Molecular analysis of argininosuccinate synthetase deficiency in human fibroblasts.

We have analyzed cultured skin fibroblasts derived from patients with argininosuccinate synthetase deficiency for alterations in gene structure, mRNA content, and protein structure. Genomic DNA was digested with the endonucleases EcoRI or HindIII, and the fragments were analyzed by Southern blotting and hybridization with a cDNA probe for argininosuccinate synthetase. The blot pattern is complex because there are at least 10 copies of argininosuccinate synthetase-like genes scattered over multiple human chromosomes. All nine patients studied showed patterns of DNA fragments that were indistinguishable from the normal control cell lines, and despite the possibility that the complexity could mask some changes, major deletions of the active gene(s) were not present. Blot hybridization of RNA indicated the presence of hybridizable mRNA of approximately normal size in seven of seven individuals examined with a suggestion of some heterogeneity. Analysis of enzyme antigen by protein transfer from NaDodSO4 containing polyacrylamide gels revealed considerable heterogeneity. This analysis revealed no cross-reacting material (CRM) in nine cell lines, CRM of normal molecular weight in one cell line, and CRM of reduced molecular weight in one cell line. These findings suggest that the genes for argininosuccinate synthetase in most citrullinemia patients are transcribed and produce stable mRNA. These mRNA either are not translated, or the translation product (enzyme) is rapidly degraded or is immunologically nonreactive. Defective gene expression in this disorder appears to involve abnormal mRNA, which may be altered by point mutations, frame shift mutations, deletions, insertions or particularly by abnormal RNA processing.

Characterization of point mutations in the same arginine codon in three unrelated patients with ornithine transcarbamylase deficiency.

Point mutations in the X-linked ornithine transcarbamylase (OTC) gene have been detected at the same Taq I restriction site in 3 of 24 unrelated probands with OTC deficiency. A de novo mutation could be traced in all three families to an individual in a prior generation, confirming independent recurrence. The DNA sequence in the region of the altered Taq I site was determined in the three probands. In two unrelated male probands with neonatal onset of severe OTC deficiency, a guanine (G) to adenine (A) mutation on the sense strand (antisense cytosine [C] to thymine [T]) was found, resulting in glutamine for arginine at amino acid 109 of the mature polypeptide. In the third case, where the proband was a symptomatic female, C to T (sense strand) transition converted residue 109 to a premature stop. These results support the observation that Taq I restriction sites, which contain an internal CG, are particularly susceptible to C to T transition mutation due to deamination of a methylated C in either the sense or antisense strand. The OTC gene seems especially sensitive to C to T transition mutation at arginine codon 109 because either a nonsense mutation or an extremely deleterious missense mutation will result.

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.

Patient selection may affect gene therapy success. Dominant negative effects observed for ornithine transcarbamylase in mouse and human hepatocytes.

We have achieved significant improvement of ornithine transcarbamylase deficiency (OTCD) in a mouse model through adenoviral-mediated gene transfer of the human ornithine transcarbamylase cDNA. Substantial reduction in orotic aciduria was observed within 24 h of treatment. Metabolic correction was later associated with phenotypic correction and moderate increase in enzymatic activity. In an effort to identify the level of gene expression required to achieve wild-type levels of enzyme activity we uncovered a dominant negative effect of the endogenous mutant protein on the activity of the delivered recombinant wild-type protein. This phenomenon is relevant to homomultimeric protein defects such as OTCD, represent a challenging category of disorders for gene therapy. Thus, although our findings indicate that adenoviral-mediated gene transfer may have potential as a short-term treatment for OTCD in humans and may be effective especially during catabolic crisis, the observations in this study suggest that careful patient selection based on mutation class may be essential for initial OTCD gene therapy trials, and perhaps, for other homomultimeric enzyme deficiencies being considered as gene therapy targets.

Arginine-mediated regulation of an argininosuccinate synthetase minigene in normal and canavanine-resistant human cells.

