- Email: [email protected]
58 Barth syndrome – Mitochondrial phospholipid disorder
420
Abstracts / Mitochondrion 7 (2007) 404–433
the FFE-system indicated that the purification was selective on the organelle level documenting the suitability of this separation technique in proteomic research.
is not degraded in fibroblasts. Pathogenesis of MTS await for more research. doi:10.1016/j.mito.2007.08.060
doi:10.1016/j.mito.2007.08.058
55 Reactive oxygen species mediated regulation of mitochondrial biogenesis in the yeast Saccharomyces cerevisiae Cyrille Chevtzoff, Anne Galinier, Louis Casteilla, Bertrand DaignanFornier, Michel Rigoulet, Anne Devin * CNRS, Universite Victor Segalen Mitochondrial biogenesis is a complex process. It necessitates the participation of both the nuclear and the mitochondrial genomes. This process is highly regulated and mitochondrial content within a cell varies according to energy demand. In the yeast Saccharomyces cerevisiae, the cAMP pathway is involved in the regulation of mitochondrial biogenesis. An overactivation of this pathway leads to an increase in mitochondrial enzymatic content. Out of the three yeast cAMP protein kinases, we have previously shown that Tpk3p is the one involved in the regulation of mitochondrial biogenesis. In this paper, we investigated the molecular mechanisms that govern this process. We show that in the absence of Tpk3p, mitochondria produce large amounts of ROS that signal to the HAP2/ 3/4/5 nuclear transcription factors involved in mitochondrial biogenesis. We clearly establish that an increase in mitochondrial ROS production down-regulates mitochondrial biogenesis. It is the first time that a redox sensitivity of the transcription factors involved in yeast mitochondrial biogenesis is shown. Such a process could be seen as a mitochondria qualitycontrol process. doi:10.1016/j.mito.2007.08.059
56 A de novo nonsense mutation in the DDP1 gene in a patient with Mohr– Tranebjaerg syndrome: New insights into phenotype and molecular findings Jose´ R. Blesa, Paz Briones, Jesu´s A. Prieto-Ruiz, Francisco Coria, Jose´ Herna´ndez-Yago, Abelardo Solano * Centro de Investigacio´n Prı´ncipe Felipe, Valencia, Institut de Bioquı´mica Clı´nica, Barcelona, Spain; Hospital Son Dureta, Palma de Mallorca, Spain We describe a case of Mohr–Tranebjaerg syndrome (MTS) in a Spanish male with a de novo, new nonsense mutation in the DDP1/TIMM8A gene that truncates prematurely the 97-amino acid wild type protein, in residue number 37. Diagnosis of the syndrome can be difficult to achieve because of the difference of age at symptoms’ onset, and the fact that few kindreds have been described so far. Our patient, a 34-year-old man, was diagnosed with progressive sensorineural deafness at age 3 and progressive visual loss was noticed at age 15. At age 30 he developed sudden, repeated painful spasms on the neck, walking difficulties, and behavioral changes, including irritability and childish manners. Her mother is a noncarrier of the mutation. No deficiencies of the respiratory chain complexes I–IV were found in the proband’s muscle or fibroblasts, as has been informed for other MTS cases and in contrast to most mitochondrial hereditary and sporadic diseases. We performed molecular genetics studies of the TIMM8A gene, its partner TIMM13 and other TIMM genes of the intermembrane space as well as mitochondrial DNA. Different mitochondrial haplogroup backgrounds could be detected in our patient and other cases published with MTS. We also performed a comparative study of TIMM8A and TIMM13 mRNA expression in cultured fibroblasts of the patient and in control fibroblasts. Interestingly, TIMM8A mRNA with the truncating mutation
