Characterization of MB creatine kinase isoform conversion in vitro and in vivo in dogs.

Time-dependent removal of the COOH-terminal lysine residue from each subunit of tissue MM creatine kinase by plasma carboxypeptidase N produces two additional isoforms that are readily separated, thereby permitting sensitive, early detection of acute myocardial infarction. Only two isoforms of MB creatine kinase have been detected in plasma leading to speculation that the COOH-terminal lysine on the B subunit is resistant to hydrolysis. To define the biochemical changes resulting in MB creatine kinase isoform conversion, we incubated highly purified MB creatine kinase from canine myocardium with plasma carboxypeptidase N. Quantitative anion-exchange chromatography of incubation mixtures and serial plasma samples from dogs subjected to coronary occlusion revealed a second, more acidic form evolved with time that was separated from the tissue isoform. Cyanogen bromide digestion of the two isoforms followed by amino acid sequencing of COOH-terminal peptides showed that MB creatine kinase undergoes removal of the COOH-terminal lysine residue from both M and B subunits. An intermediate form lacking lysine on the M subunit was delineated during incubations by the combined use of anion-exchange chromatography and conventional electrophoretic techniques. Thus, sequential cleavage of lysine from subunits of MB creatine kinase produces an intermediate isoform that has not been detected previously because of difficulties separating it from the tissue and fully converted isoforms.

Two alpha subunit donor splice site mutations cause human trifunctional protein deficiency.

Human trifunctional protein catalyzes three steps in mitochondrial beta-oxidation of fatty acids, including the long chain 3-hydroxyacyl-CoA dehydrogenase step. Deficiency of this heterocomplex, which contains 4 alpha and 4 beta 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 alpha 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 alpha subunit protein, and complete loss of trifunctional protein. This is the initial molecular characterization of trifunctional protein deficiency.

A novel mutation in medium chain acyl-CoA dehydrogenase causes sudden neonatal death.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common known genetic disorder of fatty acid oxidation. Most (approximately 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.

Mild trifunctional protein deficiency is associated with progressive neuropathy and myopathy and suggests a novel genotype-phenotype correlation.

Human mitochondrial trifunctional protein (TFP) is a heterooctamer of four alpha- and four beta-subunits that catalyzes three steps in the beta-oxidation spiral of long-chain fatty acids. TFP deficiency causes a Reye-like syndrome, cardiomyopathy, or sudden, unexpected death. We delineated the molecular basis for TFP deficiency in two patients with a unique phenotype characterized by chronic progressive polyneuropathy and myopathy without hepatic or cardiac involvement. Single-stranded conformation variance and nucleotide sequencing identified all patient mutations in exon 9 of the alpha-subunit. One patient is homozygous for the T845A mutation that substitutes aspartic acid for valine at residue 246. The second patient is a compound heterozygote for the T914A that substitutes asparagine for isoleucine at residue 269 and a C871T that creates a premature termination at residue 255. Allele-specific oligonucleotide hybridization studies revealed undetectable levels of the mRNA corresponding to the mutant allele carrying the termination codon. This study suggests a novel genotype-phenotype correlation in TFP deficiency; that is, mutations in exon 9 of the alpha-subunit, which encodes a linker domain between the NH2-terminal hydratase and the COOH-terminal 3-hydroxyacyl-CoA dehydrogenase, result in a unique neuromuscular phenotype.

Lack of mitochondrial trifunctional protein in mice causes neonatal hypoglycemia and sudden death.

Mitochondrial trifunctional protein (MTP) is a hetero-octamer of four alpha and four beta subunits that catalyzes the final three steps of mitochondrial long chain fatty acid beta-oxidation. Human MTP deficiency causes Reye-like syndrome, cardiomyopathy, or sudden unexpected death. We used gene targeting to generate an MTP alpha subunit null allele and to produce mice that lack MTP alpha and beta subunits. The Mtpa(-/-) fetuses accumulate long chain fatty acid metabolites and have low birth weight compared with the Mtpa(+/-) and Mtpa(+/+) littermates. Mtpa(-/-) mice suffer neonatal hypoglycemia and sudden death 6-36 hours after birth. Analysis of the histopathological changes in the Mtpa(-/-) pups revealed rapid development of hepatic steatosis after birth and, later, significant necrosis and acute degeneration of the cardiac and diaphragmatic myocytes. This mouse model documents that intact mitochondrial long chain fatty acid oxidation is essential for fetal development and for survival after birth. Deficiency of MTP causes fetal growth retardation, neonatal hypoglycemia, and sudden death.