Regulation of argininosuccinate synthetase (AS) was studied by using minigenes containing 3 kilobases of DNA upstream from the TATAA box and 9 kilobases downstream (including the first four exons of the AS gene) ligated to either the cDNA for AS or to the chloramphenicol acetyltransferase (CAT) gene. Unlike the endogenous AS gene, expression of the CAT minigene was not elevated in Canr1 cells, which overproduce AS compared with parental RPMI-2650 cells. Expression of the CAT minigene in both stable and transient analyses was four- to five-fold higher in RPMI-2650 cells grown in citrulline medium than in cells grown in arginine medium. Although endogenous AS activity is not subject to metabolite regulation in Canr1 cells and expression of the CAT minigene in Canr1 cells was not increased when cells were grown in citrulline medium, expression of the CAT minigene was 10- to 22-fold greater when intracellular arginine pools were depleted by transient starvation for arginine and citrulline.

Molecular structure of the human argininosuccinate synthetase gene: occurrence of alternative mRNA splicing.

The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.

Generation of a mouse model for arginase II deficiency by targeted disruption of the arginase II gene.

Mammals express two isoforms of arginase, designated types I and II. Arginase I is a component of the urea cycle, and inherited defects in arginase I have deleterious consequences in humans. In contrast, the physiologic role of arginase II has not been defined, and no deficiencies in arginase II have been identified in humans. Mice with a disruption in the arginase II gene were created to investigate the role of this enzyme. Homozygous arginase II-deficient mice were viable and apparently indistinguishable from wild-type mice, except for an elevated plasma arginine level which indicates that arginase II plays an important role in arginine homeostasis.

Long-term expression of human argininosuccinate synthetase in mice following bone marrow transplantation with retrovirus-transduced hematopoietic stem cells.

Amphotropic and ecotropic packaging cell lines were used to obtain high titers (greater than 10(6) colony forming units/ml) of retroviruses encoding human argininosuccinate synthetase, and these viruses were used to transduce murine bone marrow cells using cocultivation in vitro. The bone marrow cells were transplanted into lethally irradiated recipient mice, and argininosuccinate synthetase activity was measured in peripheral blood. Transduction with amphotropic retrovirus resulted in short-term expression for a period of 1-8 weeks, and no animals expressed the human gene after 25 weeks. Over 60% of the animals transplanted with cells transduced with ecotropic retrovirus expressed the human gene 44 weeks post-transplant, although the level of expression varied over a wide range. Analysis of the DNA from transplanted animals demonstrated the presence of the human sequence in expressing animals, and S1 nuclease analysis of RNA confirmed the presence of the human RNA transcripts. Analysis of granulocyte/macrophage (GM) colonies derived from the bone marrow of transplanted, expressing animals revealed a correlation between the level of expression of the transduced gene with the percentage of GM colonies carrying the human gene sequence. These data demonstrate the feasibility of obtaining long-term expression of genes introduced into bone marrow cells using retroviral vectors and the feasibility of obtaining expression of a gene not normally expressed in bone marrow.

Morphologic evidence for L-citrulline conversion to L-arginine via the argininosuccinate pathway in porcine cerebral perivascular nerves.

