We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability.

The chemical identification of mass spectrometric signals in metabolomic applications is important to provide conversion of analytical data to biological knowledge about metabolic pathways. The complexity of electrospray mass spectrometric data acquired from a range of samples (serum, urine, yeast intracellular extracts, yeast metabolic footprints, placental tissue metabolic footprints) has been investigated and has defined the frequency of different ion types routinely detected. Although some ion types were expected (protonated and deprotonated peaks, isotope peaks, multiply charged peaks) others were not expected (sodium formate adduct ions). In parallel, the Manchester Metabolomics Database (MMD) has been constructed with data from genome scale metabolic reconstructions, HMDB, KEGG, Lipid Maps, BioCyc and DrugBank to provide knowledge on 42,687 endogenous and exogenous metabolite species. The combination of accurate mass data for a large collection of metabolites, theoretical isotope abundance data and knowledge of the different ion types detected provided a greater number of electrospray mass spectrometric signals which were putatively identified and with greater confidence in the samples studied. To provide definitive identification metabolite-specific mass spectral libraries for UPLC-MS and GC-MS have been constructed for 1,065 commercially available authentic standards. The MMD data are available at http://dbkgroup.org/MMD/.

Hypoxia is a key determinant of tissue pathology during tumor development and organ ischemia. However, little is known regarding hypoxic regulation of genes that are directly involved in cell death or death resistance. Here we report the striking induction by severe hypoxia of the anti-apoptotic protein IAP-2. Hypoxic cells with IAP-2 up-regulation became resistant to apoptosis. IAP-2 was induced by hypoxia per se rather than by the secondary effects of hypoxia, including ATP depletion and cell injury. The inductive response did not relate to alterations of cellular redox status or arrest of mitochondrial respiration. On the other hand, IAP-2 induction was attenuated by actinomycin D, suggesting a role for gene transcription. In vitro nuclear run-on assays demonstrated specific increases in IAP-2 transcriptional activity after hypoxia exposure. HIF-1, the primary transcription factor that is responsible for multiple gene activation under hypoxia, does not have a role in IAP-2 expression. HIF-1 and IAP-2 were induced by different degrees of hypoxia; severe hypoxia or anoxia was required for IAP-2 induction. Moreover, cobalt chloride and desferrioxamine activated HIF-1 but not IAP-2. Finally, IAP-2 was induced by severe hypoxia in mouse embryonic stem cells that were deficient of HIF-1. Thus, this study not only provides the first demonstration of hypoxic regulation of an anti-apoptotic gene but also suggests the participation of novel hypoxia-responsive transcription mechanisms.

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Glomerular permeability to horseradish peroxidase, a protein slightly smaller than rat albumin but similar in shape, was studied in Wistar-Furth rats by using a purified neutral isozyme (HRP; molecular radius, a(e) = 29.8 A) as well as an anionic succinyl-derivative (sHRP a(e) = 31.8 A) and a cationized enzyme (cHRP, a(e) = 30 A). The clearance rate of the proteins was determined over a 20-min period using the amounts of enzyme actually filtered (i.e., protein in the urine and protein reabsorbed by tubules). Fractional clearance of cationic HRP (0.338 +/- 0.019) exceeded that of neutral HRP (0.061 +/- 0.005) by a factor of 5.5 and that of anionic HRP (0.007 +/- 0.000) by a factor of 48. Tubular reabsorption was less than 10% of the filtered load. The experimental results indicate marked charge dependency of the filtration of proteins across the glomerulus. Fractional clearances for these proteins are significantly lower than those reported in the literature for dextrans of similar molecular radii. Other molecular properties such as shape and deformability may explain these differences.