57 General anesthesia does not alter adenosine nucleotide levels of Caenorhabditis elegans Phil G. Morgan, Margaret M. Sedensky, Ernst-Bernhard Kayser * University Hospitals of Cleveland and CWRU, Cleveland, OH, USA Trying to elucidate the mechanism of action of volatile anesthetics, we investigate mutations in Caenorhabditis elegans which modulate the nematode’s sensitivity to these drugs. One of these mutants, gas-1(fc21), renders the worms hypersensitive to all volatile anesthetics. gas-1 encodes a subunit of mitochondrial complex I, the nematode ortholog of NDUFS2/IP49 kDa. In the mutant protein the evolutionary conserved arginine290 is replaced by lysine. In gas-1(fc21) mitochondria both NADH-quinone reductase activity and complex I – dependent state 3 respiration is decreased. Investigating other mutations and knocking down expression of subunits of the respiratory chain via RNA-interference we found an (inverse) linear correlation between complex I – dependent state 3 respiration and sensitivity to the volatile anesthetic halothane. Thus, the lower the worm’s capacity to aerobically regenerate ATP, the smaller the concentration of halothane needed to anesthetize it. We hypothesized that ATP levels determine whether a worm is ‘‘awake’’ or anesthetized. Consequently, suppression of ATP regeneration below demand leads to anesthesia. Suppression would result from the combined impact of genetically predetermined complex I (dys)function plus inhibition of ATP production by the anesthetic. A likely target for the anesthetic would be complex I, which has long been known as the most susceptible of all respiratory complexes. In order to test whether insufficient ATP could cause anesthesia, we extracted nucleotides from awake and halothane-anesthetized worms. After etheno-derivatization, adenosine nucleotides were separated by TBAHS-ion-pair reverse phase HPLC, and quantitated by the fluorescence of their etheno-adenosine moiety. Within a given worm strain, ATP concentrations as well as the energy charges were indistinguishable between awake and anesthetized nematodes disproving our hypothesis. However, both ATP concentration and energy charge were lower in gas-1(fc21) than in wildtype. Thus, in the mutant animal, complex I dysfunction cannot be fully compensated by complex I – independent ATP production. doi:10.1016/j.mito.2007.08.061
58 Barth syndrome – Mitochondrial phospholipid disorder Devrim Acehan, Michael Schalme, Fre´de´ric M. Vaz, Arnold W. Strauss, Zaza Khuchua * Vanderbilt University Medical Center, Amsterdam, The Netherlands; New York University School of Medicine, Amsterdam, The Netherlands; Academic Medical Center, Amsterdam, The Netherlands Barth syndrome is an X-linked genetic disorder caused by mutations in the tafazzin (Taz) gene and characterized by cardiomyopathy, skeletal myopathy, cyclic neutropenia, and organic aciduria. The Taz gene encodes a mitochondrial protein with a high degree of homology to acyl-transferases. Cardiolipin, a mitochondrial phospholipid, is deficient in fibroblasts and lymphocytes from Barth patients and Taz-deficient yeast. The cardiolipin from Barth patients exhibits excessive
Abstracts / Mitochondrion 7 (2007) 404–433 medium chain length and fully saturated side chains, while normal cardiolipin is highly symmetric and contains four linoleic acid (C18:2) side chains. To study the role of tafazzin in heart function and development, we created a knockdown zebrafish model. Zebrafish tafazzin mRNA is first evident at 7 h post-fertilization (hpf). At 10 and 24 hpf, tafazzin mRNA is ubiquitous, with highest levels in the head. By 51 hpf, expression becomes restricted in heart. The tafazzin knockdown created by antisense morpholino yolk injection resulted in dose-dependent lethality, severe developmental and growth retardation, marked bradycardia and pericardial effusions, and generalized edema, signs that resemble human Barth syndrome heart failure. This knockdown phenotype was rescued by concomitant injection of normal tafazzin mRNA. Abnormal cardiac development, with a linear, nonlooped heart, and hypomorphic tail and eye development proves that tafazzin is essential for overall zebrafish development, especially of the heart. We simultaneously pursued a tafazzin gene ablation in mouse embryonic stem cells. We generated a conditional allele at the Taz locus in mouse embryonic stem cells. Tafazzin-deficiency in ES cell – derived embryoid body myocytes caused reduction of the cardiolipin level and accumulation of lysocardiolipins. Taz-deficient cardiomyocytes demonstrate abnormal mitochondrial morphology and reduced mitochondrial membrane potential. The tafazzin knockdown zebrafish and mouse ES cells provides a model to study human Barth syndrome, analyze the severity of human mutations and to test potential treatments. doi:10.1016/j.mito.2007.08.062
