Slowly progressive encephalopathy with hearing loss due to a mutation in the mtDNA tRNA(Leu(CUN)) gene.

Pathogenic mutations in the tRNA(Leu(UCN)) gene of mitochondrial DNA (mtDNA) have been invariably accompanied by skeletal myopathy with or without chronic progressive external ophthalmoplegia (CPEO). We report a young woman with a heteroplasmic m.12276G>A mutation in tRNA(Leu(UCN)), who had childhood-onset and slowly progressive encephalopathy with ataxia, cognitive impairment, and hearing loss. Sequencing of the 22 tRNA mitochondrial genes is indicated in all unusual neurological syndromes, even in the absence of maternal inheritance.

Longitudinal changes of mtDNA A3243G mutation load and level of functioning in MELAS.

Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), one of the most common mitochondrial multisystemic diseases, is most commonly associated with an A-to-G transition at nucleotide position 3243 (A3243G) in mitochondrial DNA. We studied 34 individuals harboring the A3243G mutation for up to 7 years; 17 had the full MELAS phenotype and 17 who were classified as "carrier relatives" because they were either asymptomatic or had some symptoms suggestive of mitochondrial disease but no seizures or strokes. Using the sensitive real-time polymerase chain reaction to quantify the A3243G mutation, we confirmed that the percent mutation decreases progressively in DNA isolated from blood: the average percent decrease was 0.5% per year for fully symptomatic patients and 0.2% per year for oligosymptomatic carrier relatives. We also correlated mutant loads with functional status estimated by the Karnofksky score: even though the mutation load decreases, the level of functioning worsens in fully symptomatic patients, whereas the level of functioning of carrier relatives remains largely unchanged. This study suggests that A3243G mutant load in DNA isolated from blood is neither useful for prognosis nor for functional assessment.

Oxidative stress biomarkers in sporadic ALS.

We aimed to investigate oxidative stress biomarkers in a cross-sectional pilot study of 50 participants with sporadic ALS (SALS) compared to 46 control subjects. We measured urinary 8-oxodeoxyguanosine (8-oxodG), urinary 15-F(2t)-isoprostane (IsoP), and plasma protein carbonyl by ELISA methods. We also determined if ELISA measurement of 8-oxodG could be validated against measures from high-pressure liquid chromatography coupled with electrochemical detection, the current standard method. We found that 8-oxodG and IsoP levels adjusted for creatinine were significantly elevated in SALS participants. These differences persisted after age and gender were controlled in regression analyses. These markers are highly and positively correlated with each other. 8-oxodG measured by the two techniques from the same urine sample were positively correlated (p<.0001). Protein carbonyl was not different between SALS participants and controls. In conclusion, using ELISA, we confirmed that certain oxidative stress biomarkers were elevated in SALS participants. ELISA may be reliable and thus useful in epidemiology studies requiring large numbers of samples to determine the significance of increased oxidative stress markers in SALS. Further studies are required.

Identification of a novel D109Y mutation in Cu/Zn superoxide dismutase (sod1) gene associated with amyotrophic lateral sclerosis.

We report a novel missense mutation (Asp109Tyr) in exon 4 of the Cu/Zn superoxide dismutase (sod1) gene in a woman with apparently sporadic amyotrophic lateral sclerosis (SALS). Signs of motor deficit appeared at the age of 51 years which progressed over the next 6 years to upper and lower motor neuron disease and death occurred by the age of 57 years. In this mutation, the base change of guanine to thymine at codon 109 of sod1 gene leads to the replacement of aspartic by tyrosine in the protein. This amino acid change in the protein however, did not alter the catalytic activity of the SOD1 enzyme as there was no change in the enzymatic activity of purified SOD1 from the patient's erythrocytes compared to control.

Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF.

The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current-voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by approximately 60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.

Cadaverine inhibition of porin plays a role in cell survival at acidic pH.

When grown at acidic pH, Escherichia coli cells secrete cadaverine, a polyamine known to inhibit porin-mediated outer membrane permeability. In order to understand the physiological significance of cadaverine excretion and the inhibition of porins, we isolated an OmpC mutant that showed resistance to spermine during growth and polyamine-resistant porin-mediated fluxes. Here, we show that the addition of exogenous cadaverine allows wild-type cells to survive a 30-min exposure to pH 3.6 better than cells expressing the cadaverine-insensitive OmpC porin. Competition experiments between strains expressing either wild-type or mutant OmpC showed that the lack of sensitivity of the porin to cadaverine confers a survival disadvantage to the mutant cells at reduced pH. On the basis of these results, we propose that the inhibition of porins by excreted cadaverine represents a novel mechanism that provides bacterial cells with the ability to survive acid stress.