The enzymatic synthesis of 5-amino-4-imidazolecarboxamide riboside triphosphate (ZTP).

5-Amino-4-imidazolecarboxamide riboside triphosphate (ZTP) is thought to play a regulatory role in cellular metabolism. Unlike other nucleoside triphosphates, ZTP is synthesized in a one-step reaction in which the pyrophosphate group of 5-phosphoribosyl-l-pyrophosphate is transferred to the riboside monophosphate (ZMP) in a reaction catalyzed by 5-phosphoribosyl-l-pyrophosphate synthetase; reversal of this reaction leads to dephosphorylation of ZTP to ZMP. This unusual route of synthesis (and catabolism) of ZTP may be important in defining its metabolic effects in the cell.

Reversal of creatine kinase translational repression by 3' untranslated sequences.

A subline of U937 cells (U937D) was obtained in which creatine kinase B (CK-B) messenger RNA was present and bound to ribosomes, but CK activity was undetectable. Transformation of U937D cells with retrovirus vectors that contain the 3' untranslated region (3' UTR) of CK-B messenger RNA exhibited CK activity with no change in abundance of CK-B mRNA. The 3' UTR formed a complex in vitro with a component of S100 extracts from wild-type cells. This binding activity was not detectable in S100 extracts from cells that expressed CK activity after transformation with the 3' UTR-containing vector. These results suggest that translation of CK-B is repressed by binding of a soluble factor or factors to the 3' UTR.

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.

Studies on the exaggerated natriuretic response to a saline infusion in the hypothyroid rat.

The exaggerated natriuresis of hypothyroid rats receiving a 5% saline infusion was studied to determine the mechanism and the site within the nephron responsible for this increase in sodium excretion. Sodium clearance (C(Na)) and fractional sodium excretion were both demonstrated to be greater in hypothyroid rats for any amount of sodium infused. The rate of increase in fractional sodium excretion in response to saline loading was 3.4 times greater in hypothyroid animals. At the conclusion of the diuresis some of the hypothyroid animals excreted greater than 45% of the filtered sodium load, while no control animal excreted more than 12% of the filtered sodium load. The mean clearance of insulin during the saline diuresis was 36.6% lower (P < 0.001) in the hypothyroid rats. D-Aldosterone given to hypothyroid animals 3 hr before the experiment did not alter the magnitude or rate of increase in fractional sodium excretion. Inulin space determinations in nephrectomized rats revealed that extracellular fluid volume was contracted by 17.1% in the hypothyroid rats (P < 0.01). Plasma sodium was not significantly different in hypothyroid and control animals.A limit on solute free water reabsorption (T(e) (H2O)) per osmolar clearance (C(Osm)) was demonstrated in the hypothyroid rats when these animals excreted greater than 12% of the filtered osmotic load. The limit on T(e) (H2O) formation was associated with an acceleration in the rate of sodium excretion and a decline in the rate of potassium excretion. Early in the diuresis when C(Osm), C(Na), and T(e) (H2O) were comparable in hypothyroid and control rats, the filtered sodium load was 31% lower (P < 0.01) in the hypothyroid animals. These findings indicate that diminished thyroid hormone activity decreases renal sodium reabsorptive capacity. Indirect evidence suggests that the distal and possibly the proximal tubules are the sites of this diminished sodium reabsorption in hypothyroid animals.

Inosinic acid dehydrogenase activity in the Lesch-Nyhan syndrome.

Inosinic acid dehydrogenase was evaluated in normal subjects and in patients with the Lesch-Nyhan syndrome. A significant difference in activity was found between erythrocytes derived from normal controls (1.21+/-0.47 pmoles/hr per mg protein) and from 15 patients with the Lesch-Nyhan syndrome (6.72+/-6.23 pmoles/hr per mg protein). However, no difference in activity was demonstrable in muscle or leukocytes derived from normal and Lesch-Nyhan patients. The increased activity of inosinic acid dehydrogenase in erythrocytes from patients with the Lesch-Nyhan syndrome is due to stabilization of the enzyme in vivo as well as the absence of an inhibitor which is present in erythrocytes from normal subjects.

Basis for the control of purine biosynthesis by purine ribonucleotides.

