Normal root elongation requires arginine produced by argininosuccinate lyase in rice.

Plant roots play an important role in the uptake of water and nutrients, structural support and environmental sensing, but the molecular mechanisms involved in root development are poorly understood in rice (Oryza sativa), which is characterized by a dense fibrous root system. Here we report a rice mutant (red1 for root elongation defect 1) with short roots. Morphological and physiological analyses showed that the mutant had a shorter length from the quiescent center (QC) to the starting point of the elongation zone but a similar cell size and number of lateral and crown roots compared with the wild type. Furthermore, the mutant had similar radial structure and nutrient uptake patterns to the wild type. Map-based cloning revealed that the mutant phenotype was caused by a point mutation of a gene encoding an argininosuccinate lyase (ASL), catalyzing the last step of arginine biosynthesis. The OsASL1 gene has two distinct transcripts, OsASL1.1 and OsASL1.2, which result from different transcription start sites, but only OsASL1.1 was able to complement the mutant phenotype. OsASL1.1 was expressed in both the roots and shoots. The protein encoded by OsASL1.1 showed ASL activity in yeast. OsALS1.1 was localized to the plastid. The short root of the mutant was rescued by exogenous addition of arginine, but not by other amino acids. These results indicate that arginine produced by ASL is required for normal root elongation in rice.

Activated microglia cells express argininosuccinate synthetase and argininosuccinate lyase in the rat brain after transient ischemia.

Argininosuccinate-synthetase (ASS), argininosuccinate-lyase (ASL) and nitric oxide synthase (NOS) act in the l-arginine-NO-l-citrulline cycle. In the rat brain, ASS is expressed in neurons, ASL in neurons and astroglia in the striatum, both are co-expressed with nNOS in medium-sized neurons. Microglia cells express iNOS and ASS after activation but no information is available on ASL and on ASS/ASL/iNOS co-expression in this glial population. The present aim was to ascertain, by immunohistochemistry, whether the microglia cells of the rat striatum and fronto-parietal cortex express ASL and ASS in control conditions and after transient ischemia induced by middle cerebral artery occlusion, and whether ASL and ASS are co-expressed with iNOS. The study was conducted 24, 72 and 144 h after reperfusion in two groups of ischemic rats with different tissue damage and survival. ASS and ASL are not expressed by microglia cells in controls while are present in most of the activated microglia cells in the ischemic rats. In those animals with longer survival, ASS and ASL were no more detectable at 144 h, while, in the animals with shorter survival, they were co-expressed with iNOS, but only at 72 h. In the cortex, at variance with the striatum, almost all of nNOS-positive neurons co-expressed ASS and ASL. In conclusion, only activated microglia cells express ASS and ASL, this expression precedes that of iNOS and does not necessarily imply its appearance. Therefore, local factors such as the NO produced by nNOS/ASS/ASL-positive neurons, could influence ASS/ASL-positive microglia cells avoiding or allowing the induction, in these cells, of iNOS.

Prenatal diagnosis of citrullinemia and argininosuccinic aciduria: evidence for a transmission ratio distortion in citrullinemia.

BACKGROUND: In the course of 25 years, we have experienced a high rate of affected fetuses in the prenatal diagnosis of citrullinemia. METHODS AND RESULTS: Ninety-one pregnancies at 1 in 4 risk were tested; 36 were diagnosed as affected (39.5%; P = 0.0015). The high rate of positive diagnoses was found both after chorionic villus sampling (24/68 = 35.3%) and amniocentesis (12/23 = 52.2%) despite the completely different and independent techniques used. Using exactly the same (indirect) enzyme assay for argininosuccinic aciduria on chorionic villi and a similar method on amniotic fluid, the expected rate of affected fetuses was found: 13/53 = 24.5%. Technical and genetic causes for the unexpected results were excluded by confirmatory studies performed on independent fetal material, which was available for 27 of the 36 fetuses affected with citrullinemia. Biochemical confirmation was obtained in the 27 cases, whereas in 18 fetuses homozygosity or compound heterozygosity for disease-causing mutations were retrospectively demonstrated in the stored fetal cells. CONCLUSION: The results suggest the occurrence of preferential transmission of the mutant allele. An explanation for this phenomenon may be found in a protective role of argininosuccinic acid synthetase deficiency in mutant sperm cells against the possibly detrimental or apoptotic effect of nitric oxide produced normally from arginine by nitric oxide synthase.

