Studies on enzymes of nitrogen metabolism, and nitrogen end products in the developing chick (Gallus domesticus) embryo.

1. A total of 450 fertilized eggs were used to study the concentrations of uric acid, urea and ammonia in allantoic and amniotic fluids, and some enzymes of nitrogen metabolism in the liver and kidney during the development of the chick embryo from the 5th to 21st day of incubation. 2. Concentrations of the compounds studied were higher in allantoic fluid. The molar concentration of allantoic uric acid increased steadily with time. The pattern of urea and ammonia in both allantoic and amniotic fluids were the same. 3. Arginase (E.C.3.5.3.1) activity in both embryonic kidney and definitive kidney was higher than that in the liver. The specific activity of arginase (mumole urea formed/hr per g wet wt kidney) dropped during development. 4. Little arginine synthetase activity (argininosuccinate synthetase, E.C.6.3.4.5; and argininosuccinate lyase, E.C.4.3.2.1) was found in kidney, but none in the liver. 5. The complete urea cycle function was absent in both the liver and the kidney of the chick embryo.     

Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.

The synthesis of ¹⁴C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-¹⁴C]guanine or [8-¹⁴C]hypoxanthine, but not from [8-¹⁴C]adenine, was demonstrated in a cell-free extract from N₂-fixing nodules of cowpea (Walp.). The ¹⁴C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the ¹⁴C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.     

Effect of urea and uric acid on the hemolytic activity of Sendai virus

It was found that haemolytic activity of Fushimi strain of Sendai virus multiplied in allantoic cavity of chicken embryos is independent on its haemagglutinating titer and also on allantoic fluid urea and uric acid content. It was shown in experiments with embryonated eggs that these two compounds have no also influence on haemolytic activity induction in Sendai virus. Moreover, the results of an experiment in which allantoic fluid was replaced by Eagle's liquid suggest that most probably the other components present in allantoic fluid do not also influence the appearance of haemolytic activity of this virus.     

Nitrogen excretion during embryonic development of the green iguana, Iguana iguana (Reptilia; Squamata)

Development within the cleidoic egg of birds and reptiles presents the embryo with the problem of accumulation of wastes from nitrogen metabolism. Ammonia derived from protein catabolism is converted into the less toxic product urea or relatively insoluble uric acid. The pattern of nitrogen excretion of the green iguana, Iguana iguana, was determined during embryonic development using samples from allantoic fluid and from the whole homogenized egg, and in hatchlings and adults using samples of blood plasma. Urea was the major excretory product over the course of embryonic development. It was found in higher concentrations in the allantoic sac, suggesting that there is a mechanism present on the allantoic membrane enabling the concentration of urea. The newly hatched iguana still produced urea while adults produced uric acid. The time course of this shift in the type of nitrogen waste was not determined but the change is likely to be related to the water relations associated with the terrestrial habit of the adult. The green iguana produces parchment-shelled eggs that double in mass during incubation due to water absorption; the eggs also accumulate 0.02 mM of urea, representing 82% of the total measured nitrogenous residues that accumulate inside the allantois. The increase in egg mass and urea concentration became significant after 55 days of incubation then were unchanged until hatching.     

Urease Is Not Essential for Ureide Degradation in Soybean

The hypothesis that soybean (Glycine max L. [Merrill]) catabolizes ureides to urea to a physiologically significant extent was tested and rejected. Urease-negative (eu3-e1/eu3-e1) plants were supported by fixed N2 or by 2 mM NH4NO3, so that xylem-borne nitrogen contained predominantly ureides (allantoin and allantoic acid) or amide amino acids, respectively. Seed nitrogen yield was equal on either nitrogen regime, although 35-d-old fixing plants accumulated about 6 times more leaf urea. In callus, lack of an active urease reduced growth on either arginine or allantoin as the sole nitrogen source, but the reduction was greater on arginine (73%) than on allantoin (39%). Furthermore, urease-negative cells accumulated 17 times more urea than urease-positive cells on arginine; for allantoin the ratio was 1.8. Urease-negative callus accumulated urea at 3% the rate of seedlings. To test whether urea accumulating in urease-negative seedlings was derived from ureides, seeds were first allowed to imbibe in 1 mM allopurinol, an inhibitor of ureide formation. Seedling ureides were decreased by 90%, but urea levels were unchanged. Thus, ureides are poor precursors of urea, which was confirmed in seedlings that converted no more than 5% of seed-absorbed [14C-ureido]allantoate to [14C]urea, whereas 40 to 70% of [14C-guanido]arginine was recovered as [14C]urea.     