An animal model was used to determine the basis for the increase in purine biosynthesis that results from hepatic depletion of purine nucleotides, such as seen in patients with type I glycogen storage disease or following fructose administration. Mice were injected intravenously with glucose or fructose, 2.5 mg/g of body weight, and the animals were killed at 0, 3, and 30 min following carbohydrate infusion. Fructose, but not glucose, administration led to a threefold increase in [14C]glycine incorporation into hepatic purine nucleotides documenting an increase in the rate of purine biosynthesis in the liver of fructose-treated animals. In the fructose, but not the glucose-treated animals, there was a reduction in the hepatic content of purine nucleotides that are inhibitory for amidophosphoribosyltransferase, the enzyme that catalyzes the first reaction unique to the pathway of purine biosynthesis. PP-ribose-P, an important metabolite in the control of purine biosynthesis, was increased 2,3-fold in liver following fructose, but not glucose administration. In conjunction with the decrease in inhibitory nucleotides and increase in PP-ribose-P 29% of amidophosphoribosyltransferase was shifted from the large inactive to the small active form of the enzyme. Results of these studies demonstrate that the end-products of the pathway, purine nucleotides, control the activity of the enzyme that catalyzes the first reaction leading to purine nucleotide synthesis either through a direct effect of purine nucleotides on the enzyme, through an indirect effect of the change in nucleotides on PP-ribose-P synthesis, or a combination of these effects. The resultant changes in amidophosphoribosyltransferase conformation and activity provide a basis for understanding the increase in purine biosynthesis that results from hepatic depletion of purine nucleotides.

Alternative splicing: a mechanism for phenotypic rescue of a common inherited defect.

Approximately 2% of Caucasians and African-Americans are homozygous for a nonsense mutation in exon 2 of the AMPD1 (AMP deaminase) gene. These individuals have a high grade deficiency of AMPD activity in their skeletal muscle. More than 100 patients with AMPD1 deficiency have been reported to have symptoms of a metabolic myopathy, but it is apparent many individuals with this inherited defect are asymptomatic given the prevalence of this mutant. Results of the present study provide a potential molecular explanation for "correction" of this genetic defect. Alternative splicing eliminates exon 2 in 0.6-2% of AMPD1 mRNA transcripts in adult skeletal muscle. Expression studies document that AMPD1 mRNA, which has exon 2 deleted, encodes a functional AMPD peptide. A much higher percentage of alternatively spliced transcripts are found during differentiation of human myocytes in vitro. Transfection studies with human minigene constructs demonstrate that alternative splicing of the primary transcript of human AMPD1 is controlled by tissue-specific and stage-specific signals. Alternative splicing of exon 2 in individuals who have inherited this defect provides a mechanism for phenotypic rescue and variations in splicing patterns may contribute to the variability in clinical symptoms.

Disruption of the purine nucleotide cycle. A potential explanation for muscle dysfunction in myoadenylate deaminase deficiency.

A patient with symptoms of easy fatigability, postexercise myalgias, and delayed recovery of muscle strength after activity is described. Skeletal muscle from this patient had <1.0% normal myoadenylate deaminase activity and NH(3) was not released from muscle after ischemic exercise. In association with this enzyme deficiency, exercise led to a >90% reduction in muscle content of adenine nucleotides. No inosine monophosphate accumulated after exercise and total purine content of the muscle fell to 21% of control. Repletion of the adenine nucleotide pool in this patient was delayed compared to controls, and ATP content had only returned to 68% of control at 165 min after exercise. These studies demonstrate that disruption of the purine nucleotide cycle as a consequence of myoadenylate deaminase deficiency results in marked alterations in ATP content of muscle, and potentially, these changes in ATP content could account for muscle dysfunction in this patient.

Defective adenosine triphosphate synthesis. An explanation for skeletal muscle dysfunction in phosphate-deficient mice.

