Synthesis and apoenzyme attachment of lipoic acid have emerged as a new complex metabolic pathway. Mutations in several genes involved in the lipoic acid de novo pathway have recently been described (i.e., LIAS, NFU1, BOLA3, IBA57), but no mutation was found so far in genes involved in the specific process of attachment of lipoic acid to apoenzymes pyruvate dehydrogenase (PDHc), α-ketoglutarate dehydrogenase (α-KGDHc) and branched chain α-keto acid dehydrogenase (BCKDHc) complexes.

Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and are associated with a wide range of clinical symptoms. We report a subject, born to consanguineous parents, with growth retardation and neurological deterioration. Multiple respiratory chain deficiency was found in muscle and fibroblasts of the subject as well as abnormal assembly of complexes I and IV. A microsatellite genotyping of the family members detected only one region of homozygosity on chromosome 17q24.2-q25.3 in which we focused our attention to genes involved in mitochondrial translation. We sequenced MRPL12, encoding the mitochondrial ribosomal protein L12 and identified a c.542C>T transition in exon 5 changing a highly conserved alanine into a valine (p.Ala181Val). This mutation resulted in a decreased steady-state level of MRPL12 protein, with altered integration into the large ribosomal subunit. Moreover, an overall mitochondrial translation defect was observed in the subject's fibroblasts with a significant reduction of synthesis of COXI, COXII and COXIII subunits. Modeling of MRPL12 shows Ala181 positioned in a helix potentially involved in an interface of interaction suggesting that the p.Ala181Val change might be predicted to alter interactions with the elongation factors. These results contrast with the eubacterial orthologues of human MRPL12, where L7/L12 proteins do not appear to have a selective effect on translation. Therefore, analysis of the mutated version found in the subject presented here suggests that the mammalian protein does not function in an entirely analogous manner to the eubacterial L7/L12 equivalent.

Mitochondria are the source of internal heat which influences all cellular processes. Hence, monitoring mitochondrial temperature provides a unique insight into cell physiology. Using a thermosensitive fluorescent probe MitoThermo Yellow (MTY), we have shown recently that mitochondria within human cells are maintained at close to 50 °C when active, increasing their temperature locally by about 10 °C. Initially reported in the HEK293 cell line, we confirmed this finding in the HeLa cell line. Delving deeper, using MTY and MTX (MitoThermo X), a modified version of MTY, we unraveled some caveats related to the nature of these charged fluorophores. While enabling the assessment of mitochondrial temperature in HEK and HeLa cell lines, the reactivity of MTY to membrane potential variations in human primary skin fibroblasts precluded local temperature monitoring in these cells. Chemical modification of MTY into MTX did not result in a temperature probe unresponsive to membrane potential variations that could be universally used in any cell type to determine mitochondrial temperature. Thus, the cell-type dependence of MTY in measuring mitochondrial temperature, which is likely due to the variable binding of this dye to specific internal mitochondrial components, should imply cautiousness while using these nanothermometers for mitochondrial temperature analysis.

UDP-glucose (UDP-Glc) is synthesized by UGP2-encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism. The UGP-deficient cells display < 5% control levels of UDP-Glc/UDP-Gal and > 100-fold reduction of [6-³H]galactose incorporation into UDP-[6-³H]galactose, as well as multiple deficits in glycoconjugate biosynthesis. Cultivation of these cells in the presence of galactose leads to partial restoration of UDP-Glc levels, galactose metabolism and glycoconjugate biosynthesis. The V_max for recombinant human UGP(G116D) with Glc1P is 2000-fold less than that of the wild-type protein, and UGP(G116D) displayed a mildly elevated K_m for Glc1P, but no activity of the mutant enzyme towards Gal1P was detectable. To conclude, although the mechanism behind UDP-Glc/Gal production in the UGP-deficient cells remains to be determined, the capacity of this cell line to change its glycosylation status as a function of extracellular galactose makes it a useful, reversible model with which to study different aspects of galactose metabolism and glycoconjugate biosynthesis.

In the last ten years, the use of fluorescent probes developed to measure oxygen has resulted in several marketed devices, some unreasonably expensive and with little flexibility. We have explored the use of the effective, versatile, and inexpensive Redflash technology to determine oxygen uptake by a number of different biological samples using various layouts. This technology relies on the use of an optic fiber equipped at its tip with a membrane coated with a fluorescent dye (www.pyro-science.com). This oxygen-sensitive dye uses red light excitation and lifetime detection in the near infrared. So far, the use of this technology has mostly been used to determine oxygen concentration in open spaces for environmental studies, especially in aquatic media. The oxygen uptake determined by the device can be easily assessed in small volumes of respiration medium and combined with the measurement of additional parameters, such as lactate excretion by intact cells or the membrane potential of purified mitochondria. We conclude that the performance of by this technology should make it a first choice in the context of both fundamental studies and investigations for respiratory chain deficiencies in human samples.