59 Modulation of the W748S mutation in DNA polymerase gamma by the E1143G polymorphism in mitochondrial disorders Matthew J. Longley, William C. Copeland, Sherine S.L. Chan * NIEHS/NIH, Research Triangle Park, NC 27709, USA DNA polymerase gamma (pol gamma) is required for replication and repair of mitochondrial DNA. Over 80 mutations in POLG, the gene encoding the catalytic subunit of pol gamma, have been linked with disease. The W748S mutation in POLG is the most common mutation in ataxia-neuropathy spectrum disorders and is generally found in cis with the common E1143G polymorphism. It has been unclear whether E1143G participates in the disease process. We investigated the biochemical consequences of pol gamma proteins containing W748S or E1143G, or both. W748S pol gamma exhibited low DNA polymerase activity, low processivity and a severe DNAbinding defect. However, interactions between the catalytic and accessory subunits were normal. Despite the benefits derived from binding with the accessory subunit, catalytic activities did not reach wild-type (WT) levels. Also, nucleotide selectivity decreased 2.1-fold compared with WT. Surprisingly, pol gamma containing only E1143G was 1.4-fold more active than WT, and this increased polymerase activity could be due to higher thermal stability for E1143G pol gamma. The E1143G substitution partially rescued the deleterious effects of the W748S mutation, as DNA binding, catalytic activity and fidelity values were intermediate for W748S–E1143G. However, W748S– E1143G had a notably lower change in enthalpy for protein folding than W748S alone. We suggest that when E1143G is in cis with other pathogenic mutations, it can modulate the effects of these mutations. For W748S–E1143G pol gamma, the benefits bestowed by E1143G include increased DNA binding and polymerase activity; however, E1143G was somewhat detrimental to protein stability. doi:10.1016/j.mito.2007.08.063
421
60 Renal disease associated with coenzyme Q deficiency A.L. Lunceford a,*, R. Saiki c, J. Pachuski g, M. Kawamukai c, E. Polyak d, M.J. Falk e, D.L. Gasser b, C.F. Clarke f a Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA; b Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104, USA; c Department of Life Science and Biotechnology, Shimane University, Matsue, Japan; d Children’s Hospital of Philadelphia, Philadelphia, PA, USA; e Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA; f Pennsylvania Hospital, University of Pennsylvania, Philadelphia, PA, USA; g Department of Medical Sciences, University of Pennsylvania, Philadelphia, PA, USA Homozygous mice that carry the kidney disease allele of the Pdss2 locus (Pdss2kd/kd) develop interstitial nephritis and eventually die of end-stage renal disease. The Pdss2 enzyme localizes in mitochondria to generate the polyisoprenyl tail of coenzyme Q, which is an essential component of the respiratory chain and a potent lipid-soluble antioxidant. In mice, a heterotetramer composed of the Pdss1 and Pdss2 polypeptides is required to synthesize the nonaprenyl diphosphate tail precursor, solanesyl diphosphate. Young Pdss2kd/kd mice have normal Q9 levels when compared to age-matched wild-type mice. However, Pdss2kd/kd Q9 levels are significantly lower than age-matched wild-type mice by day of life 75. Although renal disease is the major disease phenotype, coenzyme Q9 deficiency is not isolated to the kidney. Indeed, polarography of freshly isolated mitochondria from the livers of Pdss2kd/kd mice shows significantly impaired complex I- and complex II-dependent OXPHOS capacity, with significantly increased complex IV-dependent OXPHOS capacity compared to age-matched wild-type controls, at least 4 months old. Co-expression of Pdss2 and Pdss1 in Escherichia coli results in the synthesis of Q9, a Q isoform distinct from the Q8 normally produced by E. coli. Co-expression of Pdss1 and Pdss2kd (harboring the V117M amino acid substitution mutation of the mutant allele), results in a decreased production of Q9. doi:10.1016/j.mito.2007.08.064
