein identification was made using the TurboSequest search algorithm and a recent version of the nonredundant human protein database downloaded from the National Center for Biotechnology Information (NCBI). RESULTS: Eighty-seven (87) primary proteins and 93 variants of these proteins were identified. A website was created (TM proteome) that combines data such as graphic spot location within the gel, peptide sequence, apparent and calculated pI, apparent and calculated mass, percentage of coverage, and protein informatic website links. CONCLUSIONS: Proteomic analysis of a transformed human TM cell line has been initiated combining preparative two-dimensional PAGE separation, LC/MS/MS analysis of major proteins, and bioinformatic cataloging of the data. Further investigation of data from the transformed cell strain will be used in a comparative fashion for spot identification of analytical proteomic gels of human TM tissue and cultured normal cells. These initial data will form the base from which the characterization of protein expression in the normal and glaucomatous TM can be accomplished.

Cell Line, Transformed↗
PubMed2005

Freeze-fracture examination of cultured human trabecular meshwork cells: effect of dexamethasone.

Glucocorticoids can alter the trabecular meshwork in the aqueous outflow pathway of the eye leading to the development of ocular hypertension and glaucoma. Previous studies have shown biochemical and ultrastructural changes in cultured human trabecular meshwork (TM) cells treated with dexamethasone (DEX). In order to assess how the membranes of these treated cells were responding to this glucocorticoid, we compared DEX-treated and control TM cells using the freeze-fracture technique. Human TM cells were grown to confluence on either Millipore HA filter inserts or glass coverslips and then treated for 14 days with or without 0.1 microM dexamethasone. The cell cultures were then aldehyde fixed and prepared for electron microscopy and freeze-fracture. Junctional complexes in control and DEX-treated cells consisted of gap junctions of various shapes and sizes. No tight junctional complexes were apparent in either control or DEX-treated cells. The majority of vesicle fusion sites on the control cells appeared to be randomly distributed and were few in number. In contrast, DEX-treated cells had a significantly greater density of fusion sites, and the majority of the vesicles were aligned into linear arrays. These findings support our previous findings of increased secretory activity in DEX-treated cultured human TM cells. These in vitro model system data appear to correlate with previous in vivo biochemical, ultrastructural and freeze-fracture data.

Adolescent↗
PubMed2003

Aqueous outflow-enhancing effect of tert-butylhydroquinone: involvement of AP-1 activation and MMP-3 expression.

PURPOSE: To test the effect of stimulators of activator protein (AP)-1, on expression of stromelysin (MMP-3) in human TM cells and on aqueous outflow in perfused human anterior segments. METHODS: Change in MMP-3 expression was determined by immunoassay of proMMP-3 levels in the media of cultured human TM cells. Anterior segments of human donor eyes with or without glaucoma were perfused with vehicle or the AP-1 stimulator tert-butylhydroquinone (tBHQ). The outflow rates or intraocular pressure (IOP), and proMMP-3 levels in the perfusate were monitored. RESULTS: AP-1 stimulators, such as beta-naphthoflavone, 3-methylcholanthrene, and tBHQ, significantly upregulated (2-4-fold) TM cell expression of MMP-3. The stimulatory effect of tBHQ was concentration dependent, with an EC(50) of approximately 3 micro M, and was blocked by concomitant treatment with 100 nM SR11302, which sequesters AP-1. When nonglaucomatous human eyes were perfused with tBHQ (10 micro M), both outflow rates and perfusate proMMP-3 level increased significantly within the first 24 hours. The outflow effect of tBHQ was suppressed when SR11302 (100 nM) was added in the perfusate. tBHQ also lowered the IOP by more than 40% in perfused glaucomatous eyes. CONCLUSIONS: An AP-1 activator, tBHQ, upregulated expression of MMP-3 in cultured human TM cells and perfused human eyes and enhanced outflow ex vivo. These effects were blocked by sequestering AP-1, suggesting that activation of AP-1 can lead to increased MMP-3 production in the TM, which in turn improves outflow facility. This unique mechanism may provide a novel therapy for glaucoma.

Anterior Eye Segment↗