Harold Sims - Academia.edu (original) (raw)

Papers by Harold Sims

Circulation, 1999

Background —Genetic defects are being increasingly recognized in the etiology of primary cardiomy... more Background —Genetic defects are being increasingly recognized in the etiology of primary cardiomyopathy (CM). Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first step in the β-oxidation spiral of fatty acid metabolism, the crucial pathway for cardiac energy production. Methods and Results —We studied 37 patients with CM, nonketotic hypoglycemia and hepatic dysfunction, skeletal myopathy, or sudden death in infancy with hepatic steatosis, features suggestive of fatty acid oxidation disorders. Single-stranded conformational variance was used to screen genomic DNA. DNA sequencing and mutational analysis revealed 21 different mutations on the VLCAD gene in 18 patients. Of the mutations, 80% were associated with CM. Severe CM in infancy was recognized in most patients (67%) at presentation. Hepatic dysfunction was common (33%). RNA blot analysis and VLCAD enzyme assays showed a severe reduction in VLCAD mRNA in patients with frame-shift or splice-site mutations and absent ...

The Journal of biological chemistry, Jun 23, 2017

Cardiolipin (CL) is a dimeric phospholipid with critical roles in mitochondrial bioenergetics and... more Cardiolipin (CL) is a dimeric phospholipid with critical roles in mitochondrial bioenergetics and signaling. Recently, inhibition of the release of oxidized fatty acyl chains from CL by the calcium-independent phospholipase A2γ (iPLA2γ)-selective inhibitor (R)-BEL suggested that iPLA2γ is responsible for the hydrolysis of oxidized CL and subsequent signaling mediated by the released oxidized fatty acids. However, chemical inhibition by BEL is subject to off-target pharmacologic effects. Accordingly, to unambiguously determine the role of iPLA2γ in the hydrolysis of oxidized CL, we compared alterations in oxidized CLs and the release of oxidized aliphatic chains from CL in experiments with purified recombinant iPLA2γ, germ-line iPLA2γ-/- mice, cardiac myocyte-specific iPLA2γ transgenic mice, and wild-type mice. Using charge-switch high mass accuracy LC-MS/MS with selected reaction monitoring and product ion accurate masses, we demonstrated that iPLA2γ is the major enzyme responsible ...

Proceedings of the National Academy of Sciences, 1995

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defect... more beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changin...

Proceedings of the National Academy of Sciences, 1995

Mitochondrial long chain fatty acid beta-oxidation provides the major source of energy in the hea... more Mitochondrial long chain fatty acid beta-oxidation provides the major source of energy in the heart. Deficiencies of human beta-oxidation enzymes produce sudden, unexplained death in childhood, acute hepatic encephalopathy, skeletal myopathy, or cardiomyopathy. Long chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD; long-chain-(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.211] catalyzes the third step in beta-oxidation, and this activity is present on the C-terminal portion of the alpha subunit of mitochondrial trifunctional protein. We used single-stranded conformation variance analysis of the exons of the human LCHAD (alpha subunit) gene to determine the molecular basis of LCHAD deficiency in three families with children presenting with sudden unexplained death or hypoglycemia and abnormal liver enzymes (Reye-like syndrome). In all families, the mothers had acute fatty liver and associated sever complications during pregnancies with the affected infants. The analysis in two affec...

New England Journal of Medicine, 1999

Background Acute fatty liver of pregnancy and the HELLP syndrome (hemolysis, elevated liver-enzym... more Background Acute fatty liver of pregnancy and the HELLP syndrome (hemolysis, elevated liver-enzyme levels, and a low platelet count) are serious hepatic disorders that may occur during pregnancy in women whose fetuses are later found to have a deficiency of long-chain 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase. This enzyme resides in the mitochondrial trifunctional protein, which also contains the active site of long-chain 2,3-enoyl-CoA hydratase and long-chain 3-ketoacyl-CoA thiolase. We undertook this study to determine the relation between mutations in the trifunctional protein in infants with defects in fatty-acid oxidation and acute liver disease during pregnancy in their mothers.