61 Long-term treatment of children with congenital lactic acidosis with dichloroacetate P.W. Stacpoole a,b,d,*, L.R. Gilbert a, R.E. Neiberger c, P.R. Carne c, D.W. Theriaque d, J.J. Shuster d a University of Florida College of Medicine; b Biochemistry and Molecular Biology; c Pediatrics; d General Clinical Research Center We report results on long-term administration of dichloroacetate (DCA) in 36 children with congenital lactic acidosis (CLA) who participated in the DCA/CLA Clinical Trial [Pediatrics 117, pp. 1519–1521, 2006]. This was a double-blind, randomized, controlled trial of oral DCA (12.5 mg/kg every 12 h). Data were analyzed for each patient from the time they began treatment through May 2005. Subject exposure to DCA totaled 110.42 years. Median height and weight increased over time, but the standardized values declined slightly and remained below the first percentile (Z = 2.33). There were no significant changes in serum levels of glucose. Beta-hydroxybutyrate, electrolytes or tests of renal or hepatic function, except for a 2% rise in total protein (p = 0.04). Venous pH, white blood count, hematocrit and platelet count did not change. Median 24 h urinary oxalate increased 22% (p = 0.003). Pre-treatment basal lactate levels were modestly elevated and increased further following carbohydrate meal challenge, but both basal (p < 0.001) and the meal-induced (p = 0.004) rise in lactate were blunted by DCA. The median pre-treatment cerebrospinal fluid lactate was 3.8 mmol/l and decreased over time with DCA exposure (p = 0.04). Serial nerve conduction measures were mostly unchanged; however, conduction velocity decreased and distal latency increased in peroneal nerves (both <0.001). Mean ± SE 3-year sur-
Abstracts / Mitochondrion 7 (2007) 404–433
the FFE-system indicated that the purification was selective on the organelle level documenting the suitability of this separation technique in proteomic research.
is not degraded in fibroblasts. Pathogenesis of MTS await for more research. doi:10.1016/j.mito.2007.08.060
doi:10.1016/j.mito.2007.08.058
55 Reactive oxygen species mediated regulation of mitochondrial biogenesis in the yeast Saccharomyces cerevisiae Cyrille Chevtzoff, Anne Galinier, Louis Casteilla, Bertrand DaignanFornier, Michel Rigoulet, Anne Devin * CNRS, Universite Victor Segalen Mitochondrial biogenesis is a complex process. It necessitates the participation of both the nuclear and the mitochondrial genomes. This process is highly regulated and mitochondrial content within a cell varies according to energy demand. In the yeast Saccharomyces cerevisiae, the cAMP pathway is involved in the regulation of mitochondrial biogenesis. An overactivation of this pathway leads to an increase in mitochondrial enzymatic content. Out of the three yeast cAMP protein kinases, we have previously shown that Tpk3p is the one involved in the regulation of mitochondrial biogenesis. In this paper, we investigated the molecular mechanisms that govern this process. We show that in the absence of Tpk3p, mitochondria produce large amounts of ROS that signal to the HAP2/ 3/4/5 nuclear transcription factors involved in mitochondrial biogenesis. We clearly establish that an increase in mitochondrial ROS production down-regulates mitochondrial biogenesis. It is the first time that a redox sensitivity of the transcription factors involved in yeast mitochondrial biogenesis is shown. Such a process could be seen as a mitochondria qualitycontrol process. doi:10.1016/j.mito.2007.08.059