Carnitine supplementation induces long-chain acylcarnitine production--studies in the VLCAD-deficient mouse.

Carnitine supplementation does not affect carnitine concentrations in tissues of wild-type and very long-chain acyl-CoA dehydrogenase-deficient mice, but results in an increase in long-chain acylcarnitine production.

Potential of fibrates in the treatment of fatty acid oxidation disorders: revival of classical drugs?

Exposure to fibrates leads to normalization of fatty acid oxidation (FAO) in fibroblasts from patients with myopathic forms of CPT2 deficiency or VLCAD deficiency. Correction of FAO is related to a drug-induced increase of residual enzyme activity, and this could provide a new treatment strategy for these disorders.

Expression of a Nagao-type, phosphatidylinositol-glycan anchored alkaline phosphatase in human choriocarcinomas.

The alkaline phosphatase (AP) synthesized by human tumor cells closely resembles human placental AP (PLAP). Little is known about the molecular events that lead to the expression of a placenta-like AP in tumor cells. The complementary DNA encoding the AP expressed by a choriocarcinoma cell line, BeWo, was isolated and characterized. The complementary DNA is the product of the germ cell AP (Nagao isozyme) gene and not of the term PLAP gene. Like placental AP, the tumor AP can be released from the cell membrane by a phosphaditylinositol-specific phospholipase C and has a phosphaditylinositol-glycan (PI-glycan) moiety at the COOH terminus. Immunoprecipitation of phosphaditylinositol-specific phospholipase C-treated AP and analysis by polyacrylamide gel electrophoresis or isoelectric focusing demonstrates that at least 95% of the AP contains PI-glycan. Two-dimensional gel electrophoresis reveals two precursors of the mature AP. One of these does not bind an antibody against the Trypanosoma variable surface glycoprotein cross-reacting determinant and probably does not contain PI-glycan. This precursor had a shorter half-life than the more prominent PI-glycan-containing precursor in pulse-chase experiments, suggesting a precursor-product relationship between the two proteins. These data demonstrate that BeWo AP is the product of a gene normally expressed in testis, thymus, and germ cells, but not in placenta. Thus, the expression of BeWo AP results from the repression of the PLAP gene and derepression of the germ cell AP gene and, as such, the expression is ectopic. The BeWo AP (Nagao isozyme) is modified with PI-glycan that is added soon after translation, not cotranslationally.

Developmental expression of sarcomeric and ubiquitous mitochondrial creatine kinase is tissue-specific.

Creatine kinase (CK) isoenzymes play prominent roles in myocardial energy metabolism. Two nuclear genes encode mitochondrial creatine kinase (MtCK), are tissue-specific in their expression, and are thus designated as sarcomeric MtCK (sMtCK) and ubiquitous MtCK (uMtCK). Quantitative analysis of the mRNA expression of both MtCKs in developing rat tissues demonstrates tissue-specific developmental regulation. sMtCK mRNA in heart is undetectable prenatally but is dramatically upregulated by 28 d postnatally. sMtCK mRNA in skeletal muscle is also extremely low prenatally but is markedly upregulated at birth and doubles by 28 d postnatally. uMtCK mRNA expression is present at low levels in fetal brain and intestine. Brain uMtCK mRNA continues to rise from -4 d prenatally until 28 d postnatally (6-fold increase), but intestinal uMtCK mRNA increases immediately prior to birth, falls, and is upregulated again at 28 d (20-fold). uMtCK mRNA is undetectable in fetal skeletal muscle or heart, but increases to low levels in skeletal muscle at birth and remains at this level into adulthood. uMtCK is not detectable in heart, lung, testes, or liver at any stage examined. We conclude that sMtCK and uMtCK are developmentally regulated in a tissue-specific manner. Unlike cytosolic muscle CK and brain CK, there is no isoenzyme switch between sMtCK and uMtCK in the developing animal. Our results suggest that specific trans-acting factors regulate the different developmental and tissue-specific expression of the MtCK genes.

Molecular cloning and expression of a cDNA encoding the membrane-associated rat intestinal alkaline phosphatase.