Molecular Genetics and Metabolism, 1999

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of a family of nuclear-encoded enzymes that... more Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of a family of nuclear-encoded enzymes that catalyze the initial step in mitochondrial fatty acid ␤-oxidation (FAO). Previous studies have indicated that two other members of the AD gene family (medium-chain AD and long-chain AD) are controlled at the transcriptional level by nuclear hormone receptors. In this study, we have cloned and characterized the human VLCAD gene promoter region to identify cisacting elements involved in its transcriptional control. VLCAD gene promoter-luciferase reporter (VLCAD-Luc) constructs were found to be transcriptionally active in a variety of mammalian cell lines and in primary rat cardiomyocytes when driven by varying lengths of the VLCAD promoter region. Removal of a 20-bp DNA segment of the proximal VLCAD gene promoter markedly reduced the transcriptional activity of VLCAD-Luc constructs. Gel mobility shift assays identified a DNA-binding activity in nuclear extracts prepared from human hepatoma G2 cells that interacted with the 20-bp regulatory region. Competition studies revealed that this DNA-binding activity could be abolished by a molar excess of unlabeled specific oligonucleotide as well as a DNA fragment containing an activator protein 2 (AP-2)-binding site but not by an unrelated nonspecific DNA fragment. These results provide an initial characterization of the human VLCAD gene promoter, identify AP-2 as a candidate activator of VLCAD gene transcription, and suggest that VLCAD gene transcription may be regulated by pathways distinct from that of other AD genes.

Journal of Lipid Research, 2013

Supplementary key words lipidome • cardiolipin • tafazzin • phospholipase • mitochondria • cardio... more Supplementary key words lipidome • cardiolipin • tafazzin • phospholipase • mitochondria • cardiolipin synthase • transgenic • electron transport chain Barth syndrome is an X-linked complex metabolic disorder caused by mutations in the gene tafazzin (1-4). Clinical manifestations of Barth syndrome include growth delay, hypertrophic or dilated cardiomyopathy, exercise intolerance (5), chronic or cyclic neutropenia, and 3-methylglutaconic aciduria type II, as well as accumulation in monolysocardiolipin (MLCL) which has characteristically defi ned Barth syndrome as a mitochondrial lipid disorder (6, 7). Tafazzin is a transacylase which sculpts the mitochondrial lipidome, most notably cardiolipin, to maintain a stereoelectronic environment conducive for effi cient bioenergetic function (8). However, the pleiotropic roles of tafazzin and cardiolipin (as well as its downstream sequelae) in regulating cellular signaling through modulation of metabolic effi ciency, membrane dynamics, and multiple other mitochondrial functions have remained enigmatic (9). Recently, an inducible tafazzin shRNA knockdown (Taz KD) mouse model of Barth syndrome was generated to gain mechanistic insights into the multifaceted roles of tafazzin and cardiolipin, to increase our understanding of the complexity of alterations in Barth syndrome, and to develop Abstract Barth syndrome is a complex metabolic disorder caused by mutations in the mitochondrial transacylase tafazzin. Recently, an inducible tafazzin shRNA knockdown mouse model was generated to deconvolute the complex bioenergetic phenotype of this disease. To investigate the underlying cause of hemodynamic dysfunction in Barth syndrome, we interrogated the cardiac structural and signaling lipidome of this mouse model as well as its myocardial bioenergetic phenotype. A decrease in the distribution of cardiolipin molecular species and robust increases in monolysocardiolipin and dilysocardiolipin were demonstrated. Additionally, the contents of choline and ethanolamine glycerophospholipid molecular species containing precursors for lipid signaling at the sn-2 position were altered. Lipidomic analyses revealed specifi c dysregulation of HETEs and prostanoids, as well as oxidized linoleic and docosahexaenoic metabolites. Bioenergetic interrogation uncovered differential substrate utilization as well as decreases in Complex III and V activities. Transgenic expression of cardiolipin synthase or iPLA 2 ␥ ablation in tafazzin-defi cient mice did not rescue the observed phenotype. These results underscore the complex nature of alterations in cardiolipin metabolism mediated by tafazzin loss of function. Collectively, we identifi ed specifi c lipidomic, bioenergetic, and signaling alterations in a murine model that parallel those of Barth syndrome thereby providing novel insights into the pathophysiology of this debilitating disease.