56 A de novo nonsense mutation in the DDP1 gene in a patient with Mohr– Tranebjaerg syndrome: New insights into phenotype and molecular findings Jose´ R. Blesa, Paz Briones, Jesu´s A. Prieto-Ruiz, Francisco Coria, Jose´ Herna´ndez-Yago, Abelardo Solano * Centro de Investigacio´n Prı´ncipe Felipe, Valencia, Institut de Bioquı´mica Clı´nica, Barcelona, Spain; Hospital Son Dureta, Palma de Mallorca, Spain We describe a case of Mohr–Tranebjaerg syndrome (MTS) in a Spanish male with a de novo, new nonsense mutation in the DDP1/TIMM8A gene that truncates prematurely the 97-amino acid wild type protein, in residue number 37. Diagnosis of the syndrome can be difficult to achieve because of the difference of age at symptoms’ onset, and the fact that few kindreds have been described so far. Our patient, a 34-year-old man, was diagnosed with progressive sensorineural deafness at age 3 and progressive visual loss was noticed at age 15. At age 30 he developed sudden, repeated painful spasms on the neck, walking difficulties, and behavioral changes, including irritability and childish manners. Her mother is a noncarrier of the mutation. No deficiencies of the respiratory chain complexes I–IV were found in the proband’s muscle or fibroblasts, as has been informed for other MTS cases and in contrast to most mitochondrial hereditary and sporadic diseases. We performed molecular genetics studies of the TIMM8A gene, its partner TIMM13 and other TIMM genes of the intermembrane space as well as mitochondrial DNA. Different mitochondrial haplogroup backgrounds could be detected in our patient and other cases published with MTS. We also performed a comparative study of TIMM8A and TIMM13 mRNA expression in cultured fibroblasts of the patient and in control fibroblasts. Interestingly, TIMM8A mRNA with the truncating mutation
57 General anesthesia does not alter adenosine nucleotide levels of Caenorhabditis elegans Phil G. Morgan, Margaret M. Sedensky, Ernst-Bernhard Kayser * University Hospitals of Cleveland and CWRU, Cleveland, OH, USA Trying to elucidate the mechanism of action of volatile anesthetics, we investigate mutations in Caenorhabditis elegans which modulate the nematode’s sensitivity to these drugs. One of these mutants, gas-1(fc21), renders the worms hypersensitive to all volatile anesthetics. gas-1 encodes a subunit of mitochondrial complex I, the nematode ortholog of NDUFS2/IP49 kDa. In the mutant protein the evolutionary conserved arginine290 is replaced by lysine. In gas-1(fc21) mitochondria both NADH-quinone reductase activity and complex I – dependent state 3 respiration is decreased. Investigating other mutations and knocking down expression of subunits of the respiratory chain via RNA-interference we found an (inverse) linear correlation between complex I – dependent state 3 respiration and sensitivity to the volatile anesthetic halothane. Thus, the lower the worm’s capacity to aerobically regenerate ATP, the smaller the concentration of halothane needed to anesthetize it. We hypothesized that ATP levels determine whether a worm is ‘‘awake’’ or anesthetized. Consequently, suppression of ATP regeneration below demand leads to anesthesia. Suppression would result from the combined impact of genetically predetermined complex I (dys)function plus inhibition of ATP production by the anesthetic. A likely target for the anesthetic would be complex I, which has long been known as the most susceptible of all respiratory complexes. In order to test whether insufficient ATP could cause anesthesia, we extracted nucleotides from awake and halothane-anesthetized worms. After etheno-derivatization, adenosine nucleotides were separated by TBAHS-ion-pair reverse phase HPLC, and quantitated by the fluorescence of their etheno-adenosine moiety. Within a given worm strain, ATP concentrations as well as the energy charges were indistinguishable between awake and anesthetized nematodes disproving our hypothesis. However, both ATP concentration and energy charge were lower in gas-1(fc21) than in wildtype. Thus, in the mutant animal, complex I dysfunction cannot be fully compensated by complex I – independent ATP production. doi:10.1016/j.mito.2007.08.061
