Analysis of mRNA transcripts improves the success rate of molecular genetic testing in OTC deficiency.

BACKGROUND: Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of urea metabolism that can lead to hyperammonemic crises and orotic aciduria. To date, a total of 341 causative mutations within the OTC gene have been described. However, in about 20% of the patients with enzymatically confirmed OTC deficiency no mutation can be detected when sequencing of genomic DNA analyzing exons and adjacent intronic segments of the OTC gene is performed. METHODS: Standard genomic DNA analysis of the OTC gene in five consecutive patients from five families revealed no mutation. Hence, liver tissue was obtained by needle sampling or open biopsy and RNA extracted from liver was analyzed. RESULTS: Complex rearrangements of the OTC transcript (three insertions and two deletions) were found in all five patients. CONCLUSION: In patients with a strong suspicion of OTC deficiency despite normal results of sequencing exonic regions of the OTC gene, characterization of liver OTC mRNA is highly effective in resolving the genotype. Liver tissue sampling by needle aspiration allows for both enzymatic analysis and RNA based diagnostics of OTC deficiency.

Mitochondrial steps of arginine biosynthesis are conserved in the hydrogenosomes of the chytridiomycete Neocallimastix frontalis.

Arginine biosynthesis in eukaryotes is divided between the mitochondria and the cytosol. The anaerobic chytridiomycete Neocallimastix frontalis contains highly reduced, anaerobic modifications of mitochondria, the hydrogenosomes. Hydrogenosomes also occur in the microaerophilic flagellate Trichomonas vaginalis, which does not produce arginine but uses one of the mitochondrial enzymes, ornithine transcarbamoylase, in a cytosolic arginine dihydrolase pathway for ATP generation. EST sequencing and analysis of the hydrogenosomal proteome of N. frontalis provided evidence for two mitochondrial enzymes of arginine biosynthesis, carbamoylphosphate synthase and ornithine transcarbamoylase, while activities of the arginine dehydrolase pathway enzymes were not detectable in this fungus.

Biochemical responses of two erythrinidae fish to environmental ammonia.

The non-ionized form of ammonia is very toxic to many aquatic species. It is especially important in several aspects of fish biology. A large range of organismal strategies for coping with environmental stressors is usually observed in living organisms. Among those, the responses for managing chemical stressors are well studied. The present work compares biochemical responses of two evolutionarily close species, Hoplias malabaricus and Hoplerythrinus unitaeniatus, exposed to environmental ammonia. Adult fish were submitted to 1.0 mg/L of ammonium chloride for 24 hours, and plasma ammonia and urea levels were determined. The activities of OUC enzymes OCT and ARG, and the accessory enzyme GS, were quantified in liver extract and are expressed below in mumol/min/mg of wet tissue. Increases in OUC enzymes (GS from 1.14 to 2.43, OCT from 0.81 to 1.72, and ARG from 3.15 to 4.23), plasma ammonia (from 0.95 to 1.42 mmol/L), and plasma urea (from 0.82 to 1.53 mmol/L) were observed (p < 0.05) in H. malabaricus exposed to 1 mg/L of ammonia chloride. The GS in H. unitaeniatus increased from 1.43 to 1.84, however the OCT, ARG, and plasma urea from H. unitaeniatus did not change. These data indicate that each species responds differently to the same environmental stressor.

Mosaic analysis of small intestinal development using the spf(ash)-heterozygous female mouse.

Mosaic analysis using the spf(ash)-heterozygous female mouse was performed to clarify the cell lineage and cell behavior during small intestinal development with special attention given to the villus and crypt formation. The spf(ash) mutation, located on the X-chromosome, causes ornithine transcarbamylase (OTC) deficiency, which leads to mosaic expression of this enzyme in the small intestine of the heterozygous female mouse. In the small intestine in heterozygous fetuses, very small patches, which were aggregates of OTC-positive cells or negative cells, with no definite orientation to the villus structures were observed. In the neonatal small intestine, the intervillus region (the presumptive crypts) was polyclonal, and the majority of crypts were comprised exclusively cells of either genotype in 2-week-old small intestine. These results suggest that extensive migration and cell mixing of small intestinal epithelial cells, which have no definite correlation with the villus formation, occur in fetal stages of development, and that the crypt morphogenesis commences after birth independently of the monoclonality of the epithelial cells. Our data with the mosaic mice also reconfirmed the monoclonality of the adult small intestinal crypts demonstrated in mouse aggregation chimeras.

Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the Arginine Deiminase system of Streptococcus pyogenes.

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42 degrees C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42 degrees C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them.

Biochemical analysis of arginase and ornithine carbamoyltransferase in human vitreous humor.

BACKGROUND: The presence of arginase and ornithine carbamoyltransferase (OTC'ase) enzymes in human vitreous humor after death was investigated in this study. To the best of our knowledge, there is no prior report on the activity of arginase or ornithine carbamoyltransferase in human vitreous humor. METHODS: The presence of arginase and OTC'ase activities were examined in the human vitreous humor of 19 samples. Spectrophotometric methods were used to determine activities of arginase and OTC'ase. RESULTS: Arginase activity was detected in human vitreous humor, whereas OTC'ase activity was below the detection limit. Therefore, we focused on biochemical analysis of arginase in human vitreous humor. Kinetic properties of arginase activity in vitreous humor were optimized. In contrast to other arginases, optimal preincubation temperature and pH were 40 degrees C and 8.8, respectively. Km of vitreous arginase for L-arginine was 6 mM. Preincubation of the enzyme with Mn(2)+ ions caused a significant increase (33%) in arginase activity. CONCLUSIONS: The activity and presence of arginase as well as its kinetics in human vitreous are documented in this study. Biochemical functions and the importance of arginase in vitreous humor are not well understood. However, its presence may be explained by means of its involvement in polyamine biosynthesis as observed in the other extrahepatic tissues.

Buffer effect on the kinetics of ornithine carbamyl transferase by HPLC.

A kinetic study of ornithine carbamyl transferase (OCT) from Streptococcus faecalis has been carried out, employing a new HPLC method based on the direct determination of citrulline. A different kinetic pattern was observed in tris(hydroxymethyl)-aminomethane (TRIS) and triethanolamine (TEA) buffers: a ping-pong or an ordered sequential mechanism are suggested, respectively. The inhibition of Tris buffer on OCT reaction has also been demonstrated. The inhibition of Tris buffer on OCT reaction has also been demonstrated.

Mosaic pattern of ornithine transcarbamylase expression in spfash mouse liver.

Mosaicism of ornithine transcarbamylase expression was immunohistochemically examined in hepatocytes of spfash heterozygous female mouse livers. An immunohistochemical method using polyclonal antibodies against ornithine transcarbamylase visualized the mosaicism in serial paraffin sections. Very complicated mosaic patterns consisting of positive and negative hepatocytes were obtained in a section of adult liver, but a computer-aided three-dimensional analysis of serial sections demonstrated that patches with complicated shapes and various sizes, which are contiguous groups of positive or negative hepatocytes and are isolated in sections, connected very well with one another. No definite orientation such as portal-central was observed in the three-dimensional images of each patch. In balanced regions of the mosaicism, most individual plates along their straight portal-central lengths did not appear to be composed of only marker-positive or marker-negative hepatocytes. By contrast, in unbalanced regions of the mosaicism, individual plates along their total portal-central lengths often consisted entirely of a major type of hepatocytes. A patch appeared to be present in more than two lobules, but each patch did not constitute a complete lobule. Complicated mosaic patterns of patches were also seen in neonatal and postnatal livers. These results suggest that, although hepatocytes proliferate and migrate extensively during development, they might allocate their daughter cells contiguously and the orientation of their allocation might be random, leading to the formation of three-dimensionally large contiguous quasiclones of hepatocytes, the shapes of which are very complicated.

Characterization of vacuolar arginine uptake and amino acid efflux in Neurospora crassa using cupric ion to permeabilize the plasma membrane.

Treatment of Neurospora crassa mycelia with cupric ion has been shown to permeabilize the plasma and mitochondrial membranes. Permeabilized mycelia were shown to take up arginine into the vacuoles. Uptake was ATP-independent and appeared to be driven by an existing K+-gradient. The kinetic characteristics of the observed uptake were similar to those observed using vacuolar membrane vesicles: the Km for arginine uptake was found to be 4.2-4.5 mM. Permeabilized mycelia were used to study the regulation of arginine uptake into vacuoles. The results suggest that uptake is relatively indifferent to the contents of the vacuoles and is not affected by growth of mycelia in amino acid-supplemented medium. Efflux of arginine, lysine, and ornithine from vacuoles was also measured using mycelia permeabilized with cupric ion. Arginine release was shown to be specifically enhanced by cytosolic ornithine and/or increases in the vacuolar pool of arginine or ornithine. Lysine efflux was shown be indifferent to the presence of other amino acids. These observations emphasize the importance of vacuolar compartmentation in controlling arginine and ornithine metabolism and suggest that vacuolar compartmentation may play an important role in nitrogen homeostasis of filamentous fungi.