Clinical, enzymatic, and molecular genetic characterization of a biochemical variant type of argininosuccinic aciduria: prenatal and postnatal diagnosis in five unrelated families.

A biochemical variant of argininosuccinate lyase deficiency, found in five individuals, is introduced. In comparison to classical patients, the variant cases of argininosuccinate lyase deficiency were characterized by residual enzyme activity as measured by the incorporation of [14C]citrulline into proteins. The five patients of different ethnic backgrounds presented with relatively mild clinical symptoms, variable age of onset, marked argininosuccinic aciduria and severe, but not complete, deficiency of argininosuccinate lyase. [14C]Citrulline incorporation into proteins, which is completely blocked in classical argininosuccinic aciduria, was only partially reduced in fibroblasts of these patients. Further investigation showed that previous standard conditions of the assay were not optimal. Higher concentrations of citrulline in the incubation medium strongly stimulated 14C incorporation in normal cells, but not in the patients; as a result, the relative incorporation level in the patients dropped to 6-28% compared to 18-75% of normal in the original procedure. Prenatal diagnosis was successfully performed in three of the families. Affected pregnancies were indicated by (partial) deficiency of [14C]citrulline incorporation in chorionic villi and/or increased levels of argininosuccinate in amniotic fluid. Analysis of the ASL gene in the five patients revealed a considerable allelic heterogeneity. Three novel mutations--R385C (2 patients), V178M and R379C--were detected in homozygous states, whereas one patient was compound heterozygous for the known mutations R193Q and Q286R. In conclusion, there are patients of different ethnic backgrounds who are characterized by residual activity of argininosuccinate lyase and who present with less severe clinical courses. In addition, we present an improved biochemical assay for accurate prenatal and postnatal diagnosis.

NADPH-diaphorase and cytosolic urea cycle enzymes in the rat accessory olfactory bulb.

The nitric oxide cycle consists of nitric oxide synthase, argininosuccinate synthetase and argininosuccinate lyase to form nitric oxide. We have examined the colocalization of nitric oxide synthase and the cytosolic urea cycle enzymes (argininosuccinate synthetase, argininosuccinate lyase and arginase) in the accessory olfactory bulb of the rat by using a double labeling procedure combining reduced-nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) reaction with fluorescent immunocytochemistry. Each glomerulus showed a different NADPH-d activity, and those with the strongest NADPH-d activities were assembled in the caudomedial part of the accessory olfactory bulb. Argininosuccinate synthetase-like immunoreactive glomeruli were distributed in the caudomedial part of the accessory olfactory bulb, and most of them were also strongly NADPH-d positive. The mitral or tufted cells were argininosuccinate synthetase-, argininosuccinate lyase- and arginase-like immunoreactive, but were not NADPH-d positive. The granule cells were NADPH-d positive or argininosuccinate lyase-like immunoreactive, but were not argininosuccinate synthetase- or arginase-like immunoreactive. Some granule cells were both NADPH-d positive and argininosuccinate lyase-like immunoreactive. The results indicate the heterogeneity of glomeruli of the accessory olfactory bulb with respect to the distribution of these enzymes. The granule cells have nitric oxide synthase and argininosuccinate lyase, and thus may efficiently produce nitric oxide.

Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver.

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were colocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.

Intracellular localization of the mRNAs of argininosuccinate synthetase and argininosuccinate lyase around liver mitochondria, visualized by high-resolution in situ reverse transcription-polymerase chain reaction.