Journal of Clinical Investigation, 1995

Human trifunctional protein catalyzes three steps in mitochondrial ,B-oxidation of fatty acids, i... more Human trifunctional protein catalyzes three steps in mitochondrial ,B-oxidation of fatty acids, including the long chain 3-hydroxyacyl-CoA dehydrogenase step. Deficiency of this heterocomplex, which contains 4a and 4.8 subunits, causes sudden unexplained infant death, a Reye-like syndrome, cardiomyopathy, or skeletal myopathy. We determined the molecular basis of this deficiency in a patient with neonatal presentation and later sudden death using reverse transcription and PCR amplification of his a subunit mRNA. We demonstrated a universal deletion of exon 3 (71 bp) in his mRNA. This deletion causes a frameshift and very early premature termination. Amplification of genomic DNA demonstrated that the patient was a compound heterozygote with two different mutations in the 5' donor splice site following exon 3: a paternally inherited G to A transversion at the invariant position + 1 and a maternally inherited A to G mutation at position +3. Both allelic mutations apparently cause exon 3 skipping, resulting in undetectable levels of a subunit protein, and complete loss of trifunctional protein. This is the initial molecular characterization of trifunctional protein deficiency.

Journal of Clinical Investigation, 1994

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common known genetic disorder o... more Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common known genetic disorder of fatty acid oxidation. Most (-80%) cases are homozygous for a single mutation: A to G replacement at nucleotide 985 (A985G). MCAD deficiency typically presents in the second year of life as hypoketotic hypoglycemia associated with fasting and may progress to liver failure, coma, and death. Prompt diagnosis and management may prevent long-term sequelae. MCAD deficiency was verified by analysis of urinary acylglycine and serum acylcarnitine species from two neonates referred for diagnosis. Full-length cDNA and MCAD exon 7 and 11 genomic clones were prepared for sequence analysis. Normal and mutant cDNAs were expressed in bacteria, and enzymatic activity was assayed by the ferricenium hexaflurophosphate method. Four compound heterozygote individuals from two unrelated families with A985G on one allele and a novel G to A mutation at nucleotide 583 (G583A) as the second mutant allele presented with MCAD deficiency in the first week of life. The expressed G583A mutant protein lacks enzymatic activity. This novel mutation, G583A, is associated with severe MCAD deficiency causing hypoglycemia or sudden, unexpected neonatal death. This previously unrecognized phenotype of MCAD deficiency may contribute significantly to preventable infant deaths. (J. Clin. Invest. 1994. 94:1477-1483.) Key words: fatty acid oxidation * medium chain acyl-CoA dehydrogenase deficiency * sudden infant death * amplification refractory mutation system * point mutation

Journal of Biological Chemistry, 2004

Journal of Biological Chemistry, 2005

Annals of Neurology, 1998

Very long chain acyl‐coenzyme A (acyl‐CoA) dehydrogenase (VLCAD) deficiency is a severe disorder ... more Very long chain acyl‐coenzyme A (acyl‐CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial β‐oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified stongly elevated levels of tetradecenoic acid, 14:1(n‐9), tetradecadienoic acid, 14:2(n‐6), and hexadecadienoic acid, 16:2(n‐6). Palmitoyl‐CoA and behenoyl‐CoA dehydrogenase in fibroblasts were deficient. Muscle VLCAD activity was very low. DNA analysis revealed compound heterozygosity for two missense mutations in the VLCAD gene. The relatively mild clinical course may be due to residual enzyme activity as a consequence of the two missense mutations. Treatment with L‐carnitine and medium chain triglycerides in the diet did not reduce the attacks of rhabdomyolysis.