Subcellular localization of the enzymes of the arginine dihydrolase pathway in Trichomonas vaginalis and Tritrichomonas foetus.

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56-80%) in the non sedimentable fraction of both species. A large proportion (35-40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non-ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.

Explanation for different types of regulation of arginine biosynthesis in Escherichia coli B and Escherichia coli K12 caused by a difference between their arginine repressors.

In Escherichia coli K12, formation of the enzymes of arginine biosynthesis are controlled by arginine, with complete repression during growth with added arginine, severe repression (about 95%) during growth without added arginine and complete derepression during arginine-limited growth. In E. coli B, the degree of repression is not correlated with arginine concentrations. Under all conditions of growth enzyme formation is repressed, with repression being somewhat less in a medium with arginine than in a medium without arginine. These differences in repressibility between the two strains have been shown previously to be due to the presence of different alleles of argR, the gene for the arginine repressor. Here we have compared the binding of the two repressors to the operator sites of argF (ARG boxes). In DNase I footprinting and gel retardation experiments with argF ARG boxes we have shown that the arginine repressor of E. coli K12 bound to arginine (ArgRK-arg) has a greater affinity than the arginine repressor of E. coli B bound to arginine (ArgRB-arg), whereas free ArgRB (ArgRBf) has a much stronger affinity than free ArgRK (ArgRKf). The stronger binding of ArgRBf can explain the repression seen in E. coli B during arginine-limited growth and indicates that ArgRBf, but not ArgRKf, is able to repress enzyme synthesis under physiological conditions. The weaker repression of E. coli B than of E. coli K12 seen in the presence of arginine can be explained by the lower affinity of ArgRB-arg for operator sites as compared to ArgRK-arg. Another contributing cause for the weaker repression is the reduction of ArgRBf concentration due to autoregulation of the gene for the repressor. Thus the combined effects of repression by ArgRBf, but not ArgRKf, with the weaker repression by ArgRB-arg as compared to ArgRK-arg, convert the arginine dependent regulation in E. coli K12 to arginine independent regulation in E. coli B.

Effect of L-carnitine on cerebral and hepatic energy metabolites in congenitally hyperammonemic sparse-fur mice and its role during benzoate therapy.

Sparse-fur (spf) mutant mice with X-linked ornithine transcarbamylase deficiency were used to study the effect of L-carnitine on energy metabolites in congenital hyperammonemia. L-Carnitine was used at doses of 2, 4, 8, or 16 mmol/kg body weight (BW), and levels of ammonia, glutamine, glutamate, and some intermediates of energy metabolism were measured in brain and liver of spf/Y mice. Cerebral and hepatic levels of ammonia were decreased with 4 mmol L-carnitine (P < .001), whereas other doses did not seem to have any effect on this metabolite. Cerebral levels of glutamine were decreased following administration of L-carnitine at doses of up to 4 mmol/kg BW, whereas hepatic glutamine levels remained unaltered at all doses of L-carnitine. Both cerebral and hepatic levels of pyruvate, lactate, and alpha-ketoglutarate were decreased at doses of up to 8 mmol L-carnitine/kg BW. L-Carnitine treatment elevated adenosine triphosphate (ATP), free coenzyme A (CoA), and acetyl CoA levels in both brain and liver of spf/Y mice. Cytosolic and mitochondrial redox ratios of spf/Y mice, which were altered by congenital chronic hyperammonemia, were partially corrected by L-carnitine administration. L-Carnitine supplementation to spf/Y mice during sodium benzoate therapy also restored the availability of free CoA and ATP, thus counteracting the adverse effects of higher doses of sodium benzoate. These changes in free CoA and acetyl CoA levels could be due to the deinhibition of pantothenate kinase and stimulation of fatty acid oxidation by L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)

Arabidopsis chloroplasts dissimilate L-arginine and L-citrulline for use as N source.