Results from biochemical and pharmacologic studies suggest that Lcitrulline is taken up by cerebral perivascular nerves and is converted to Larginine for synthesizing nitric oxide (NO). The current study was designed using morphologic techniques to determine whether Lcitrulline is taken up into axoplasm of perivascular nerves and to explore the possibility that conversion of Lcitrulline to Larginine in these nerves is through the argininosuccinate pathway in porcine cerebral arteries. Results from light and electron microscopic autoradiographic studies indicated that dense silver grains representing L-[3H] citrulline uptake were found in cytoplasm of perivascular nerves, smooth muscle cells, and endothelial cells. The neuronal silver grains were significantly decreased in arteries pretreated with glutamine, which has been shown biochemically to block neuronal uptake of Lcitrulline. Results from light and electron microscopic immunohistochemical and histochemical studies indicate that dense nitric oxide synthase-immunoreactive (NOS-I), argininosuccinate synthetase-immunoreactive (ASS-I), and argininosuccinate lyase-immunoreactive (ASL-I) fibers were found in the adventitia of cerebral arteries. NOS-, ASS-, and ASL-immunoreactivities fibers were found in the axoplasm and in the endothelium. In whole-mount preparations, the NOS-I, ASS-I, and ASL-I fibers were completely coincident with NADPH diaphorase fibers, suggesting that axoplasmic ASS, ASL, and NOS were co-localized in the same neurons. These studies provide the first morphologic evidence indicating that Lcitrulline is taken up into cytoplasm of cerebral perivascular nerves and that the axoplasmic enzymes catalyzing the conversion of Lcitrulline to Larginine (for synthesizing NO) by argininosuccinate pathway always are co-localized in same neurons. These results support the hypothesis that Lcitrulline, the by-product of NO synthesis, is recycled to form Larginine for synthesizing NO in perivascular nerves to mediate cerebral neurogenic vasodilation. Results of the current morphologic studies also support the presence of Lcitrulline-Larginine cycle in cerebral vascular endothelium.

Molecular characterization of the murine argininosuccinate synthetase locus.

The cDNA and gene encoding murine argininosuccinate synthetase were cloned and characterized. The cDNA sequence predicts a peptide of 412 amino acids (aa) including the initiator methionine. There is 98% identity with the aa sequence of the human enzyme. The 3'-untranslated region of the cDNA includes two regions of sequence which are conserved between mouse, rat, human and cow. The murine gene contains 16 exons with the start codon occurring in exon 3. Although alternative splicing occurs in primates to include or exclude exon 2, exon 2 sequences were included in the murine mRNA in all tissues and developmental stages examined. The inclusion of exon 2 in murine mRNA, compared to the usual exclusion in human mRNA, may be explained by differences in the donor splice sequences for exon 2.

Immunohistochemical localization of argininosuccinate synthetase in the rat brain in relation to nitric oxide synthase-containing neurons.

The distribution of the urea cycle enzyme, argininosuccinate synthetase, in the rat brain was determined using immunohistochemistry. This enzyme participates in the only known metabolic pathway for citrulline, its condensation with aspartate to form argininosuccinate, which can then be cleaved to fumarate and arginine. It may thus provide a mechanism to recycle citrulline, formed in the nervous system via nitric oxide synthase activity, back to the nitric oxide precursor, L-arginine. Argininosuccinate synthetase immunoreactivity was detected in discrete populations of neurons throughout the brain. Double-staining with nicotinamide adenine dinucleotide phosphate (reduced form)-diaphorase histochemistry for the localization of nitric oxide synthase demonstrated that argininosuccinate synthetase coexists with nitric oxide synthase in some brain regions. However, many neurons were found that contained one of these two enzymes, but not the other. Thus some nitric oxide synthase-containing neurons appear able to recycle citrulline via argininosuccinate, while others do not. Additional roles for argininosuccinate synthetase in the brain are discussed.

Recycling of L-citrulline to sustain nitric oxide-dependent enteric neurotransmission.

Neurons that synthesize nitric oxide from arginine produce stoichiometric amounts of citrulline. We investigated whether nitric oxide-releasing enteric neurons have the capacity to recycle citrulline to arginine and thereby sustain nitrergic neurotransmission. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity, enzymes capable of citrulline to arginine conversion, were both localized in discrete populations of myenteric and submucosal neurons in the canine proximal colon. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity co-localized with neuronal beta-nicotinamide adenine dinucleotide phosphate diaphorase staining, a marker for nitric oxide synthase. The functional significance of argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity was shown by testing the effects of exogenous citrulline on responses to enteric inhibitory nerve stimulation, which were assessed by measuring contractions, inhibitory junction potentials and electrical slow waves. As shown previously, arginine analogues (L-nitroarginine methyl ester or L-nitroarginine; 100 microM) inhibited nitric oxide-dependent responses, and excess L-arginine restored inhibitory responses. Citrulline alone (0.1-2 mM) had no effect on nitrergic transmission under control conditions, but in the presence of L-nitroarginine methyl ester or L-nitroarginine, citrulline (0.1-2 mM) restored nitrergic transmission in a concentration-dependent manner. Other neutral amino acids (L-serine, L-leucine) did not mimic the effects of citrulline. Taken together, these data suggest that enteric nitrergic neurons have the enzymatic apparatus and functional capability of recycling citrulline to arginine.