Argininosuccinate synthetase and argininosuccinate lyase, two cytoplasmic enzymes of the urea cycle, are released into the soluble phase in the absence of detergent when cells are disrupted. Yet previous biochemical studies, as well as immunocytochemistry at the electron microscope level, have shown that these enzymes are localized around mitochondria in situ. Such intracellular localization of soluble enzymes requires mechanisms to deliver the proteins to the appropriate sites, where they may then be anchored by specific protein-protein interactions. A method was developed to examine the intracellular distribution of the mRNA of argininosuccinate synthetase and argininosuccinate lyase in intact rat liver at the ultrastructural level by in situ reverse transcription and the polymerase chain reaction, using primers targeting regions of the coding sequences of the rat enzymes, digoxigenin-dUTP as the label, and anti-digoxigenin/1 nm [corrected] gold plus silver enhancement as the detection method. The tissue was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl. Examination of the numbers and the location of the silver grains, coupled with morphometric analysis of the electron micrographs, permitted the calculation of the silver "enrichment ratio" for each type of cell structure. These ratios showed that the mRNAs for argininosuccinate synthetase and argininosuccinate lyase were located next to the cytoplasmic side of the mitochondrial membrane and in the nearby endoplasmic reticulum. Most of the silver grains that were observed in the endoplasmic reticulum were within 200 nm of the mitochondria; it was not possible, however, to determine if those grains were actually associated with the reticular membranes. These studies demonstrate that the mRNAs of these two soluble cytoplasmic proteins are localized to the same limited regions where the proteins are situated. Translation of the proteins, therefore, must occur at these specific sites. The targeting of argininosuccinate synthetase and argininosuccinate lyase mRNAs to the immediate vicinity of the mitochondria may be the first step of the mechanisms by which the spatial organization of these soluble proteins in situ is accomplished. The targeting of mRNAs for soluble cytoplasmic proteins of organized metabolic pathways has not been demonstrated previously. These studies also show that in situ reverse transcription and the polymerase chain reaction at the ultrastructural level, which has not been previously reported, can be used to detect specific mRNAs; it should be extremely valuable for the intracellular detection of low-abundance mRNAs.

Argininosuccinate synthetase and argininosuccinate lyase are localized around mitochondria: an immunocytochemical study.

Argininosuccinate synthetase and argininosuccinate lyase are soluble cytoplasmic enzymes of the urea cycle. Previous biochemical studies using permeabilized hepatocytes showed that these enzymes are organized in situ, and function as if they are located next to the outer membrane of mitochondria. We have now confirmed and extended those observations in intact liver by means of immunocytochemistry at the electron microscope level. Morphometric analysis of the electron micrographs shows that argininosuccinate synthetase and argininosuccinate lyase are located in the immediate vicinity of the mitochondria, predominantly next to the cytoplasmic surface of the outer membrane. Some immuno-specific protein is also observed in the endoplasmic reticulum in the immediate vicinity of the mitochondria. These results support our previous biochemical findings, and additionally suggest that virtually all of the argininosuccinate synthetase and argininosuccinate lyase of the liver parenchymal cell are located just outside the mitochondria.

Differential expression of the two delta-crystallin genes in lens and non-lens tissues: shift favoring delta 2 expression from embryonic to adult chickens.