When aseptically grown on defined medium with either L-arginine, L-citrulline, or nitrate as the sole N source, Arabidopsis plants grew and developed normally. Three catabolic activities, L-arginine iminohydrolase, L-ornithine carbamoyltransferase, and carbamate kinase, were found in stromal fractions of purified Arabidopsis chloroplasts. These activities dissimilate L-arginine and/or L-citrulline into L-ornithine, ammonium, bicarbonate, and ATP. In physiological tests with purified, intact Arabidopsis chloroplasts, L-[guanido-14C]arginine was rapidly taken up and about 10% was decomposed, releasing 14CO2. Therefore, chloroplasts can take up and dissimilate L-arginine. In principle, chloroplast arginine dissimilation allows Arabidopsis to use L-arginine and/or L-citrulline as general N sources for growth. However, plants rarely encounter exogenous L-arginine and/or L-citrulline in amounts exceeding their biosynthetic needs. Therefore, L-arginine and L-citrulline might serve as endogenous N sources.

Selection of variant hepatoma cells in liver-specific growth media: regulation at the mRNA level.

Three liver-specific growth media, respectively free of arginine (Arg-), tyrosine (Tyr-) and glucose (G-), have been used to characterize cells of the rat H4IIEC3, human HepG2 and mouse BW hepatoma lines. Cells of clone FaO, a derivative of line H4IIEC3, freely grew in Tyr- and G- media, and gave rise to stable variants in Arg- conditions. Cells of line HepG2 and clone BWTG3, a derivative of line BW, degenerated in all three media. Arg and tyr variants were however derived from HepG2 cells; their genesis appeared to be pathway specific, illustrating the complexity of the regulatory loops that are implicated in the control of the differentiated state. No variant was ever obtained with BWTG3 cells, demonstrating the stability of their deficiency in the post-natal hepatic functions that are involved in Arg-, Tyr- and G- selections. Variant clones of HepG2 and FaO cells that have been isolated in Arg- medium were characterized in details for liver-specific urea-cycle enzyme activities and mRNA. These variants were shown to be controlled at the mRNA level, most likely at transcription. Isolation of stable FaO and HepG2 variant clones as well as the converse demonstration of the stable deficiency of BWTG3 cells in post-natal hepatic functions were aimed at expression cloning. Our results are thus discussed in terms of transfection with full-length cDNA expression libraries and cloning of regulatory genes that could activate or extinguish liver specific genes.

Dexamethasone and glucagon cause synergistic increases of urea cycle enzyme activities in livers of normal but not adrenalectomized rats.

Adrenalectomized and intact rats were given constant high-dose infusions of glucagon, 0.3 mg/kg per day for 7 days, with or without low-dose dexamethasone, 0.01 mg/kg daily, to test whether glucocorticoids potentiate glucagon induction of the 5 urea cycle enzymes as they do in cultured rat hepatocytes. Glucagon did not induce any of the urea cycle enzymes in adrenalectomized Sprague-Dawley rats and only induced argininosuccinate lyase (EC 4.3.2.1) in adrenalectomized inbred Wistar-Furth rats. Dexamethasone alone induced arginase in adrenalectomized and in intact Wistar-Furth rats and restored the other enzymes to normal levels in adrenalectomized rats. In intact Wistar-Furth rats, the combination of hormones gave synergistic increases of all 5 enzymes over the responses to each hormone alone, but in adrenalectomized rats the combination was only additive or less than additive compared with the sum of single hormone responses. The lack of synergism between the two hormones in adrenalectomized rats suggest that other factors play a role in glucagon induction of this cycle.

Molecular analysis of a human liver mitochondrial ornithine transcarbamylase deficiency.

The liver of a young girl which had been successfully transplanted was investigated at the ornithine transcarbamylase (OTC, EC 2.1.3.3) gene expression level. Northern blot hybridization using a human OTC cDNA probe showed a greater than 80% decrease in specific OTC mRNA although having the same molecular size as a normal control. OTC polypeptide was simultaneously synthesized with a normal molecular size but at a low level (20%) as shown by immunoblotting. The OTC enzyme from the deficient liver exhibited very little catalytic activity (7.2% as compared to the normal subject). These results may support several explanations of this disease such as mutation of the OTC gene promoter leading to a low transcriptional activity or mutation of the encoding sequence which results in a modified translation product but with a normal size. mRNA instability may also occur.