Rat intestinal alkaline phosphatase (IAP) has been purified and proteolytic fragments sequenced. A cDNA library was constructed from duodenal poly(A) + RNA and screened for IAP positive clones by a full-length cDNA clone-encoding human IAP. A full length rat IAP clone (2237 bp) was isolated and sequenced, revealing a predicted primary sequence of 519 amino acids (61.974 kDa) with an additional signal peptide of 20 amino acids. 80% of amino acids from residues 1-474 were identical when compared with the human IAP, but there was only 31% identity in the COOH-terminal 45 amino acids. The homology diverges just before the putative binding site for the phosphatidylinositol-glycan (PI-glycan) anchor. The resulting peptide in rat AP contains five hydrophilic amino acids not present in the primary structure of human IAP. Binding of a synthetic 48-mer encoding a portion of this unique and divergent region (residues 476-491) was compared with that of the full-length clone on Northern blots of rat intestinal RNA. Two mRNAs, 3.0 and 2.7 kb, were detected by both probes, confirming earlier results, but the 48-mer bound preferentially to the 3.0 kb mRNA. The protein product of the full-length cDNA in a cell-free system was 62 kDa, corresponding with the smaller of the two IAP proteins produced by rat duodenal RNA. The cDNA transfected into COS-1 cells produced a membrane-bound IAP that was released by phosphatidylinositol-specific phospholipase (PI-PLC). These data provide definitive evidence that IAP is anchored by PI-glycan and conclusively demonstrate that the unique COOH-terminal structure encoded by this rat mRNA supports the addition of a PI-glycan anchor.

Tissue specific and developmental expression of rat long-and medium-chain acyl-CoA dehydrogenases.

Utilization of fatty acids for energy varies among mammalian tissues and during development due to changes in expression of enzymes of mitochondrial beta oxidation. To discern whether two related nuclear genes are expressed similarly, the tissue distribution and developmental profile of the rat long- and medium-chain acyl-CoA dehydrogenase (LCAD and MCAD) mRNAs were compared. A 1451 base full-length LCAD cDNA from neonatal rat aorta was used to study mRNA accumulation in adult and fetal rat tissues. LCAD and MCAD mRNAs were expressed in aorta, heart, and brown fat at levels 8-40 fold greater than in liver, kidney, and duodenum. Brain, placenta, ovary, testes, and skeletal muscle showed the least mRNA. Western blots of adult tissues with anti-rat LCAD antiserum showed corresponding amounts of LCAD protein subunits. LCAD mRNA was detectable in heart, liver, kidney, and brain of fetal rats and increased with age. LCAD and MCAD mRNAs were present in brown fat in 2-10 fold greater amounts compared to other tissues from the newborn period to the end of the weaning period. The high level of expression of LCAD and MCAD mRNA in aorta, heart, and brown fat likely reflects the high energy requirements of those tissues. Differential expression of LCAD and MCAD mRNAs reflects not only inherent gene prescribed programs, but also external influences such as hormones and diet.

Structural characterization and tissue-specific expression of the mRNAs encoding isoenzymes from two rat mitochondrial creatine kinase genes.

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy transduction. Mitochondrial CK (MtCK) reversibly catalyzes the transfer of high energy phosphate to creatine and exists, in the human, as two isoenzymes encoded by separate genes. We report here the cDNA sequences of the two isoenzymes of MtCK in the rat. Rat sarcomeric MtCK has 87% nucleotide identity in the 1257 bp coding region and 82% in the 154 bp 3' untranslated region as compared with human sarcomeric MtCK. Rat ubiquitous MtCK has 92% nucleotide identity over the 1254 bp coding region with human ubiquitous MtCK and 81% identity of the 148 by 3' untranslated region. Nucleotide identity between the rat sarcomeric and ubiquitous MtCK coding regions is 70%, with no conservation of their 3' untranslated regions. Thus, MtCK sequence is conserved in a tissue-specific, rather than species-specific, manner. Conservation of the 3' untranslated regions is highly unusual and suggests a regulatory function for this region. The NH2-terminal transit peptide sequences share 82% amino acid homology between rat and human sarcomeric MtCKs and 92% homology between rat and human ubiquitous MtCKs, but have only 41% homology to each other. This tissue-specific conservation of the transit peptides suggests receptor specificity in mitochondrial uptake. Rat sarcomeric MtCK mRNA is expressed only in skeletal muscle and heart, but rat ubiquitous MtCK mRNA is expressed in many tissues, with highest levels in brain, gut and kidney. Ubiquitous MtCK mRNA levels are dramatically regulated in uterus and placenta during pregnancy. Coexpression of sarcomeric and ubiquitous MtCK with their cytosolic counterparts, MCK and BCK, respectively, supports the creatine phosphate shuttle hypothesis and suggests that expression of these genes is coordinately regulated.

Molecular heterogeneity of creatine kinase isoenzymes.