58 Barth syndrome – Mitochondrial phospholipid disorder Devrim Acehan, Michael Schalme, Fre´de´ric M. Vaz, Arnold W. Strauss, Zaza Khuchua * Vanderbilt University Medical Center, Amsterdam, The Netherlands; New York University School of Medicine, Amsterdam, The Netherlands; Academic Medical Center, Amsterdam, The Netherlands Barth syndrome is an X-linked genetic disorder caused by mutations in the tafazzin (Taz) gene and characterized by cardiomyopathy, skeletal myopathy, cyclic neutropenia, and organic aciduria. The Taz gene encodes a mitochondrial protein with a high degree of homology to acyl-transferases. Cardiolipin, a mitochondrial phospholipid, is deficient in fibroblasts and lymphocytes from Barth patients and Taz-deficient yeast. The cardiolipin from Barth patients exhibits excessive
Abstracts / Mitochondrion 7 (2007) 404–433 medium chain length and fully saturated side chains, while normal cardiolipin is highly symmetric and contains four linoleic acid (C18:2) side chains. To study the role of tafazzin in heart function and development, we created a knockdown zebrafish model. Zebrafish tafazzin mRNA is first evident at 7 h post-fertilization (hpf). At 10 and 24 hpf, tafazzin mRNA is ubiquitous, with highest levels in the head. By 51 hpf, expression becomes restricted in heart. The tafazzin knockdown created by antisense morpholino yolk injection resulted in dose-dependent lethality, severe developmental and growth retardation, marked bradycardia and pericardial effusions, and generalized edema, signs that resemble human Barth syndrome heart failure. This knockdown phenotype was rescued by concomitant injection of normal tafazzin mRNA. Abnormal cardiac development, with a linear, nonlooped heart, and hypomorphic tail and eye development proves that tafazzin is essential for overall zebrafish development, especially of the heart. We simultaneously pursued a tafazzin gene ablation in mouse embryonic stem cells. We generated a conditional allele at the Taz locus in mouse embryonic stem cells. Tafazzin-deficiency in ES cell – derived embryoid body myocytes caused reduction of the cardiolipin level and accumulation of lysocardiolipins. Taz-deficient cardiomyocytes demonstrate abnormal mitochondrial morphology and reduced mitochondrial membrane potential. The tafazzin knockdown zebrafish and mouse ES cells provides a model to study human Barth syndrome, analyze the severity of human mutations and to test potential treatments. doi:10.1016/j.mito.2007.08.062
59 Modulation of the W748S mutation in DNA polymerase gamma by the E1143G polymorphism in mitochondrial disorders Matthew J. Longley, William C. Copeland, Sherine S.L. Chan * NIEHS/NIH, Research Triangle Park, NC 27709, USA DNA polymerase gamma (pol gamma) is required for replication and repair of mitochondrial DNA. Over 80 mutations in POLG, the gene encoding the catalytic subunit of pol gamma, have been linked with disease. The W748S mutation in POLG is the most common mutation in ataxia-neuropathy spectrum disorders and is generally found in cis with the common E1143G polymorphism. It has been unclear whether E1143G participates in the disease process. We investigated the biochemical consequences of pol gamma proteins containing W748S or E1143G, or both. W748S pol gamma exhibited low DNA polymerase activity, low processivity and a severe DNAbinding defect. However, interactions between the catalytic and accessory subunits were normal. Despite the benefits derived from binding with the accessory subunit, catalytic activities did not reach wild-type (WT) levels. Also, nucleotide selectivity decreased 2.1-fold compared with WT. Surprisingly, pol gamma containing only E1143G was 1.4-fold more active than WT, and this increased polymerase activity could be due to higher thermal stability for E1143G pol gamma. The E1143G substitution partially rescued the deleterious effects of the W748S mutation, as DNA binding, catalytic activity and fidelity values were intermediate for W748S–E1143G. However, W748S– E1143G had a notably lower change in enthalpy for protein folding than W748S alone. We suggest that when E1143G is in cis with other pathogenic mutations, it can modulate the effects of these mutations. For W748S–E1143G pol gamma, the benefits bestowed by E1143G include increased DNA binding and polymerase activity; however, E1143G was somewhat detrimental to protein stability. doi:10.1016/j.mito.2007.08.063
