Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-⌬EEVD) or ATPase function (hsp70-⌬ATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury. In contrast, mutants lacking either the chaperone function (hsp70-⌬EEVD) or the ATP hydrolytic domain (hsp70-⌬ATPase) failed to prevent mitochondrial AIF release. Although hsp70-⌬EEVD did not inhibit bax activation or mitochondrial membrane injury after cell stress, this hsp70 mutant co-immunoprecipitated with leaked AIF in injured cells and decreased nuclear AIF accumulation. In contrast, hsp70-⌬ATPase did not interact with AIF either in intact cells or in a cell-free system and furthermore, failed to prevent nuclear AIF accumulation. These results demonstrate that mitochondrial protection against bax-mediated injury requires both intact chaperone and ATPase functions, whereas the ATPase domain is critical for sequestering AIF in the cytosol.Disruption of the outer mitochondrial membrane releases toxic proteins, including cytochrome c and apoptosis-inducing factor (AIF) 3 that activate caspase-dependent and -independent pathways responsible for apoptotic cell death. In a prior study in our laboratory, neither a pancaspase inhibitor nor a specific caspase 3 inhibitor completely prevented apoptosis in renal cells exposed to metabolic inhibitors (1), suggesting that at least a portion of the observed apoptosis is mediated by non-caspase-dependent factors including AIF. Nuclear translocation of AIF activates endogenous endonucleases leading to degradation of native DNA into 50-kilobase fragments, peripheral chromatin condensation, and nuclear shrinkage (2-4) that is sufficient to cause apoptosis (5, 6). AIF mediates apoptosis in multiple cell lines after diverse insults including ischemia (7-10), oxidant stress (11), ethanol (12), or p53 exposure (8).AIF is synthesized in the cytosol as a 67-kDa precursor protein that contains a mitochondrial-localizing sequence (13,14). After being imported into mitochondria, the mitochondrial-localizing sequence is cleaved, resulting in the accumulation of the mature, 57-kDa AIF protein (13). Under normal circumstances AIF may facilitate electron transport, since it exhibits robust oxidoreductase activity (14). AIF-mediated cell death is a two-step process that involves initial release from the intramembranous mitochondrial space into the cytosol followed by nuclear uptake and accumulation (5,13,15,16).hsp70, an inducible cytoprotectant protein, antagonizes apoptosis by interfering with multiple ch...
Silica impregnated polymer monolithic columns may provide a simple method for lysing and extracting DNA from bacteria inside of microfluidic chips. Here we use Escherichia coli as a test organism for a point of care thermoplastic microfluidic module designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis and solid phase extraction of nucleic acids from the sample. To demonstrate proof-of-concept, we doped human hematuric urine samples with E. coli at concentrations ranging from 101–105 colony-forming units/mL (CFU/mL) to simulate patient samples. We then performed on-chip lysis and DNA extraction. The bacterial DNA was amplified using real-time PCR demonstrating lysis and isolation down to 101 CFU/mL. Results were comparable to a commercial kit at higher concen trations and performed better at recovering DNA at lower concentrations.
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