Effect of folic acid plus glycine supplement on uterine prostaglandin and endometrial granulocyte-macrophage colony-stimulating factor expression during early pregnancy in pigs

The objective was to determine the effects of folic acid+glycine supplement on uterine metabolism of prostaglandin and mRNA expression of endometrial granulocyte-macrophage colony-stimulating factor (GM-CSF) in nulliparous (NYL) and multiparous Yorkshire-Landrace (YL) sows, and in multiparous Meishan-Landrace sows (ML). In each of these three groups, sows were randomly assigned to two treatments: 15 ppm folic acid+0.6% glycine or no supplement. The dietary supplement was given from the estrus before mating to slaughter on Day 25 of pregnancy. At slaughter, endometrial tissue was collected to determine endometrial expression levels of GM-CSF mRNA, cyclooxygenase-1 (COX1) and -2 (COX2) and to evaluate in vitro endometrial secretion of prostaglandin E2 (PGE2) secretion. Allantoic fluid samples were also collected to determine the concentration of PGE2, prostaglandin F2alpha (PGF2alpha), estradiol-17beta (E2), progesterone (P4), and transforming-growth factor-beta2 (TGF-beta2). The allantoic contents of PGF2alpha, E2 and P4, and endometrial in vitro secretion of PGE2 were not significantly influenced by the folic acid+glycine supplement. The folic acid+glycine supplement tended (P<0.07) to increase allantoic content of PGE2 and TGF-beta2 in all sows and increased (P<0.05) endometrial expression of COX2, especially in NYL sows. The endometrial expression of COX1 was decreased (P<0.05) by folic acid+glycine supplement, especially in multiparous YL sows. The allantoic contents of PGE2 and PGF2alpha were not significantly affected by sow type. However, NYL sows had higher (P<0.05) endometrial in vitro secretion of PGE2 and allantoic content of P4 than multiparous YL and ML sows. The allantoic content of E2 was also higher (P<0.05) in NYL sows than in multiparous ML sows only. The allantoic content of TGF-beta2 was lower (P<0.05) in multiparous ML than in multiparous YL only sows. Finally, in YL and NYL sows, folic acid+glycine supplement decreased (P<0.05) the endometrial expression of GM-CSF but not in ML sows. In summary, folic acid+glycine supplement altered endometrial expression of GM-CSF and uterine metabolism of prostaglandins during the post-attachment period of porcine embryos but some of these effects were manifest only in Meishan and nulliparous sows.     

The all2 gene is required for the induction of the purine deamination pathway in Schizosaccharomyces pombe

Five mutants were isolated at the all2 gene on the basis of their inability to utilize hypoxanthine as a sole source of nitrogen. These mutants failed to utilize the purines adenine, hypoxanthine, xanthine, uric acid, allantoin and allantoic acid, although they could utilize urea and ammonium. The all2 mutants appeared to be defective in purine induction of uricase, allantoinase, allantoicase and ureidoglycollase activities but retained wild-type activity of the constitutively synthesized urease. The all2 mutations were recessive.     

The metabolism of caffeine by a Pseudomonas putida strain

1) A bacterium capable of growing aerobically with caffeine (1,3,7-trimethylxanthine) as sole source of carbon and nitrogen was isolated from soil. The morphological and physiological characteristics of the bacterium were examined. The organism was identified as a strain of Pseudomonas putida and is referred to as Pseudomonas putida C1. 15 additional caffeine-degrading bacteria were isolated, and all of them were also identified as Pseudomonas putida strains. The properties of the isolates are discussed in comparison with 6 Pseudomonas putida strains of the American Type Culture Collection. 2) The degradation of caffeine by Pseudomonas putida C1 was investigated; the following 14 metabolites were identified: 3,7-dimethylxanthine (theobromine), 1,7-dimethylxanthine, 7-methylxanthine, xanthine, 3,7-dimethyluric acid, 1,7-dimethyluric acid, 7-methyluric acid, uric acid, allantoin, allantoic acid, ureidoglycolic acid, glyoxylic acid, urea, and formaldehyde. Formaldehyde has been demonstrated to be the product of oxidative N-demethylation mediated by an inducible demethylase. A pathway of caffeine degradation is proposed.     