Hepatic polyamine metabolism in children with Reye's syndrome.

Acute mitochondrial insult has been suggested as a primary reason for the clinical, histopathological and biochemical abnormalities seen in Reye's syndrome. However, the etiology of mitochondrial dysfunction has not been identified. Polyamines have been known to alter the mitochondrial structure and function. Influenza infection may cause an increase in ornithine decarboxylase activity and thereby channel ornithine for polyamine biosynthesis, leading to mitochondrial dysfunction in Reye's syndrome. To test this hypothesis, the hepatic concentrations of polyamines, polyamine-metabolizing enzymes and urea cycle enzyme activities in Reye's syndrome patients were determined and compared with patients who died from illnesses other than Reye's syndrome. The hepatic concentration of putrescine, spermidine and spermine were increased in Reye's syndrome patients. The activity of ornithine decarboxylase was elevated but, due to the small number of samples, these values did not reach statistical significance. Ornithine carbamoyltransferase activity was decreased in the liver of Reye's syndrome patients. Our results suggest that increased synthesis of polyamines from ornithine may initiate mitochondrial injury in Reye's syndrome.

Immunological characterization of plant ornithine transcarbamylases.

Pea (Pisum sativum L.) ornithine transcarbamylase (OTC) antisera were used to investigate the immunological relatedness of several plant and animal OTC enzymes. The antisera immunoprecipitated OTC activity in all monocot and dicot species tested, and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of immunoprecipitated protein revealed monomeric proteins ranging from 35,200 to 36,800 daltons in size. Pea OTC antisera did not recognize mammalian OTC protein. OTC activity and protein levels detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblots from homogenates of green leaf, etiolated epicotyl and cotyledon, and root tissues of pea were poorly correlated. This might result from differences in amounts of enzymatically active OTC protein in the homogenates. Alternatively, the antisera may fail to recognize different isozyme forms of OTC, which have been reported for some plant species. A putative cytosolic precursor OTC (pOTC) polypeptide exhibiting and Mr = 39,500 to 40,000 daltons was immunoprecipitated from in vitro translation mixtures of total pea leaf poly(A)+ RNA. The size of the pOTC polypeptide, as compared with mature OTC monomer (36,000 daltons), suggests that a 4 kilodalton N-terminal leader sequence, like that responsible for mitochondrial targeting of the mammalian enzyme, may be involved in organellar import of the plant enzyme.

Carbamyl phosphate synthetase and ornithine transcarbamylase activities in enzyme-deficient human liver measured by radiochromatography and correlated with outcome.

Sensitive and specific radiochromatographic methods to measure enzymatic activities of carbamyl phosphate synthetase I (CPS I) and ornithine transcarbamylase (OTC) were developed. The activities of these enzymes were assayed in frozen liver tissue obtained from 23 individuals with hyperammonemia caused by CPS I (five patients) and OTC deficiency (18 patients). In addition, livers of one aborted fetus with OTC deficiency and four normal individuals were studied. The assays use radioactive ornithine as a substrate followed by separation of citrulline formed in the reactions by HPLC and quantitation of the radioactivity in both amino acids by a radioactivity flow monitor or by a scintillation counter. Both CPS I and OTC assays were linear with respect to length of incubation time and concentration of tissue homogenate. The sensitivity of the methods allowed measurements of CPS I and OTC activities as low as 0.1 mumol/g/min on 5 mg of liver tissue and the diagnosis of CPS I or OTC deficiency could be established on as low as 0.5 and 0.05 mg of tissue, respectively. CPS I activity in different sections of four normal livers was 3.01 +/- 0.16 mumol/g/min (mean +/- SEM, n = 19) and OTC activity was 93.4 +/- 6.3 (mean +/- SEM, n = 19). Residual enzymatic activity could be detected and measured in the liver tissues of one of the five subjects with CPS I deficiency and in 14 of 19 subjects with OTC deficiency. OTC/CPS I activity ratio in normal liver tissue was 31.2 +/- 1.3 (mean +/- SEM, n = 19), whereas this ratio ranged from 343 to greater than 5000 in CPS I deficient livers and from less than 0.02 to 1.55 in OTC deficient livers.(ABSTRACT TRUNCATED AT 250 WORDS)