421
60 Renal disease associated with coenzyme Q deficiency A.L. Lunceford a,*, R. Saiki c, J. Pachuski g, M. Kawamukai c, E. Polyak d, M.J. Falk e, D.L. Gasser b, C.F. Clarke f a Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA; b Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104, USA; c Department of Life Science and Biotechnology, Shimane University, Matsue, Japan; d Children’s Hospital of Philadelphia, Philadelphia, PA, USA; e Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA; f Pennsylvania Hospital, University of Pennsylvania, Philadelphia, PA, USA; g Department of Medical Sciences, University of Pennsylvania, Philadelphia, PA, USA Homozygous mice that carry the kidney disease allele of the Pdss2 locus (Pdss2kd/kd) develop interstitial nephritis and eventually die of end-stage renal disease. The Pdss2 enzyme localizes in mitochondria to generate the polyisoprenyl tail of coenzyme Q, which is an essential component of the respiratory chain and a potent lipid-soluble antioxidant. In mice, a heterotetramer composed of the Pdss1 and Pdss2 polypeptides is required to synthesize the nonaprenyl diphosphate tail precursor, solanesyl diphosphate. Young Pdss2kd/kd mice have normal Q9 levels when compared to age-matched wild-type mice. However, Pdss2kd/kd Q9 levels are significantly lower than age-matched wild-type mice by day of life 75. Although renal disease is the major disease phenotype, coenzyme Q9 deficiency is not isolated to the kidney. Indeed, polarography of freshly isolated mitochondria from the livers of Pdss2kd/kd mice shows significantly impaired complex I- and complex II-dependent OXPHOS capacity, with significantly increased complex IV-dependent OXPHOS capacity compared to age-matched wild-type controls, at least 4 months old. Co-expression of Pdss2 and Pdss1 in Escherichia coli results in the synthesis of Q9, a Q isoform distinct from the Q8 normally produced by E. coli. Co-expression of Pdss1 and Pdss2kd (harboring the V117M amino acid substitution mutation of the mutant allele), results in a decreased production of Q9. doi:10.1016/j.mito.2007.08.064
61 Long-term treatment of children with congenital lactic acidosis with dichloroacetate P.W. Stacpoole a,b,d,*, L.R. Gilbert a, R.E. Neiberger c, P.R. Carne c, D.W. Theriaque d, J.J. Shuster d a University of Florida College of Medicine; b Biochemistry and Molecular Biology; c Pediatrics; d General Clinical Research Center We report results on long-term administration of dichloroacetate (DCA) in 36 children with congenital lactic acidosis (CLA) who participated in the DCA/CLA Clinical Trial [Pediatrics 117, pp. 1519–1521, 2006]. This was a double-blind, randomized, controlled trial of oral DCA (12.5 mg/kg every 12 h). Data were analyzed for each patient from the time they began treatment through May 2005. Subject exposure to DCA totaled 110.42 years. Median height and weight increased over time, but the standardized values declined slightly and remained below the first percentile (Z = 2.33). There were no significant changes in serum levels of glucose. Beta-hydroxybutyrate, electrolytes or tests of renal or hepatic function, except for a 2% rise in total protein (p = 0.04). Venous pH, white blood count, hematocrit and platelet count did not change. Median 24 h urinary oxalate increased 22% (p = 0.003). Pre-treatment basal lactate levels were modestly elevated and increased further following carbohydrate meal challenge, but both basal (p < 0.001) and the meal-induced (p = 0.004) rise in lactate were blunted by DCA. The median pre-treatment cerebrospinal fluid lactate was 3.8 mmol/l and decreased over time with DCA exposure (p = 0.04). Serial nerve conduction measures were mostly unchanged; however, conduction velocity decreased and distal latency increased in peroneal nerves (both <0.001). Mean ± SE 3-year sur-