American Journal of Physiology-Cell Physiology, 2010

The endothelium comprises a cellular barrier between the circulation and tissues. We have previou... more The endothelium comprises a cellular barrier between the circulation and tissues. We have previously shown that activation of protease-activated receptor 1 (PAR-1) and PAR-2 on the surface of human coronary artery endothelial cells by tryptase or thrombin increases group VIA phospholipase A2(iPLA2β) activity and results in production of multiple phospholipid-derived inflammatory metabolites. We isolated cardiac endothelial cells from hearts of iPLA2β-knockout (iPLA2β-KO) and wild-type (WT) mice and measured arachidonic acid (AA), prostaglandin I2(PGI2), and platelet-activating factor (PAF) production in response to PAR stimulation. Thrombin (0.1 IU/ml) or tryptase (20 ng/ml) stimulation of WT endothelial cells rapidly increased AA and PGI2release and increased PAF production. Selective inhibition of iPLA2β with ( S)-bromoenol lactone (5 μM, 10 min) completely inhibited thrombin- and tryptase-stimulated responses. Thrombin or tryptase stimulation of iPLA2β-KO endothelial cells did no...

American Journal of Physiology-Heart and Circulatory Physiology, 2010

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that ... more Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A2β (iPLA2β) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA2β+/+) mice and those lacking the β-isoform (iPLA2β−/−) both ex vivo and in vivo. Vessels isolated from iPLA2β−/−mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA2β+/+mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA2β+/+vessels. In contrast, vessels from iPLA2β−/−constricted by 54% after d...

Journal of Biological Chemistry, 2007

2 The abbreviations used are: iPLA 2 ␥, calcium-independent phospholipase A 2 ␥; TGiPLA 2 ␥, tran... more 2 The abbreviations used are: iPLA 2 ␥, calcium-independent phospholipase A 2 ␥; TGiPLA 2 ␥, transgenic mice overexpressing iPLA 2 ␥; BEL, (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one; COX, cytochrome c oxidase 1V;

American Journal of Obstetrics and Gynecology, 1989

Circulation, 1999

Background —Genetic defects are being increasingly recognized in the etiology of primary cardiomy... more Background —Genetic defects are being increasingly recognized in the etiology of primary cardiomyopathy (CM). Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first step in the β-oxidation spiral of fatty acid metabolism, the crucial pathway for cardiac energy production. Methods and Results —We studied 37 patients with CM, nonketotic hypoglycemia and hepatic dysfunction, skeletal myopathy, or sudden death in infancy with hepatic steatosis, features suggestive of fatty acid oxidation disorders. Single-stranded conformational variance was used to screen genomic DNA. DNA sequencing and mutational analysis revealed 21 different mutations on the VLCAD gene in 18 patients. Of the mutations, 80% were associated with CM. Severe CM in infancy was recognized in most patients (67%) at presentation. Hepatic dysfunction was common (33%). RNA blot analysis and VLCAD enzyme assays showed a severe reduction in VLCAD mRNA in patients with frame-shift or splice-site mutations and absent ...

The Journal of biological chemistry, Jun 23, 2017

Cardiolipin (CL) is a dimeric phospholipid with critical roles in mitochondrial bioenergetics and... more Cardiolipin (CL) is a dimeric phospholipid with critical roles in mitochondrial bioenergetics and signaling. Recently, inhibition of the release of oxidized fatty acyl chains from CL by the calcium-independent phospholipase A2γ (iPLA2γ)-selective inhibitor (R)-BEL suggested that iPLA2γ is responsible for the hydrolysis of oxidized CL and subsequent signaling mediated by the released oxidized fatty acids. However, chemical inhibition by BEL is subject to off-target pharmacologic effects. Accordingly, to unambiguously determine the role of iPLA2γ in the hydrolysis of oxidized CL, we compared alterations in oxidized CLs and the release of oxidized aliphatic chains from CL in experiments with purified recombinant iPLA2γ, germ-line iPLA2γ-/- mice, cardiac myocyte-specific iPLA2γ transgenic mice, and wild-type mice. Using charge-switch high mass accuracy LC-MS/MS with selected reaction monitoring and product ion accurate masses, we demonstrated that iPLA2γ is the major enzyme responsible ...