Chicken argininosuccinate lyase (ASL)/delta-crystallin, a lens enzyme-crystallin, is encoded in two linked genes (delta 1 and delta 2); only the delta 2 polypeptide contains ASL activity. Here we have quantified delta 1- and delta 2-crystallin mRNA in the lens, cornea, neural retina, heart, and brain at different stages of embryonic development and in 1-wk-old and 1-yr-old chickens by the polymerase chain reaction using internal delta 1 and delta 2 RNA standards. The delta 1/delta 2 mRNA ratio differed for every tissue and was regulated during development. In the embryo there was more delta 1 than delta 2 mRNA in the lens (50-100 times), cornea (3-4 times), and neural retina (2-20 times), about equal amounts of delta 1 and delta 2 mRNA in the heart, and more delta 2 mRNA in the brain (15 times). delta 1-Crystallin mRNA differentially decreased in every tissue after hatching; by contrast, the delta 2 mRNA remained about the same except for the lens, where it decreased 50-fold between 1 wk and 1 yr after hatching. In the 1-yr-old chicken, the delta 2/delta 1 mRNA ratios were 7 in the lens, 175 in the cornea, 22 in the neural retina, 107 in the heart, and 136 in the brain, indicating that delta 2-crystallin is strongly favored in all adult tissues of the chicken. The excess of delta 1 to delta 2 mRNA in the embryonic lens, cornea, and neural retina is intriguing, and suggests some connection with developing transparent eye tissues. Finally, we raise the possibility that expression of both delta-crystallin genes may create tetrameric ASL isoenzymes (perhaps with different specific activities). The unexpected predominance of delta 2 mRNA in the 1-yr-old lens suggests that both the enzymatic and refractive functions of ASL/delta-crystallin are operative and spatially separated, with the enzymatic role present in the cortical fibers and the refractive role in the center of the lens.

Argininosuccinic aciduria: clinical and biochemical findings in three children with the late onset form, with special emphasis on cerebrospinal fluid findings of amino acids and pyrimidines.

Three children with the late onset form of argininosuccinic aciduria are presented. The first two are sisters. The clinical features are characterized by mild retardation and ataxia, complicated by episodes of hyperammonemia. All patients showed elevated concentrations of argininosuccinic acid and its anhydrides in all body fluids, most pronounced in cerebrospinal fluid (CSF). Moreover, in Cases 1 and 2, we found elevated concentrations of pseudouridine and uridine limited to CSF, which was not reported before. In Case 3, with some residual activity of argininosuccinate lyase (ASL), we found normal values of these compounds. In urine we found elevated concentrations of uracil in Cases 1 and 2, and orotic acid in Case 2. Plasma showed an elevated concentration of orotic acid in all three patients, uracil was elevated in Case 2, cytidine was elevated in Cases 2 and 3. The results are being discussed and indicate that CSF values of pyrimidines reveal new biochemical abnormalities of brain tissue in urea cycle disorders.

Staining of argininosuccinate lyase activity in polyacrylamide gel.

A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to L-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of L-malate to give CO2 and pyruvate with concomitant reduction of NADP+ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer.

Taxon-specific recruitment of enzymes as major soluble proteins in the corneal epithelium of three mammals, chicken, and squid.

Studies of others have shown that class 3 aldehyde dehydrogenase is a major component of the epithelial cells of the mammalian cornea. Here we demonstrate by peptide sequencing that other major proteins of the corneal epithelium are also identical or related to enzymes in the human, mouse, kangaroo, chicken, and squid. Aldehyde dehydrogenase class 3 was found to be the major protein of human, mouse, and kangaroo corneal epithelial cells. Peptidyl prolyl cis-trans isomerase (cyclophilin) or a homologue thereof is strikingly abundant in the corneal epithelial cells of chicken, but not mammals, and appears to be absent from the cornea of squid. By contrast, enolase or its homologue is relatively abundant in both the mammalian and chicken corneal epithelial cells. In some instances, abundant enzymes are common to cornea and lens in the same species--for example, arginino-succinate lyase/delta 1-crystallin in the chicken and glutathione S-transferase-like protein in the squid; in other cases, the abundant proteins in the cornea have not been found as lens crystallins in any species--for example, aldehyde dehydrogenase class 3 and cyclophilin. These data suggest that enzymes and certain enzyme-crystallins have been recruited as major corneal proteins in a taxon-specific manner and may serve structural rather than, or as well as, enzymatic roles in corneal epithelial cells.

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.

Differential cellular localization of enzymes of L-arginine metabolism in the rat brain.