The basis for skeletal muscle dysfunction in phosphate-deficient patients and animals is not known, but it is hypothesized that intracellular phosphate deficiency leads to a defect in ATP synthesis. To test this hypothesis, changes in muscle function and nucleotide metabolism were studied in an animal model of hypophosphatemia. Mice were made hypophosphatemic through restriction of dietary phosphate intake. Gastrocnemius function was assessed in situ by recording isometric tension developed after stimulation of the nerve innervating this muscle. Changes in purine nucleotide, nucleoside, and base content of the muscle were quantitated at several time points during stimulation and recovery. Serum concentration and skeletal muscle content of phosphorous are reduced by 55 and 45%, respectively, in the dietary restricted animals. The gastrocnemius muscle of the phosphate-deficient mice fatigues more rapidly compared with control mice. ATP and creatine phosphate content fall to a comparable extent during fatigue in the muscle from both groups of animals; AMP, inosine, and hypoxanthine (indices of ATP catabolism) appear in higher concentration in the muscle of phosphate-deficient animals. Since total ATP use in contracting muscle is closely linked to total developed tension, we conclude that the comparable drop in ATP content in association with a more rapid loss of tension is best explained by a slower rate of ATP synthesis in the muscle of phosphate-deficient animals. During the period of recovery after muscle stimulation, ATP use for contraction is minimal, since the muscle is at rest. In the recovery period, ATP content returns to resting levels more slowly in the phosphate-deficient than in the control animals. In association with the slower rate of ATP repletion, the precursors inosine monophosphate and AMP remain elevated for a longer period of time in the muscle of phosphate-deficient animals. The slower rate of ATP repletion correlates with delayed return of normal muscle contractility in the phosphate-deficient mice. These studies suggest that the slower rate of repletion of the ATP pool may be the consequence of a slower rate of ATP synthesis and this is in part responsible for the delayed recovery of normal muscle contractility.

Disruption of the purine nucleotide cycle by inhibition of adenylosuccinate lyase produces skeletal muscle dysfunction.

Controversy exists as to whether the purine nucleotide cycle is important in normal skeletal muscle function. Patients with disruption of the cycle from a deficiency of AMP deaminase exhibit variable degrees of muscle dysfunction. An animal model was used to examine the effect of inhibition of the purine nucleotide cycle on muscle function. When the compound 5-amino-4-imidazolecarboxamide riboside (AICAriboside) is phosphorylated to the riboside monophosphate in the myocyte it is an inhibitor of adenylosuccinate lyase, one of the enzymes of the purine nucleotide cycle. AICAriboside was infused in 28 mice, and 22 mice received saline. Gastrocnemius muscle function was assessed in situ by recording isometric tension developed during stimulation. The purine nucleotide content of the muscle was measured before and after stimulation. Disruption of the purine nucleotide cycle during muscle stimulation was evidenced by a greater accumulation of adenylosuccinate, the substrate for adenylosuccinate lyase, in the animals receiving AICAriboside (0.60 +/- 0.10 vs. 0.05 +/- 0.01 nmol/mumol total creatine, P less than 0.0001). There was also a larger accumulation of inosine monophosphate in the AICAriboside vs. saline-treated animals at end stimulation (73 +/- 6 vs. 56 +/- 5 nmol/mumol total creatine, P less than 0.03). Inhibition of flux through the cycle was accompanied by muscle dysfunction during stimulation. Total developed tension in the AICAriboside group was 40% less than in the saline group (3,023 +/- 1,170 vs. 5,090 +/- 450 g . s, P less than 0.002). An index of energy production can be obtained by comparing the change in total phosphagen content per unit of developed tension in the two groups. This index indicates that less high energy phosphate compounds were generated in the AICAriboside group, suggesting that interruption of the purine nucleotide cycle interfered with energy production in the muscle. We conclude from these studies that defective energy generation is one mechanism whereby disruption of the purine nucleotide cycle produces muscle dysfunction.

Myoadenylate deaminase deficiency. Functional and metabolic abnormalities associated with disruption of the purine nucleotide cycle.