Proceedings of the National Academy of Sciences, 1995

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defect... more beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changin...

Proceedings of the National Academy of Sciences, 1995

Mitochondrial long chain fatty acid beta-oxidation provides the major source of energy in the hea... more Mitochondrial long chain fatty acid beta-oxidation provides the major source of energy in the heart. Deficiencies of human beta-oxidation enzymes produce sudden, unexplained death in childhood, acute hepatic encephalopathy, skeletal myopathy, or cardiomyopathy. Long chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD; long-chain-(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.211] catalyzes the third step in beta-oxidation, and this activity is present on the C-terminal portion of the alpha subunit of mitochondrial trifunctional protein. We used single-stranded conformation variance analysis of the exons of the human LCHAD (alpha subunit) gene to determine the molecular basis of LCHAD deficiency in three families with children presenting with sudden unexplained death or hypoglycemia and abnormal liver enzymes (Reye-like syndrome). In all families, the mothers had acute fatty liver and associated sever complications during pregnancies with the affected infants. The analysis in two affec...

New England Journal of Medicine, 1999

Background Acute fatty liver of pregnancy and the HELLP syndrome (hemolysis, elevated liver-enzym... more Background Acute fatty liver of pregnancy and the HELLP syndrome (hemolysis, elevated liver-enzyme levels, and a low platelet count) are serious hepatic disorders that may occur during pregnancy in women whose fetuses are later found to have a deficiency of long-chain 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase. This enzyme resides in the mitochondrial trifunctional protein, which also contains the active site of long-chain 2,3-enoyl-CoA hydratase and long-chain 3-ketoacyl-CoA thiolase. We undertook this study to determine the relation between mutations in the trifunctional protein in infants with defects in fatty-acid oxidation and acute liver disease during pregnancy in their mothers.

Molecular Genetics and Metabolism, 1999

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of a family of nuclear-encoded enzymes that... more Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of a family of nuclear-encoded enzymes that catalyze the initial step in mitochondrial fatty acid ␤-oxidation (FAO). Previous studies have indicated that two other members of the AD gene family (medium-chain AD and long-chain AD) are controlled at the transcriptional level by nuclear hormone receptors. In this study, we have cloned and characterized the human VLCAD gene promoter region to identify cisacting elements involved in its transcriptional control. VLCAD gene promoter-luciferase reporter (VLCAD-Luc) constructs were found to be transcriptionally active in a variety of mammalian cell lines and in primary rat cardiomyocytes when driven by varying lengths of the VLCAD promoter region. Removal of a 20-bp DNA segment of the proximal VLCAD gene promoter markedly reduced the transcriptional activity of VLCAD-Luc constructs. Gel mobility shift assays identified a DNA-binding activity in nuclear extracts prepared from human hepatoma G2 cells that interacted with the 20-bp regulatory region. Competition studies revealed that this DNA-binding activity could be abolished by a molar excess of unlabeled specific oligonucleotide as well as a DNA fragment containing an activator protein 2 (AP-2)-binding site but not by an unrelated nonspecific DNA fragment. These results provide an initial characterization of the human VLCAD gene promoter, identify AP-2 as a candidate activator of VLCAD gene transcription, and suggest that VLCAD gene transcription may be regulated by pathways distinct from that of other AD genes.

