Characterization of mouse spleen cells by subtractive proteomics.

Major analytical challenges encountered by shotgun proteome analysis include both the diversity and dynamic range of protein expression. Often new instrumentation can provide breakthroughs in areas where other analytical improvements have not been successful. In the current study, we utilized new instrumentation (LTQ FT) to characterize complex protein samples by shotgun proteomics. Proteomic analyses were performed on murine spleen tissue separated by magnetic beads into distinct CD45- and CD45+ cell populations. Using shotgun protein analysis we identified approximately 2,000 proteins per cell group by over 12,000 peptides with mass deviations of less than 4.5 ppm. Datasets obtained by LTQ FT analysis provided a significant increase in the number of proteins identified and greater confidence in those identifications and improved reproducibility in replicate analyses. Because CD45- and not CD45+ cells are able to regenerate functional pancreatic islet cells in a mouse model of type I diabetes, protein expression was further compared by a subtractive proteomic approach in search of an exclusive protein expression profile in CD45- cells. Characterization of the proteins exclusively identified in CD45- cells was performed using gene ontology terms via the Javascript GoMiner. The CD45- cell subset readily revealed proteins involved in development, suggesting the persistence of a fetal stem cell in an adult animal.

Nuclear factor kappaB activity determines the sensitivity of kidney epithelial cells to apoptosis: implications for mercury-induced renal failure.

Nuclear factor kappa B (NF-kappaB) is a thiol-dependent transcriptional factor that promotes cell survival and protects cells from apoptotic stimuli. Numerous studies have demonstrated increased sensitivity to apoptosis associated with inhibition of NF-kappaB activation in various cell types. We have previously demonstrated that mercuric ion (Hg(2+)), one of the strongest thiol-binding agents known, impairs NF-kappaB activation and DNA binding at low microM concentrations in kidney epithelial cells. In the present studies we investigated the hypothesis that inhibition of NF-kappaB activation by Hg(2+) and other selective NF-kappaB inhibitors would increase the sensitivity of kidney epithelial (NRK52E) cells to apoptogenic agents to which these cells are normally resistant. Fewer than 10% of untreated cells in culture were found to be apoptotic when evaluated by DNA fragmentation (TUNEL) assay. Treatment of cells with Hg(2+) in concentrations up to 5 microM or with tumor necrosis factor-alpha (TNF) (300 units/ml) did not significantly increase the proportion of apoptotic cells, compared with untreated controls. However, when TNF was given following Hg(2+) pretreatment (0.5 to 5 microM for 30 min), the proportion of cells undergoing apoptosis increased by 2- to 6-fold over that seen in untreated controls. Kidney cells pretreated with specific NF-kappaB inhibitors (Bay11-7082 or SN50) prior to TNF also showed a significant increase in apoptosis. Increased sensitivity to apoptotic cell death following these treatments was significantly attenuated in cells transfected with a p65 expression vector. In studies in vivo, rats pretreated by intraperitoneal injection with Hg(2+) (0.75 mg/kg) 18 h prior to administration of bacterial lipopolysaccharide (LPS) (10 mg/kg) displayed impaired NF-kappaB activation and an increased mitochondrial cytochrome c release in kidney cortical cells. These findings are consistent with the view that prevention of NF-kappaB activity in vitro or in vivo enhances the sensitivity of kidney cells to apoptotic stimuli to which these cells are otherwise resistant. Since apoptosis is known to play a seminal role in the pathogenesis of renal failure caused by toxicant injury to tubular cells, the present findings suggest that inhibition of NF-kappaB activity may define a molecular mechanism underlying the pathogenesis of Hg(2+) toxicity in kidney cells.

Attenuation of nuclear factor kappa B (NF-kappaB) promotes apoptosis of kidney epithelial cells: a potential mechanism of mercury-induced nephrotoxicity.

Nuclear factor kappa B (NF-kappaB), a pleiotropic transcriptional factor that promotes cell survival and protects cells from apoptosis, requires reduced thiols at critical steps in its activation pathway. Mercuric ion (Hg(2+)), one of the strongest thiol-binding agents known, impairs NF-kappaB activation and transcriptional activity in normal rat kidney epithelial (NRK52E) cells at concentrations as low as 0.5 microM by binding to specific reduced thiol moieties in the NF-kappaB activation pathway. We hypothesized that prevention of NF-kappaB activation by Hg(2+) will increase the sensitivity of kidney cells to the apoptosis-inducing effects of other toxicants to which these cells are otherwise resistant by virtue of their NF-kappaB-activating capacity. Fewer than 5% of untreated kidney cells in culture (70-90% confluent) were found to be apoptotic when evaluated by DNA fragmentation (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling) or flow cytometric DNA profile analyses. Hg(2+) (5 microM) treatment for 24 hr increased this proportion by 1.5- to 2-fold. Neither lipopolysaccharide (LPS) (1 microg/mL) nor tumor necrosis factor-alpha (TNF-alpha; 300 U/mL), both potent activators of NF-kappaB in kidney cells, significantly altered the proportion of apoptotic cells, compared with untreated controls, when applied without Hg(2+) pretreatment. However, when LPS or TNF-alpha was administered after Hg(2+) pretreatment (5 microM for 30 min), the proportion of cells undergoing apoptosis 22 hr later increased by 4- to 6-fold compared with untreated controls. In contrast, Hg(2+) pretreatment did not increase the amount of apoptosis caused by apoptosis-inducing agents that do not activate NF-kappaB (staurosporine, Fas ligand). These findings suggest that Hg(2+) enhances the sensitivity of kidney cells to apoptotic stimuli as a consequence of inhibition of NF-kappaB activity. Because apoptosis is known to play a key role in the pathogenesis of renal failure resulting from toxicant injury to proximal tubular cells, promotion of apoptosis via inhibition of NF-kappaB activity may define a novel molecular mechanism by which Hg(2+) toxicity is initiated in kidney cells.