Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.

We constructed a bioprocess environment enabling automatic sampling from a bioreactor combined with a compact on-line high performance liquid chromatography (HPLC) unit. This setup allowed us to measure extracellular glucose, ethanol, glycerol, and acetate concentrations automatically at 5 min intervals during the cultivation. This environment also provides mechanical measurement of the optical density (OD) of cells and enables us to collect and store (−35 o C) samples for further off-line analyses. Among the available devices, the performance of the sampling-analysis unit is by far the best with regard to speed and number of analytes. Both the sampling and analysis phases are easily controlled by software; thus, providing a unique environment to perform various bioprocess activity tasks, whether they would be cell line screening or optimisation of conditions for growth and productivity. Complex research set-ups can be created and continuous automated measurements empower long-term cultivations with a time series. We provide evidence for the applicability of this environment by performing three comparable batch cultivations with Saccharomyces cerevisiae yeast and show that both the on-line sampling and analysis modes produce reliable data for further use in the monitoring and controlling of bioprocesses. On-line data provided new insight into the dynamics of the diauxic shift during aerobic glucose batch cultivation. When cell growth and carbon dioxide production ceased for the first time during the diauxic shift, acetate accumulation and consumption of the remaining glucose below 0.15 g/L continued to occur for 1 h. At the same time, glycerol and ethanol began to be consumed. Samples were also collected during cultivation for later analysis of intracellular metabolites and to collect more valuable information about metabolism.