Journal of Lipid Research, 2013

Supplementary key words lipidome • cardiolipin • tafazzin • phospholipase • mitochondria • cardio... more Supplementary key words lipidome • cardiolipin • tafazzin • phospholipase • mitochondria • cardiolipin synthase • transgenic • electron transport chain Barth syndrome is an X-linked complex metabolic disorder caused by mutations in the gene tafazzin (1-4). Clinical manifestations of Barth syndrome include growth delay, hypertrophic or dilated cardiomyopathy, exercise intolerance (5), chronic or cyclic neutropenia, and 3-methylglutaconic aciduria type II, as well as accumulation in monolysocardiolipin (MLCL) which has characteristically defi ned Barth syndrome as a mitochondrial lipid disorder (6, 7). Tafazzin is a transacylase which sculpts the mitochondrial lipidome, most notably cardiolipin, to maintain a stereoelectronic environment conducive for effi cient bioenergetic function (8). However, the pleiotropic roles of tafazzin and cardiolipin (as well as its downstream sequelae) in regulating cellular signaling through modulation of metabolic effi ciency, membrane dynamics, and multiple other mitochondrial functions have remained enigmatic (9). Recently, an inducible tafazzin shRNA knockdown (Taz KD) mouse model of Barth syndrome was generated to gain mechanistic insights into the multifaceted roles of tafazzin and cardiolipin, to increase our understanding of the complexity of alterations in Barth syndrome, and to develop Abstract Barth syndrome is a complex metabolic disorder caused by mutations in the mitochondrial transacylase tafazzin. Recently, an inducible tafazzin shRNA knockdown mouse model was generated to deconvolute the complex bioenergetic phenotype of this disease. To investigate the underlying cause of hemodynamic dysfunction in Barth syndrome, we interrogated the cardiac structural and signaling lipidome of this mouse model as well as its myocardial bioenergetic phenotype. A decrease in the distribution of cardiolipin molecular species and robust increases in monolysocardiolipin and dilysocardiolipin were demonstrated. Additionally, the contents of choline and ethanolamine glycerophospholipid molecular species containing precursors for lipid signaling at the sn-2 position were altered. Lipidomic analyses revealed specifi c dysregulation of HETEs and prostanoids, as well as oxidized linoleic and docosahexaenoic metabolites. Bioenergetic interrogation uncovered differential substrate utilization as well as decreases in Complex III and V activities. Transgenic expression of cardiolipin synthase or iPLA 2 ␥ ablation in tafazzin-defi cient mice did not rescue the observed phenotype. These results underscore the complex nature of alterations in cardiolipin metabolism mediated by tafazzin loss of function. Collectively, we identifi ed specifi c lipidomic, bioenergetic, and signaling alterations in a murine model that parallel those of Barth syndrome thereby providing novel insights into the pathophysiology of this debilitating disease.

Journal of Clinical Investigation, 1995

Human trifunctional protein catalyzes three steps in mitochondrial ,B-oxidation of fatty acids, i... more Human trifunctional protein catalyzes three steps in mitochondrial ,B-oxidation of fatty acids, including the long chain 3-hydroxyacyl-CoA dehydrogenase step. Deficiency of this heterocomplex, which contains 4a and 4.8 subunits, causes sudden unexplained infant death, a Reye-like syndrome, cardiomyopathy, or skeletal myopathy. We determined the molecular basis of this deficiency in a patient with neonatal presentation and later sudden death using reverse transcription and PCR amplification of his a subunit mRNA. We demonstrated a universal deletion of exon 3 (71 bp) in his mRNA. This deletion causes a frameshift and very early premature termination. Amplification of genomic DNA demonstrated that the patient was a compound heterozygote with two different mutations in the 5' donor splice site following exon 3: a paternally inherited G to A transversion at the invariant position + 1 and a maternally inherited A to G mutation at position +3. Both allelic mutations apparently cause exon 3 skipping, resulting in undetectable levels of a subunit protein, and complete loss of trifunctional protein. This is the initial molecular characterization of trifunctional protein deficiency.