Cytoplasmic creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is a dimeric enzyme exhibiting three isoenzymes (MM, MB and BB). The two subunits have been reported to have identical molecular weights (Mr) of 41 000. We have demonstrated that the M subunits from human, canine, rabbit, mouse and bovine tissue have similar apparent Mr values of 43 000 as determined by SDS-polyacrylamide gel electrophoresis. In contrast, the Mr of the B subunits was different from that of the M subunit and varied with each species (human Mr 44 500; canine Mr 46 000; rabbit Mr 44 000 and mouse Mr 49 000). Cyanogen bromide cleavage showed all M subunits to have identical fragments, while B subunits exhibited cleavage products with patterns unique for each species. Despite the differences in Mr and cyanogen bromide fragment patterns, all B subunits were capable of producing enzymatically active hybrid (MB) molecules in combination with M subunits from any species tested. Mitochondrial creatine kinase subunits exhibited identical molecular weights and were similar to the M subunits and failed to combine with either the cytosolic M or B subunits. Thus, B subunits appear less conserved during evolution compared to M subunits, but have retained the amino acid sequences essential for subunit interaction and enzymatic activity.

Regulation of the human long chain acyl-CoA dehydrogenase gene by nuclear hormone receptor transcription factors.

Mitochondrial fatty acid oxidation provides most of the energy required for myocardial function after birth. Long chain acyl-CoA dehydrogenase (LCAD) catalyzes the first step in the beta-oxidation spiral. Our objective was to define regulatory elements of the human LCAD gene required for high levels of expression in mature heart and to locate elements suppressing gene expression in the fetus. We characterized the human LCAD gene structure and used in vitro transfection into cardiomyocytes and hepatoma cells of LCAD genomic fragments fused to a reporter gene to examine the effects of putative regulatory elements on transcription. Binding of transcription factors to nuclear hormone receptor consensus DNA binding domains was studied by gel shift experiments. The 200 bp of the human LCAD gene immediately upstream of the transcription initiation site are sufficient to act as a minimal promoter for the gene and provide some tissue-specific positive regulatory elements. The region from -1800 bp to -250 bp contains elements which markedly suppress transcription, including nuclear hormone receptor response elements. The dominant interaction is with the repressor factor, chicken ovalbumin upstream promoter transcription factor. We conclude that the developmental and tissue-specific regulation of the human LCAD gene is mediated, in part, by these nuclear hormone receptor transcription factors.

Dilated cardiomyopathy in infants and children.

The outcome of medical treatment of dilated cardiomyopathy in infants and children was reviewed to develop a predictive index for selection of patients likely to benefit from cardiac transplantation. The clinical findings, laboratory investigations, treatment and outcome of 20 patients (Group 1) less than 2 years of age at presentation and 12 patients (Group 2) greater than 2 years of age at onset were compared. Of 20 Group 1 patients, 5 (25%) died. Available autopsies (four patients) showed endocardial fibroelastosis. Of 15 survivors, 10 showed improvement in cardiac status and 5 remained unchanged. Ninety-three percent of survivors had dilated cardiomyopathy consistent with endocardial fibroelastosis by angiocardiography. All 12 Group 2 patients died. In addition to age at presentation and poor outcome, Group 2 differed from Group 1 in having a higher incidence of other family members with cardiomyopathy, more significant rhythm disturbances at presentation and a more rapid course to death. Risk factors of poor outcome in both groups included persistent cardiomegaly and the development of significant arrhythmias by Holter electrocardiographic monitoring. Cardiac transplantation is recommended for children with dilated cardiomyopathy presenting after age 2 years who survive 1 month. Those patients less than 2 years old at presentation whose condition has not improved after 1 year and who have persistent cardiomegaly or complex ventricular arrhythmias may also benefit from transplantation.

Pig heart short chain L-3-hydroxyacyl-CoA dehydrogenase revisited: sequence analysis and crystal structure determination.

Short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD) is a soluble dimeric enzyme critical for oxidative metabolism of fatty acids. Its primary sequence has been reported to be conserved across numerous tissues and species with the notable exception of the pig heart homologue. Preliminary efforts to solve the crystal structure of the dimeric pig heart SCHAD suggested the unprecedented occurrence of three enzyme subunits within the asymmetric unit, a phenomenon that was thought to have hampered refinement of the initial chain tracing. The recently solved crystal coordinates of human heart SCHAD facilitated a molecular replacement solution to the pig heart SCHAD data. Refinement of the model, in conjunction with the nucleotide sequence for pig heart SCHAD determined in this paper, has demonstrated that the previously published pig heart SCHAD sequence was incorrect. Presented here are the corrected amino acid sequence and the high resolution crystal structure determined for pig heart SCHAD complexed with its NAD+ cofactor (2.8 A; R(cryst) = 22.4%, R(free) = 28.8%). In addition, the peculiar phenomenon of a dimeric enzyme crystallizing with three subunits contained in the asymmetric unit is described.