Prenatal diagnosis of cystic fibrosis by using linked DNA markers in 138 pregnancies at 1-in-4 risk.

Prenatal diagnosis was carried out in 138 pregnancies at 1-in-4 risk for cystic fibrosis (CF) by using closely linked DNA markers, including XV-2c and KM-19. In fully informative families, 25 of 123 (20%) fetuses were predicted to be affected; 16 of these 25 pregnancies were terminated and 9 were continued. Postnatal sweat tests are completed in 42 cases; the diagnoses were confirmed in 4 of 4 infants predicted to be affected and in 37 of 38 infants predicted to be unaffected. One infant predicted to be a carrier had an abnormal sweat test after birth, but the mother also had an abnormal sweat test, and there was no evidence of an error in linkage analysis. The data indicate that prenatal diagnosis using linkage analysis is fully informative in most families and is highly reliable with either chorionic villus sampling or amniocentesis. Although outcome data are available on only 42 pregnancies, based on our experience, on general principles of linkage analysis, and on the tight linkage of the known DNA markers with CF, we recommend that DNA analysis replace microvillar intestinal enzyme analysis for 1-in-4 risk pregnancies when DNA is available from the propositus.

Alterations in arginine metabolic enzymes in trauma.

Arginine is the sole substrate for nitric oxide (NO) synthesis by NO synthases (NOS) and promotes the proliferation and maturation of human T-cells. Arginine is also metabolized by the enzyme arginase, producing urea and ornithine, the precursor for polyamine production. We sought to determine the molecular mechanisms regulating arginase and NOS in splenic immune cells after trauma. C3H/HeN mice underwent laparotomy as simulated moderate trauma or anesthesia alone (n = 24 per group). Six, 12, 24, or 48 h later, 6 animals from each group were sacrificed, and splenectomy was performed and plasma collected. Six separate animals had neither surgery nor anesthesia and were sacrificed to provide resting values (t = 0 h). Spleen arginase I and II and iNOS mRNA abundance, arginase I protein expression, and arginase activity were determined. Plasma NO metabolites (nitrite + nitrate) were also measured. Trauma increased spleen arginase I protein expression and activity (P = 0.01) within 12 and for at least 48 h after injury and coincided with up-regulated arginase I mRNA abundance at 24 h. Neither arginase II nor iNOS mRNA abundance in the spleen was significantly increased by trauma at 24 h. Plasma nitrite + nitrate was decreased in animals 48 h post-injury compared to anesthesia controls (P < 0.05). Trauma induces up-regulation of arginase I gene expression in splenic immune cells within 24 h of injury. Arginase II is not significantly up-regulated at that time point. Arginase I, rather than iNOS appears to be the dominant route for arginine metabolism in splenic immune cells 24 h after trauma.

Genetic contributions of the endothelial nitric oxide synthase gene to ovulation and menopause in a mouse model.