Journal of Clinical Investigation, 1994

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common known genetic disorder o... more Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common known genetic disorder of fatty acid oxidation. Most (-80%) cases are homozygous for a single mutation: A to G replacement at nucleotide 985 (A985G). MCAD deficiency typically presents in the second year of life as hypoketotic hypoglycemia associated with fasting and may progress to liver failure, coma, and death. Prompt diagnosis and management may prevent long-term sequelae. MCAD deficiency was verified by analysis of urinary acylglycine and serum acylcarnitine species from two neonates referred for diagnosis. Full-length cDNA and MCAD exon 7 and 11 genomic clones were prepared for sequence analysis. Normal and mutant cDNAs were expressed in bacteria, and enzymatic activity was assayed by the ferricenium hexaflurophosphate method. Four compound heterozygote individuals from two unrelated families with A985G on one allele and a novel G to A mutation at nucleotide 583 (G583A) as the second mutant allele presented with MCAD deficiency in the first week of life. The expressed G583A mutant protein lacks enzymatic activity. This novel mutation, G583A, is associated with severe MCAD deficiency causing hypoglycemia or sudden, unexpected neonatal death. This previously unrecognized phenotype of MCAD deficiency may contribute significantly to preventable infant deaths. (J. Clin. Invest. 1994. 94:1477-1483.) Key words: fatty acid oxidation * medium chain acyl-CoA dehydrogenase deficiency * sudden infant death * amplification refractory mutation system * point mutation

Journal of Biological Chemistry, 2004

Journal of Biological Chemistry, 2005

Annals of Neurology, 1998

Very long chain acyl‐coenzyme A (acyl‐CoA) dehydrogenase (VLCAD) deficiency is a severe disorder ... more Very long chain acyl‐coenzyme A (acyl‐CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial β‐oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified stongly elevated levels of tetradecenoic acid, 14:1(n‐9), tetradecadienoic acid, 14:2(n‐6), and hexadecadienoic acid, 16:2(n‐6). Palmitoyl‐CoA and behenoyl‐CoA dehydrogenase in fibroblasts were deficient. Muscle VLCAD activity was very low. DNA analysis revealed compound heterozygosity for two missense mutations in the VLCAD gene. The relatively mild clinical course may be due to residual enzyme activity as a consequence of the two missense mutations. Treatment with L‐carnitine and medium chain triglycerides in the diet did not reduce the attacks of rhabdomyolysis.

American Journal of Physiology-Cell Physiology, 2010

The endothelium comprises a cellular barrier between the circulation and tissues. We have previou... more The endothelium comprises a cellular barrier between the circulation and tissues. We have previously shown that activation of protease-activated receptor 1 (PAR-1) and PAR-2 on the surface of human coronary artery endothelial cells by tryptase or thrombin increases group VIA phospholipase A2(iPLA2β) activity and results in production of multiple phospholipid-derived inflammatory metabolites. We isolated cardiac endothelial cells from hearts of iPLA2β-knockout (iPLA2β-KO) and wild-type (WT) mice and measured arachidonic acid (AA), prostaglandin I2(PGI2), and platelet-activating factor (PAF) production in response to PAR stimulation. Thrombin (0.1 IU/ml) or tryptase (20 ng/ml) stimulation of WT endothelial cells rapidly increased AA and PGI2release and increased PAF production. Selective inhibition of iPLA2β with ( S)-bromoenol lactone (5 μM, 10 min) completely inhibited thrombin- and tryptase-stimulated responses. Thrombin or tryptase stimulation of iPLA2β-KO endothelial cells did no...

American Journal of Physiology-Heart and Circulatory Physiology, 2010

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that ... more Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A2β (iPLA2β) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA2β+/+) mice and those lacking the β-isoform (iPLA2β−/−) both ex vivo and in vivo. Vessels isolated from iPLA2β−/−mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA2β+/+mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA2β+/+vessels. In contrast, vessels from iPLA2β−/−constricted by 54% after d...

Journal of Biological Chemistry, 2007

2 The abbreviations used are: iPLA 2 ␥, calcium-independent phospholipase A 2 ␥; TGiPLA 2 ␥, tran... more 2 The abbreviations used are: iPLA 2 ␥, calcium-independent phospholipase A 2 ␥; TGiPLA 2 ␥, transgenic mice overexpressing iPLA 2 ␥; BEL, (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one; COX, cytochrome c oxidase 1V;

American Journal of Obstetrics and Gynecology, 1989