To assess the role of the purine nucleotide cycle in human skeletal muscle function, we evaluated 10 patients with AMP deaminase deficiency (myoadenylate deaminase deficiency; MDD). 4 MDD and 19 non-MDD controls participated in an exercise protocol. The latter group was composed of a patient cohort (n = 8) exhibiting a constellation of symptoms similar to those of the MDD patients, i.e., postexertional aches, cramps, and pains; as well as a cohort of normal, unconditioned volunteers (n = 11). The individuals with MDD fatigued after performing only 28% as much work as their non-MDD counterparts. Muscle biopsies were obtained from the four MDD patients and the eight non-MDD patients at rest and following exercise to the point of fatigue. Creatine phosphate content fell to a comparable extent in the MDD (69%) and non-MDD (52%) patients at the onset of fatigue. Following exercise the 34% decrease in ATP content of muscle from the non-MDD subjects was significantly greater than the 6% decrease in ATP noted in muscle from the MDD patients (P = 0.048). Only one of four MDD patients had a measurable drop in ATP compared with seven of eight non-MDD patients. At end-exercise the muscle content of inosine 5'-monophosphate (IMP), a product of AMP deaminase, was 13-fold greater in the non-MDD patients than that observed in the MDD group (P = 0.008). Adenosine content of muscle from the MDD patients increased 16-fold following exercise, while there was only a twofold increase in adenosine content of muscle from the non-MDD patients (P = 0.028). Those non-MDD patients in whom the decrease in ATP content following exercise was measurable exhibited a stoichiometric increase in IMP, and total purine content of the muscle did not change significantly. The one MDD patient in whom the decrease in ATP was measurable, did not exhibit a stoichiometric increase in IMP. Although the adenosine content increased 13-fold in this patient, only 48% of the ATP catabolized could be accounted for by the combined increases of adenosine, inosine, hypoxanthine, and IMP. Studies performed in vitro with muscle samples from seven MDD and seven non-MDD subjects demonstrated that ATP catabolism was associated with a fivefold greater increase in IMP in non-MDD muscle. There were significant increases in AMP and ADP content of the muscle from MDD patients following ATP catabolism in vitro, while there was no detectable increase in AMP or ADP in non-MDD muscle. Adenosine content of MDD muscle increased following ATP catabolism, but there was no detectable increase in adenosine content of non-MDD muscle following ATP catabolism in vitro. These studies demonstrate that AMP deaminase deficiency leads to reduced entry of adenine nucleotides into the purine nucleotide cycle during exercise. We postulate that the resultant disruption of the purine nucleotide cycle accounts for the muscle dysfunction observed in these patients.

Functionally distinct elements are required for expression of the AMPD1 gene in myocytes.

AMP deaminase (AMPD) is an enzyme found in all eukaryotic cells. Tissue-specific and stage-specific isoforms of this enzyme are found in vertebrates, and expression of these different isoforms is determined by selective expression of the multiple genes. The AMPD1 gene is expressed predominantly in skeletal muscle, in which transcript abundance is controlled by stage-specific and fiber type-specific signals. This enzyme activity is presumed to be important in skeletal muscle because a metabolic myopathy develops in individuals with an inherited deficiency of AMPD1. In the present study, cis- and trans-acting factors that control expression of AMPD1 have been identified. Two cis-acting elements located within 100 nucleotides of the transcriptional start site are required for muscle-specific expression of AMPD1. One element (-100 to -79) behaves like a tissue-specific enhancer, and it interacts with protein(s) found predominantly in nuclei of myoblasts and myotubes. This element is similar in sequence to an MEF2 binding motif, and it contains an A/T core that is essential for enhancer activity and binding of a nuclear protein(s). The second element (-60 to -40) has properties of a stage-specific promoter in that it is essential for muscle-specific expression of the AMPD1 promoter, does not confer muscle-specific expression on a heterologous promoter construct, and interacts with a protein(s) restricted to nuclei of differentiated myotubes. Interaction between these functionally distinct elements may be required for regulating the expression of AMPD1 during myocyte differentiation and in different muscle fiber types.

Exon recognition and nucleocytoplasmic partitioning determine AMPD1 alternative transcript production.

Two mature transcripts are produced from the rat AMP deaminase 1 (AMPD1) gene, one that retains exon 2 and one from which exon 2 has been removed. The ratio of these two transcripts is controlled by stage-specific and tissue-specific signals (I. Mineo, P. R. H. Clarke, R. L. Sabina, and E. W. Holmes, Mol. Cell. Biol. 10:5271-5278, 1990; R. L. Sabina, N. Ogasawara, and E. W. Holmes, Mol. Cell. Biol. 9:2244-2246, 1989). By using transfection studies with native, mutant, and chimeric minigene constructs, two steps in RNA processing that determine the ratio of these two transcripts have been identified. The first step is recognition of this exon in the primary transcript. The primary transcript is subject to alternative splicing in which exon 2 is either recognized and thereby included in the mature mRNA or is ignored and retained in a composite intron containing intron 1-exon 2-intron 2. The following properties of the primary transcript influence exon recognition. (i) Exon 2 is intrinsically difficult to recognize, possibly because of its small size (only 12 bases) and/or a suboptimal 5' donor site at the exon 2-intron 2 boundary. (ii) Intron 2 plays a permissive role in recognition of exon 2 because it is removed at a relatively slow rate, presumably because of the suboptimal polypyrimidine tract in the putative 3' branch site. The second step in RNA processing that influences the ratio of mature transcripts produced from the AMPD1 gene occurs subsequent to the ligation of exon 2 to exon 1. An RNA intermediate, composed of exon 1-exon 2-intron 2-exon 3, is produced in the first processing step, but it is variably retained in the nucleus. Retention of this intermediate in the nucleus is associated with accumulation of the mature mRNA containing exon 2, while cytoplasmic escape of this intermediate is reactions, exon recognition and nucleocytoplasmic partitioning, determine the relative abundance of alternative mRNAs derived from the AMPD1 gene.