OBJECTIVE: To investigate the influence of the endothelial nitric oxide synthase gene (Nos3) on ovulatory capacity and reproductive senescence. DESIGN: Prospective, controlled study. SETTING: Academic research institution. SUBJECT(S): Laboratory mice with targeted mutagenesis of Nos3. INTERVENTION(S): Hyperstimulation protocol, oocyte culture, and ovarian histology using wild-type (Nos3(+/+); n = 20), heterozygous (Nos3(+/m); n = 39), and homozygous deficient (Nos3(m/m); n = 11) female mice; observation of reproductive outcomes. MAIN OUTCOME MEASURE(S): Number and survival of oocytes; onset of menarche and menopause. RESULT(S): The mean number of superovulated oocytes (18 +/- 36 vs. 41 +/- 4) and the 48-hour overall survival rate of embryos (65% vs. 81%) were significantly reduced for Nos3(m/m) female mice compared with Nos3(+/+) female mice. Nos3(m/m) females showed a significantly reduced number and size of antral follicles and corpora lutea compared with wild-type controls. Compared with Nos3(+/m) x Nos3(+/m) breedings, Nos3(m/m) x Nos3(m/m) breedings showed a higher female age at first litter (76.2 +/- 10.3 vs. 107.8 +/- 26.6 days), fewer litters (10.5 +/- 3.6 vs. 7. 8 +/- 4.2), and a lower female age at reproductive senescence (400.2 +/- 64.5 vs. 332.1 +/- 27.4 days), respectively. CONCLUSION(S): Our data suggest that Nos3 deficiency is associated with reduced ovulatory capacity and impaired early embryonic viability and that it influences the onset of menarche and menopause.

Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry.

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. Although rare, GAMT deficiency has been identified in children with seizures, extrapyramidal movements, developmental delay, myopathies and behavioral abnormalities. Treatment with creatine monohydrate has been proven to be effective. We describe an isotope dilution electrospray tandem mass spectrometry (ES-MS/MS) assay for the simultaneous determination of plasma GAA and creatine using multiple reaction monitoring (MRM), d(3)-creatine as the internal standard and derivatization of GAA and creatine as butyl-esters. We analysed plasma of 16 healthy adults and 20 healthy children as well as three affected children. Plasma GAA concentrations were 5.02+/-1.84 micromol/l (mean+/-S.D.) in adults, 3.91+/-0.76 micromol/l in children age 5-10 years and 11.57, 15.16, 14.36 micromol/l in children with GAMT deficiency. Plasma creatine concentrations were 34.7+/-15.25 micromol/l in adults, 58.96+/-22.30 micromol/l in children and 5.37, 8.15, 403.5 micromol/l in two untreated children and one treated child with GAMT deficiency, respectively. GAA can also be reliably measured from filter cards, which is sufficient to make the correct diagnosis while creatine is consistently falsely elevated probably secondary to liberation of red cell creatine. In nine healthy newborn infants, GAA concentrations from filter cards were 4.83+/-1.43 and 5.04+/-1.84 micromol/l in 16 healthy adults. We conclude that isotope dilution ES-MS/MS is ideal for rapid high-throughput diagnosis of GAMT deficiency both from plasma and filter paper cards. Using this technique neonatal screening is feasible for this treatable inborn error of creatine metabolism.

An endothelial nitric oxide synthase gene polymorphism is associated with preeclampsia.

OBJECTIVE: We sought to test the hypothesis that a polymorphism of the endothelial nitric oxide synthase gene (NOS3) is associated with preeclampsia. METHODS: We collected and performed polymerase chain reaction (PCR) on genomic DNA from pregnant patients with and without preeclampsia. Patient history and clinical course were evaluated. MAIN OUTCOME MEASURE(S): Frequency of the intron 4 polymorphism of NOS3 (designated allele A) among patients with preeclampsia compared with controls. Clinical features of patients with preeclampsia and the A allele compared with those patients with preeclampsia who did not have the A allele. RESULTS: The frequency of the A allele was 0.10 among controls versus 0.39 among patients with preeclampsia (p < 0.01). The odds ratio of developing preeclampsia when at least one A allele was present was 6.5 [95% confidence interval (CI): 2.1-19.7]. After adjusting for ethnic variation, the odds ratio increased to 7.2 (95% CI: 2.0-25.5). Among patients with preeclampsia, systolic blood pressure at the time of admission was higher for patients with at least one A allele compared with patients homozygous for the B allele (168 versus 156 mm Hg; p = 0.03), independent of gestational age (p = 0.01). CONCLUSION: These data provide evidence for an association between NOS3 and preeclampsia. In defined ethnic groups, this NOS3 may offer predictive information regarding the subsequent development of preeclampsia and its clinical course.

Isolation and characterization of argininosuccinate synthetase from human liver.

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.