Zinc Finger Nuclease mediated knockout of ADP dependent Glucokinase in Cancer cell lines: Effects on cell survival and Mitochondrial Oxidative Metabolism

<div><p>Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of <i>ADPGK</i>, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of <i>ADPGK,</i> and were ADPGK-null by immunoblotting. <i>ADPGK</i> knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when <i>ADPGK</i> was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of <i>ADPGK</i> in HCT116 cells caused few changes in global gene expression, knockout of <i>ADPGK</i> in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the <i>ADPGK</i> knockouts, in some cases markedly so. Collectively, the results demonstrate that <i>ADPGK</i> can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of <i>ADPGK</i> is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.</p></div

(Per)chlorate reduction by an acetogenic bacterium, Sporomusa sp., isolated from an underground gas storage

A mesophilic bacterium, strain An4, was isolated from an underground gas storage reservoir with methanol as substrate and perchlorate as electron acceptor. Cells were Gram-negative, spore-forming, straight to curved rods, 0.5–0.8 μm in diameter, and 2–8 μm in length, growing as single cells or in pairs. The cells grew optimally at 37°C, and the pH optimum was around 7. Strain An4 converted various alcohols, organic acids, fructose, acetoin, and H2/CO2 to acetate, usually as the only product. Succinate was decarboxylated to propionate. The isolate was able to respire with (per)chlorate, nitrate, and CO2. The G+C content of the DNA was 42.6 mol%. Based on the 16S rRNA gene sequence analysis, strain An4 was most closely related to Sporomusa ovata (98% similarity). The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell-free extracts

TECHNETIUM-99M-MAG3 CLEARANCE AS A PARAMETER OF EFFECTIVE RENAL PLASMA-FLOW IN PATIENTS WITH PROTEINURIA AND LOWERED SERUM-ALBUMIN LEVELS

Although the renal clearance of Tc-99m-MAG3 is about 60% of the I-131-hippurate clearance, Tc-99m-MAG3 clearance may be useful to estimate ERPF. In one study, however, proteinuria seemed to influence the MAG3/hippurate clearance ratio. In order to establish whether proteinuria or serum albumin level has influence on this ratio, a comparison was made between Tc-99m-MAG3 clearance and I-131-hippurate clearance in 14 patients. There was a good linear correlation between MAG3 and hippurate clearance, although the standard error of estimate of ERPF from MAG3 was relatively large, which remained unexplained. No correlation was found between proteinuria and MAG3/hippurate clearance ratio nor between serum albumin level, GFR, FF, ERPF and the MAG3/hippurate clearance ratio. We therefore conclude that there is no correlation between proteinuria and albumin level and the MAG3/hippurate ratio. A reasonable estimation of ERPF with MAG3 can be made in patients with proteinuria and lowered serum albumin levels although the estimation may be less accurate

COMPARISON OF COMPUTED-TOMOGRAPHY AND CO-57 BLEOMYCIN SCINTIGRAPHY IN STAGING THE MEDIASTINAL LYMPH-NODES OF PATIENTS WITH NON-SMALL-CELL LUNG-CANCER

The value of computed tomography (CT) and of Co-57-bleomycin scintigraphy (Co-57-BLM) in staging the mediastinal lymph nodes was compared in 28 patients suffering from non-small-cell lung cancer. The results were assessed against the pathological findings obtained during thoracotomy or mediastinoscopy. CT staging of the mediastinum had a sensitivity of 75%, a specificity of 80%, an accuracy of 79%, a positive predictive index of 60% and a negative predictive index of 89%. Co-57-BLM scintigraphic staging had a sensitivity of 43%, a specificity of 94%, an accuracy of 80%, a positive predictive index of 75% and a negative predictive index of 81%. In this small series these differences were not statistically significant; it thus appears that CT and Co-57-BLM are of equal value in staging the mediastinum. Mediastinoscopy is not contributory in case of a negative CT or Co-57-BLM. A positive CT or Co-57-BLM, however, indicates the need for histological verification of the mediastinal findings