Edward W. Holmes

Common Variant in AMPD1 Gene Predicts Improved Clinical Outcome in Patients With Heart Failure

BACKGROUND This study was undertaken to identify gene(s) that may be associated with improved clinical outcome in patients with congestive heart failure (CHF). The adenosine monophosphate deaminase locus (AMPD1) was selected for study. We hypothesized that inheritance of the mutant AMPD1 allele is associated with increased probability of survival without cardiac transplantation in patients with CHF. METHODS AND RESULTS AMPD1 genotype was determined in 132 patients with advanced CHF and 91 control reference subjects by use of a polymerase chain reaction-based, allele-specific oligonucleotide detection assay. In patients with CHF, those heterozygous (n=20) or homozygous (n=1) for the mutant AMPD1 allele (AMPD1 +/- or -/-, respectively) experienced a significantly longer duration of heart failure symptoms before referral for transplantation evaluation than CHF patients homozygous for the wild-type allele (AMPD1 +/+; n=111; 7.6+/-6.5 versus 3.2+/-3.6 years; P<0.001). The OR of surviving without cardiac transplantation >/=5 years after initial hospitalization for CHF symptoms was 8.6 times greater (95% CI: 3.05, 23.87) in those patients carrying >/=1 mutant AMPD1 allele than in those carrying 2 wild-type AMPD1 +/+ alleles. CONCLUSIONS After the onset of CHF symptoms, the mutant AMPD1 allele is associated with prolonged probability of survival without cardiac transplantation. The mechanism by which the presence of the mutant AMPD1 allele may modify the clinical phenotype of heart failure remains to be determined.

Human IMP dehydrogenase: Kinetics and regulatory properties

Abstract Human IMP dehydrogenase has been partially purified from placenta and characterized. The enzyme activity is located in the cytosol. The Michaelis constants for IMP and NAD are 14 and 46 μM, respectively. In addition, the enzyme requires a monovalent cation for maximal activity and the apparent K m for K + is 17 mM. The purine ribonucleotides XMP, GMP, and AMP inhibit the enzyme in a manner which is competitive with respect to IMP. The K i values are 30 μM, 100 μM, and 530 μM, respectively. The enzyme is also sensitive to inhibition to a lesser degree by pyrimidine ribonucleotides. The pharmacologic agents, 6-mercaptopurine ribonucleotide, allopurinol ribonucleotide and mycophenolic acid are also inhibitors of human IMP dehydrogenase. A kinetic analysis indicates an ordered sequential reaction mechanism in which IMP binds first and XMP is released last.

Hexokinase Isoenzymes in Human Erythrocytes: Association of Type II with Fetal Hemoglobin

Hexokinase activity in human erythrocytes is associated with three electrophoretically distinct bands. Normal adult erythrocytes contain hexokinases Types I and III. Type II hexokinase is present in the erythrocytes of newborn infants and absent from those of normal adults; it is, however, present in erythrocytes of adults with hereditary persistence of fetal hemoglobin. Type II hexokinase and fetal hemtoglobin appear to be associated.

Selective Fetal Malnutrition: the Effect of in Vivo Ethanol Exposure upon in Vitro Placental Uptake of Amino Acids in the Non-Human Primate

Summary: In vitro uptake (45 and 90 minutes) of amino acids, alpha-amino isobutyric (AIB) and valine (VAL), was measured in six placentae from the nonhuman primate, Macacca fascicularis. Three of the pregnant primates were chronically treated with ethanol before and throughout pregnancy (CHR); one during the last trimester only (LT); and two were controls (C). Compared to the C placentae, the LT placenta had significantly decreased uptake only for AIB at 45 min: 33.4 ± 6.8% reduction (mean ± S.E.) (P < 0.05). In contrast, the CHR placentae demonstrated significantly reduced uptake (P < 0.01) for both amino acids at both time points. Percent reduction at 45 and 90 min: AIB, 35.2 ± 6.5% and 32.6 ± 5.6% and VAL, 38.7 ± 2.9% and 22.1 ± 4.1%. The results indicate that chronic in vivo ethanol exposure impairs the in vitro placental uptake of two actively transported amino acids, using an animal with a placenta almost identical to the human.

Control of AMP deaminase 1 binding to myosin heavy chain.

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178-333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660-674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8-12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178-333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660-674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8-12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.

Control of the purine nucleotide cycle in extracts of rat skeletal muscle: Effects of energy state and concentrations of cycle intermediates

The enzymes of the purine nucleotide cycle-AMP deaminase, adenylosuccinate synthetase, and adenylosuccinate lyase-were examined as a functional unit in an in vitro system which simulates the purine nucleotide composition of sarcoplasm. Activity of each cycle enzyme in extracts of rat skeletal muscle was observed to increase as ATP/ADP, reflecting the energy state of the system, was lowered from approximately 50 to 1. The increase in AMP deaminase activity could be attributed to effects of energy state and factors such as AMP concentration, which are obligatorily coupled to energy state. The increases in synthetase and lyase activities were accounted for by increases in the concentration of IMP and adenylosuccinate, respectively. The inhibitory influence of IMP concentration on synthetase activity reported in other systems was not observed in this system; synthetase activity progressively increased as IMP concentration was raised to approximately 4 mM, and apparent saturation occurred at concentrations above 4 mM. Also, adenylosuccinate was found to be an activator of AMP deaminase. The results of this study document that the activities of the enzymes of the purine nucleotide cycle increase in parallel at low energy states, and the components of the cycle function as a coordinated unit with individual enzyme activities linked via concentrations of cycle intermediates.

