Sandeep Raha

Mitochondria, oxygen free radicals, disease and ageing

Superoxide is generated by the mitochondrial respiratory chain. The transformation of this superoxide into hydrogen peroxide and, under certain conditions, then into hydroxyl radicals is important in diseases where respiratory chain function is abnormal or where superoxide dismutase function is altered, as in amyotrophic lateral sclerosis. In addition, these reactive oxygen species can influence the ageing process through mechanisms involving mutagenesis of mtDNA or increased rates of shortening of telomeric DNA.

Simultaneous isolation of total cellular RNA and DNA from tissue culture cells using phenol and lithium chloride

A rapid procedure for the isolation of intact total cellular RNA from cultured cells is described. This method combines the simultaneous disruption of cells and extraction of nucleic acids in a single step with the use of phenol and a buffer containing 100 mM LiCl. Total cellular RNA can be isolated in approximately 2 hours. The yield and quality of the RNA is comparable to the more widely employed methods requiring extensive preparatory steps such as extraction using guanidinium thiocyanate and subsequent CsCl gradient centrifugation. The RNA isolated using our procedure contains transcripts up to 10 kb in length and is suitable for Northern analysis. This procedure also yields high-molecular-weight DNA, which is a suitable substrate for restriction endonucleases.

Repopulation of ?0 cells with mitochondria from a patient with a mitochondrial DNA point mutation in tRNAGly results in respiratory chain dysfunction

Familial hypertrophic ventricular cardiomyopathy has been demonstrated to be associated with a number of mitochondrial DNA (mtDNA) mutations. A fibroblast cell line carrying a mutation in its mtDNA at position 9997 in the gene encoding tRNA glycine was obtained from a patient with hypertrophic cardiomyopathy. To demonstrate that the etiology of this disease was a result of the mtDNA mutation, cybrid clones were constructed by fusion of enucleated patient skin fibroblasts to ρ0 osteosarcoma cells. Clones carrying high levels of mutant mtDNA showed predominantly cytochrome c oxidase and complex I deficiency, as well as an elevated lactate/pyruvate (L/P) ratio, a biochemical marker characteristic of respiratory chain deficiencies. Pulse‐labeling experiments demonstrated a strong negative correlation between the levels of newly synthesized mtDNA‐encoded polypeptides and glycine content. These data suggest that the T9997C mutation in mtDNA is causative of respiratory chain dysfunction when present at high levels of heteroplasmy. Hum Mutat 13:245–254, 1999.

Impairment of pyruvate dehydrogenase activity by acetaldehyde.

The facial features that are characteristic of fetal alcohol syndrome (FAS) are strikingly similar to those seen in pyruvate dehydrogenase (PDH) deficiency. Furthermore, alcohol-induced central nervous system insult results in midline anomalies such as agenesis of the corpus callosum, which has also been described in several metabolic diseases, including PDH deficiency. The purpose of this work was to examine the effect of acetaldehyde on PDH in vitro. The activity of PDH was measured in the presence of acetaldehyde (10 microM-1 mM) by measuring the formation of the reduced form of nicotinamide-adenine dinucleotide at 340 nm. Pyruvate dehydrogenase was separated by using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique after incubation with [1,2-(14)C]-acetaldehyde to detect the formation of covalent adducts autoradiographically. The effect of acetaldehyde on the phosphorylation of the complex was also determined autoradiographically after incubating of PDH with (32)P-adenosine triphosphate. The results of this study show that acetaldehyde impairs PDH activity by a mixed inhibition type mechanism (Kic=62.4+/-25.7 microM, Kiu=225+/-68 microM), which is not a result of the formation of covalent adducts with PDH, nor of a stimulation of phosphorylation or inactivation of the complex. Because PDH levels are low throughout development and that the competition between pyruvate and acetaldehyde may be enhanced due to ethanol-induced lowering of ambient pyruvate concentrations, we conclude that impairment of PDH may have a significant effect on the developing fetus.

Toward the development of optical nucleic acid biosensors based on TIRF and TCSPC for high sensitivity determinations

The results of preliminary investigations toward the design of an optical biosensor instrument for the selective and direct analysis of low copy numbers of target nucleic acids in native form are reported. A concept development prototype was constructed based on a total internal reflection fluorescence (TIRF) configuration and the use of time correlated single photon counting (TCSPC). Selective detection of interfacial hybrid formation was done by identification of luminescence of characteristic (20ns) lifetime from the intercalant fluorophore ethidium bromide associated with nucleic acid hybrids formed at the interaction surface of optical sensor elements. Results of these investigations suggest that detection limits on the order of 107 dye:dsDNA complexes can be achieved when an effective sensor interaction surface of 150 µm diameter is used. The presence of interfacial nucleic acid duplexes at a sensor surface was further verified by thermal denaturation studies. The sensitivity of this concept design prototype was found to be most limited by long lifetime fluorescence intrinsic to the detection optics in conjunction with large amounts of scatter dispersed from the sensor cartridge. Future directions for continued device development are discussed.