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.

A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5' splice donor site not present in the primary transcript of AMPD1.

AMP deaminase (AMPD) is a central enzyme in eucaryotic energy metabolism, and tissue-specific as well as stage-specific isoforms are found in many vertebrates. This study demonstrates the AMPD1 gene product in rat is alternatively spliced. The second exon, a 12-base miniexon, was found to be excluded or included in a tissue-specific and stage-specific pattern. This example of cassette splicing utilizes a unique pathway through an RNA intermediate that generates an alternative 5' splice donor site at the point where exon 2 is ligated to exon 1. In the analogous intermediate of human AMPD1, the potential 5' splice donor site created at the boundary of exon 1 and exon 2 was a poor substrate for splicing because of differences in exon 2 sequences, and human AMPD1 was not alternatively spliced. These results demonstrate that in some cases alternative splicing may proceed through an RNA intermediate that generates an alternative splice donor site not present in the primary transcript. Discrimination between alternative 5' splice donor sites in the RNA intermediate of AMPD1 is apparently controlled by tissue-specific and stage-specific signals.

Renal and hepatic output of glutathione in plasma and whole blood.

Measurement of glutathione (GSH) output by the rat kidney and liver demonstrated a substantial net release into red cells across both tissues. The results suggest important roles for kidney and liver in the maintenance of GSH concentrations in red cells and a significant role for the red cell in the interorgan transport of GSH.

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.

A rapid method for the diagnosis of acute uric acid nephropathy.

Acute uric acid nephropathy is a reversible type of renal failure that results from the deposition of uric acid crystals in the collecting tubules. The present study has compared a number of laboratory tests in 5 patients with a clinical diagnosis of this disorder and 27 patients with acute renal failure of other causes. Neither the serum creatinine, BUN, serum urate concentrations, nor the ratio of serum urate:BUN differentiated between these two groups of patients. However, the ratio of uric acid to creatinine concentration on a random urine specimen did differentiate between these two patient populations. All patients with uric acid nephropathy had a ratio greater than 1.0, while all patients with other types of acute renal failure had ratios of less than 1.0.

Repetitive episodes of brief ischaemia (12 min) do not produce a cumulative depletion of high energy phosphate compounds.

During myocardial ischaemia the purine (ATP, GTP) and pyrimidine (CTP, UTP) nucleotide content of the myocyte falls. When the ischaemic episode resolves, many hours or even days are required for restoration of nucleotide pools. These observations suggest that repetitive episodes of ischaemia might produce progressive depletion of nucleotide pools. In order to determine the effect of repetitive episodes of brief ischaemia on nucleotide pools, open-chest dogs underwent three 12 min periods of occlusion of the left anterior descending coronary artery, with each occlusion followed by 10 min of reperfusion. During the first occlusion nucleotide pools decreased by 30% (ATP); 36% (GTP), 52% (CTP), and 48% (UTP). The subsequent two occlusions produced no further decrease in nucleotide pools. The myocardial content of adenine nucleotide catabolites (adenosine + inosine + hypoxanthine) tended to be greater during the first occlusion than during the subsequent occlusions, and substrate delivery (ie regional myocardial blood flow) was similar during each of the periods of ischaemia. These results indicate that a decrease in the rate of nucleotide degradation, rather than an increase in nucleotide synthesis, accounts for the maintenance of nucleotide content during subsequent ischaemic episodes after the initial ischaemic period. Thus repetitive episodes of regional ischaemia do not produce a cumulative decrease in the high energy phosphate content of the myocardium.

Gene therapy: socioeconomic and ethical issues. A roundtable discussion.

Gene therapy research has the potential to revolutionize the way in which many human diseases are treated. Despite its enormous potential, roundtable panelists concluded that the field needs time to mature scientifically without pressure to develop a marketable therapeutic product. In addition, health care decision makers, physicians, and the lay public need to be educated on the future medical, economic, and ethical ramifications of gene therapy.