Polyclonal rabbit antisera specific to argininosuccinate synthetase (ASS), argininosuccinate lyase and arginase revealed that these enzymes of L-arginine metabolism are generally localized in different cells of the rat brain. In the main olfactory bulb and the cerebellar cortex the three immunoreactivities were localized in different cells: in the somatic motor nuclei ASS-like immunoreactivity was localized in incoming fibers, and the other two enzymes were found in the motor neurons. The results suggest that L-argininosuccinate and/or L-arginine may be transcellularly transported in the nervous system.

Molecular cloning and nucleotide sequence of rat brain argininosuccinate lyase cDNA with an extremely long 5'-untranslated sequence: evidence for the identity of the brain and liver enzymes.

Argininosuccinate lyase (EC 4.3.2.1) is an enzyme present in the brain of ureotelic animals. Using as a probe rat liver argininosuccinate lyase cDNA, already isolated and sequenced (Amaya, Y., Matsubasa, T., Takiguchi, M., Kobayashi, K., Saheki, T., Kawamoto, S. and Mori, M., J. Biochem., 103 (1988) 177-181), we screened a rat brain cDNA library constructed in the lambda gt11 expression vector and obtained a single cDNA clone. This cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,390), a 5'-untranslated sequence of 967 bp and a 3'-untranslated sequence of 74 bp. The length of the 5'-non-coding region of the cDNA seems to be one of the longest among the cDNAs heretofore isolated. A comparison of the brain cDNA sequence (2424 bp) with the corresponding region of the liver cDNA (1574 bp) revealed differences in 5 nucleotides. The brain clone contained A----G and C----G base differences from the hepatic sequence, resulting in amino acid changes from Tyr and Arg in the liver clone, to Cys and Gly in the brain clone, respectively. The other 3 nucleotide differences are silent with respect to the amino acid sequence of the protein. Therefore, the amino acid sequence of the brain argininosuccinate lyase, as deduced from the nucleotide sequence of its cDNA clone, was identical with that of the liver protein, except for two amino acid residues. These minor changes may reflect a microheterogeneity of the argininosuccinate lyase gene. The brain and liver enzymes seem to be encoded by the same structural gene.

Homology of delta crystallin and argininosuccinate lyase.

1. Delta crystallin, a major lens protein characteristic of birds and reptiles, is homologous to argininosuccinate lyase; 57% of the residues in chicken delta crystallin and human lyase are identical. 2. Even more similar (62% identical residues) to the human lyase is the sequence translated from the presumably inactive delta-2 gene of the delta crystallin locus. 3. As both delta crystallin and lyase are synthesized in birds only during the embryonic and juvenile stages, the persistence of delta crystallin in the adult lens appears to be paedomorphic. 4. Possible correlations of the origins of delta crystallin with other events in sauropsid evolution are proposed.

Argininosuccinic aciduria: prenatal studies in a family at risk.

We have monitored two successive pregnancies in a family which we found to be at risk for argininosuccinic aciduria. We measured argininosuccinic acid (ASA) concentrations in amniotic fluid and utilized an indirect assay of ASA lyase activity in cultured amniotic fluid cells. The assay procedure is based on the uptake of 14C from [14C]citrulline and of [3H]leucine into protein. ASA was easily measured in amniotic fluid from the first fetus at risk, whereas none was detectable in control fluids. Amniotic fluid cells cultured from this fetus had only 5.5% of control ASA lyase activity. The pregnancy was terminated, and hepatic ASA lyase activity in the fetus was shown to be about 1.3% of control values. In addition, eight fetal tissues were analyzed for ASA, and all had significant accumulation. ASA was not detected in amniotic fluid from the second fetus at risk, and ASA lyase activity in cultured cells was 80% of control activity. Enzymatic analysis of erythrocyte lysate confirmed the diagnosis of an unaffected child (ASA lyase = 46% of control) and indicated heterozygosity. Thus, we provide further evidence that argininosuccinic aciduria can be diagnosed successfully in utero by indirect assay of ASA lyase activity in cultured amniotic fluid cells. In addition, high amniotic fluid ASA concentrations provide strong adjunctive evidence for such a prenatal determination, and may prove to be sufficient for diagnosis.