Progression of hepatic damage during cold storage after procurement in a liver and kidney donor with HELLP syndrome.

BACKGROUND: HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome and acute fatty liver of pregnancy are associated with preeclampsia and fetal defects in fatty acid metabolism. This defect causes the accumulation of metabolites that are harmful to the maternal liver. CASE REPORT: We report a liver and kidney donor with HELLP syndrome and describe the progression of disease in the liver during cold storage. Before procurement, liver biopsy demonstrated minimal necrosis. However, after cold storage, repeat biopsy demonstrated more than 30% necrosis. The liver was not engrafted; the kidneys were transplanted without complication. CONCLUSION: Livers procured from patients with HELLP syndrome should be carefully evaluated for progression of hepatic damage during cold storage and transport.

Deletion of the N-terminus of murine map2 by gene targeting disrupts hippocampal ca1 neuron architecture and alters contextual memory.

Microtubule-associated protein-2 (MAP2) is a brain specific A-kinase anchoring protein that targets the cyclic AMP-dependent protein kinase holoenzyme (PKA) to microtubules. Phosphorylation of MAP2 by different protein kinases is crucial for neuronal growth. The N-terminus of MAP2 contains the binding site for regulatory subunit II of cAMP-dependent protein kinase (PKA-RIIbeta). Using homologous recombination, we created a mutant line of mice (delta1-158) that express truncated MAP2 lacking the N-terminal peptide and the PKA binding site. Deletion of the PKA binding site from the MAP2 gene resulted in decreased efficiency of MAP2 phosphorylation. Biochemical and immunohistochemical studies demonstrate major changes in the morphology of hippocampal neurons in delta1-158 mice. Behavioral tests indicate that delta1-158 mice were impaired (exhibited less conditioned freezing) relative to Wild-Type (WT) controls during a test of contextual, but not during auditory cue, fear conditioning when tested at 8 weeks or 8 months of age. The delta1-158 mice displayed a heightened sensitivity to shock at 8 weeks, but not at 8 months of age. We conclude that PKA binding to MAP2 and MAP2 phosphorylation is essential for the selective development of contextual memory.

Treatment of d-transposition of the great arteries: management of hypoxemia after balloon atrial septostomy.

Between 1975 and 1979, a group of 43 patients with d-transposition of the great arteries were diagnosed and underwent Rashkind balloon atrial septostomy at the time of initial catheterization. Thirty-six (88 percent) survived to the time of intraatrial baffle repair, and 31 (72 percent) are long-term survivors, 2 of them now awaiting repair. Palliative operations were performed in nine patients before definitive surgery; four of these patients are long-term survivors. Prostaglandin E1 infusion improved oxygenation and relieved acidosis in four patients. It is concluded that most patients with d-transposition of the great arteries will survive to elective intraatrial baffle repair between 6 and 12 months without surgical palliation in spite of significant hypoxemia. Prostaglandin E1 infusion may be lifesaving and provide sufficient palliation in patients with persistent hypoxemia and acidosis after balloon atrial septostomy.

Spontaneous closure of secundum atrial septal defect in infants and young children.

The records of 264 pediatric patients with uncomplicated ostium secundum atrial septal defect (ASD) were reviewed. Eighty-seven patients were younger than age 4 years at the time of cardiac catheterization. Subnormal weight gain, frequent pneumonia, cyanosis or tachypnea were present in 26 patients (30%). Of the 36 infants at catheterization, 17 (48%) had the previously described symptoms, including 12 (33%) who had congestive heart failure. Eight of the 36 infants were found to have closed their defect at a subsequent catheterization. Six of 18 patients who underwent cardiac catheterization between 1 and 2 years of age also had spontaneously closed their ASD at subsequent study. Statistical analysis of hemodynamic data revealed no difference (except a smaller shunt size) between ASDs that closed and those that did not in patients who were less than 4 years at initial catheterization. Analysis of hemodynamic data revealed no statistical differences between groups of patients with an ASD who were younger than and those older than 4 years at time of diagnostic study. Patients with ASDs that closed were significantly different from patients with atrial level shunting thought to be secondary to a valve-incompetent foramen ovale with respect to age at initial study (11 versus 2 months, p less than 0.001), mean left atrial pressure (7.7 versus 12.3 mm Hg, p less than 0.02) and difference between mean right and left atrial pressures (1.0 versus 4.2 mm Hg, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)