Expression of three stage-specific transcripts of AMP deaminase during myogenesis

AMP deaminase, a ubiquitous enzyme in eucaryotes, plays a central role in energy metabolism. In the present study, RNase protection analyses and immunoprecipitation with tissue-specific antisera were used to examine the transcripts and peptides of AMP deaminase produced during myogenesis in vivo and during myocyte differentiation in vitro. In embryonic muscle and undifferentiated myoblasts, a 3.4-kilobase (kb) transcript encoded a 78-kilodalton (kDa) AMP deaminase peptide that cross-reacted with antisera raised to the AMP deaminase isoform purified from kidney of the adult animal. In perinatal muscle and myocytes at an intermediate stage of differentiation in vitro, a 2.5-kb transcript was produced, and it encoded a 77.5-kDa AMP deaminase peptide that cross-reacted with antisera to the isoform purified from adult heart muscle. At about the time of birth, another 2.5-kb AMP deaminase transcript that encoded an 80-kDa peptide became detectable. This peptide cross-reacted with antisera to the predominant isoform purified from adult skeletal muscle.

Immunologic evidence for three isoforms of AMP deaminase (AMPD) in mature skeletal muscle

Four rabbit polyclonal antisera to purified AMP deaminase (AMPD) isozymes were used to precipitate homogenate AMPD activity from dissected gracilis, soleus and gastrocnemius muscles of the cat, rabbit, rat, mouse, Rhesus monkey, human and toad. The antisera were also tested against other unusual muscles: autonomically innervated striated muscle of the upper esophagus (UEM), skeletal muscle of patients with myo-AMPD deficiency and extraocular muscles (EOM) of humans and Rhesus monkeys. The reference antiserum, M, prepared against human psoas muscle AMPD, precipitated > 90% AMPD from all primate skeletal muscles tested, and from type-2 muscles of all mammals tested, but < 75% from cat and rodent soleus, toad gastrocnemius and primate UEM, EOM and myo-AMPD deficient muscles. Thus, a second isozyme was clearly indicated. Antibody B, against rat liver and kidney AMPD, had no effect with any muscle specimen. Antibody C, against rat heart AMPD, produced additive precipitation of AMPD from soleus of rat and mouse, while antibody E1, against human red cell (and heart) AMPD, produced additive AMPD precipitation from toad gastrocnemius, cat soleus and muscles of several AMPD-deficient humans. A second AMPD isozyme thus accounted for as much as 25% of total activity in some animal red muscles, but no more than 5% in human mixed muscles. At least one more isozyme is needed to account for muscle AMPD unreactive with all antibodies tested in rabbit soleus, toad gastrocnemius and primate UEM and EOM. A list is appended of the approximate AMPD activity in various human cells and tissues.

Long-range restriction site mapping of a syntenic segment conserved between human chromosome 1 and mouse chromosome 3

A linkage map determined from segregation analysis of 338 meiotic events in an interspecific mouse cross was utilized to help investigate genomic organization of a linkage group conserved between human chromosome 1p and mouse chromosome 3. Using pulsed-field gel electrophoresis, the genes encoding the lymphocyte adhesion molecule human CD2/murine Ly-37, the alpha 1-subunit of Na, K-ATPase, the beta-subunit of thyrotropin, the beta-subunit of nerve growth factor, and muscle adenylate deaminase were similarly positioned on long-range restriction maps in both species. These studies indicate that the development of detailed genetic maps using interspecific Mus crosses facilitates rapid analysis of murine genomic organization and may enable physical mapping of syntenic regions within the human genome. Moreover, the data suggest profound conservation of genomic organization during mammalian evolution.

Transmural Gradient in High-Energy Phosphate Content in Patients with Coronary Artery Disease

In 16 patients undergoing elective coronary artery bypass, transmural biopsies were performed during bypass but before global ischemia. Subendocardial and subepicardial halves were separately assayed in each sampled tissue. Adenosine triphosphate (ATP) levels, total adenine nucleotide content (sigma Ad), and creatine phosphate (CP) content were significantly higher (p less than 0.005) in the subepicardium than the subendocardium in regions of the heart distal to major occlusions: 35.36 +/- 2.12 nmole/mg versus 28.7 +/- 1.7 (ATP), 42.24 +/- 2.04 versus 35.6 +/- 1.6 (sigma Ad), and 29.99 +/- 4.32 +/- versus 16.35 +/- 3.48 (CP). The opposite was true in two hearts with normal coronary arteries, in which high-energy phosphates tended to be higher in the subendocardium than the subepicardium. A transmural metabolic gradient therefore exists in regions of the myocardium distal to significant coronary occlusive disease. The subendocardiums relative depression in metabolic reserve cold determine its susceptibility to ischemic damage and influence techniques designed to preserve the heart during ischemia.