Effects of Allopurinol [4-Hydroxypyrazolo(3,4-d)Pyrimidine] on the Metabolism of Allantoin in Soybean Plants

Some studies on the effects of xanthine oxidase inhibitor allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] on allantoin metabolism of soybean plants (Glycine max cv. Tamanishiki) are reported. Soybean seedlings, aseptically germinated for 96 hours on agar containing 1 millimolar allopurinol, contained only slight amounts of allantoin, allantoic acid, and urea as compared with controls. Analysis of purines and pyrimidines of the allopurinol-treated seedlings showed marked accumulation of xanthine both in the cotyledons and seedling axes. No hypoxanthine accumulation was found. Xanthine accumulation due to allopurinol treatment was relatively low after the cotyledons had fallen. For nodulated plants, allopurinol caused a significant drop in allantoin (+allantoic acid) in the stems and nodules, accompanied by a striking accumulation of xanthine in the nodules. The xanthine concentration in the nodules far exceeded that in the germinated seedlings. Allopurinol at a concentration of 50 micromolar strongly inhibited xanthine oxidase prepared from soybean nodules.

The results suggested that the main pathway of allantoin formation in soybean plants was through purine decomposition, via xanthine-uric acid. It was specially noted that a very active purine-decomposing system existed in soybean nodules.

     

Incorporation of 15N into allantoin in nodulated soybean plants supplied with 15N2

Incorporation of ¹⁵N into allantoin and allantoic acid in noduleswas higher than that in roots. This confirms that nodules produceallantoin. The ¹⁵N concentration in allantoin was slightly higherthan that in allantoic acid, suggesting that allantoin decomposedto allantoic acid. Allantoin and allantoic acid in nodules weretranslocated rapidly to roots. (Received August 25, 1976; )     

The effect of thiourea on ureide metabolism in Neurospora crassa

The wild-type strain of Neurospora crassa Em 5297a can utilize allantoin as a sole nitrogen source. The pathway of allantoin utilization is via its conversion into allantoic acid and urea, followed by the breakdown of urea to ammonia. This is shown by the inability of the urease-less mutant, N. crassa 1229, to grow on allantoin as a sole nitrogen source and by the formation of allantoate and urea by pre-formed mycelia of this mutant. In the wild strain (Em 5297a) thiourea is tenfold more toxic on an allantoin medium than on an inorganic nitrogen medium; allantoin as well as urea counteract thiourea toxicity in the allantoin nitrogen medium. This selective toxicity of thiourea for the mould utilizing allantoin nitrogen does not, however, result in an impairment of allantoin uptake, allantoinase activity or the formation of urea from allantoin. The only process affected by thiourea is the synthesis of urease; urea antagonizes this effect of thiourea in N. crassa.     

Developmental changes of amino acids in ovine fetal fluids

We recently reported an unusual abundance of arginine (4-6 mM) in porcine allantoic fluid during early gestation. However, it is not known whether such high concentrations of arginine are unique for porcine allantoic fluid or whether they represent an important physiological phenomenon for mammals. The present study was conducted to test the hypothesis that arginine is also the most abundant amino acid in ovine allantoic fluid. Allantoic and amniotic fluids, as well as fetal and maternal plasma samples, were obtained from ewes between Days 30 and 140 of gestation. Glycine was the most abundant amino acid in maternal uterine arterial plasma, representing approximately 25% of total alpha-amino acids. Alanine, glutamine, glycine, plus serine contributed approximately 50% of total alpha-amino acids in fetal plasma. Fetal:maternal plasma ratios for amino acids varied greatly, being less than 1 for glutamate during late gestation, 1.5-3 for most amino acids throughout gestation, and greater than 10 for serine during late gestation. Marked changes were observed in amino acid concentrations in amniotic and allantoic fluids associated with conceptus development. Concentrations of alanine, citrulline, and glutamine in allantoic fluid increased by 20-, 34-, and 18-fold, respectively, between Days 30 and 60 of gestation and were 24.7, 9.7, and 23.5 mM, respectively, on Day 60 of gestation (compared with 0.8 mM arginine). Remarkably, alanine, citrulline, plus glutamine accounted for approximately 80% of total alpha-amino acids in allantoic fluid during early gestation. Serine (16.5 mM) contributed approximately 60% of total alpha-amino acids in allantoic fluid on Day 140 of gestation. These novel findings of the unusual abundance of traditionally classified nonessential amino acids in allantoic fluid raise important questions regarding their roles in ovine conceptus development.     

Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.     

Uric acid metabolism during pupal-adult development of Pieris brassicae

Uric acid metabolism has been investigated during the pupal and adult stages of Pieris brassicae. Uric acid and its main metabolite, allantoic acid, have been quantified in various organs (fat body, gut, wings) during development, in order to determine synthesis, degradation, and transport phenomena. Both labelling experiments (using 2-¹⁴C uric acid, guanine, and guanosine) and enzymatic studies (xanthine dehydrogenase, guanine deaminase, and uricase) were performed.Labelled uric acid, when injected into a young pupa, accumulates preferentially into the fat body, and its degradation leads to an increase in allantoic acid, which is found chiefly in imaginal structures (wings, heads, body wall). Since uricase is present only in low levels through the pupal stage, only a small fraction of uric acid is metabolized.In the developing pharate adult, uric acid is transported via the haemolymph from fat body to the wings and gut. Male wings accumulate more uric acid than female wings. At emergence, a large amount of uric acid and most of the allantoic acid are excreted into the meconium, but not together; uric acid is excreted into the so-called ‘meconium 1’ containing ommochromes, whereas its metabolite is eliminated only after wing expansion into ‘meconium 2’, a colourless fluid. Shortly before emergence, the fat body recovers its ability to synthesize uric acid, a fraction of which is excreted within ‘meconium 1’.During adult life, the synthesis of uric acid occurs in the fat body and ovaries, where it is especially abundant. Ageing organs (wings, heads, testes) accumulate it markedly. A small fraction is excreted together with allantoic acid by the butterfly.Purine catabolism pathways have been investigated, showing that in guanine derivatives, the freebase state of guanine leads quickly to uric acid (and its metabolites), whereas ¹⁴C-guanosine may be transformed into nucleotide and incorporated efficiently into wing pteridines when it is injected at the time of adult pigmentation.Another purine derivative, identified as adenosine, has been shown to accumulate in male fat body just before adult emergence. Its amount increases during the first days of emerged adult life, and it corresponds to an alternative pathway of purine catabolism. Its absence in females is related to development of the ovaries.     

Perturbations in the biochemical composition of fetal fluids are apparent in surviving bovine somatic cell nuclear transfer pregnancies in the first half of gestation

Amniotic and allantoic fluid volumes and composition change dynamically throughout gestation. Cattle that are pregnant with somatic cell nuclear transfer (NT) fetuses show a high incidence of abnormal fluid accumulation (particularly hydrallantois) and fetal mortality from approximately midgestation. To investigate fetal fluid homeostasis in these pregnancies, Na, K, Cl, urea, creatinine, Ca, Mg, total PO(4), glucose, fructose, lactate, total protein, and osmolalities were measured in amniotic and allantoic fluids collected at Days 50, 100, and 150 of gestation from NT pregnancies and those generated by the transfer of in vitro-produced embryos or by artificial insemination. Deviations in fetal fluid composition between NT and control pregnancies were apparent after placental and fetal organ development, even when no gross morphological abnormalities were observed. Individual NT fetuses were affected to varying degrees. Elevated allantoic Na was associated with lower K and increased allantoic fluid volume or edema of the fetal membranes. Total PO(4) levels in NT allantoic and amniotic fluid were elevated at Days 100 and 150. This was not accompanied by hypophosphatemia at Day 150, suggesting that PO(4) acquisition by NT fetuses was adequate but that its readsorption by the kidneys may be impaired. Excessive NT placental weight was associated with low allantoic glucose and fructose as well as high lactate levels. However, the fructogenic ability of the NT placenta appeared to be normal. The osmolality of the fetal fluids was maintained within a narrow range, suggesting that the regulation of fluid composition, but not osmolality, was impaired in NT pregnancies.     

A Cluster of Three Genes Responsible for Allantoin Degradation in SACCHAROMYCES CEREVISIAE

Mutant strains of Saccharomyces cerevisiae unable to utilize allantoin as sole nitrogen source were isolated and divided into three groups on the basis of their biochemical and genetic characteristics. The three loci associated with these mutant classes were designated dal1 (allantoinase minus), dal2 (allantoicase minus) and dal4 (allantoin transport minus). All three loci are located in a cluster that is proximal to the lys1 locus on the right arm of chromosome IX. The gene order and intergenic distances were estimated to be: dal1--2.5 cM--dal4--1.9cM--dal2--4.6cM-lys1.     

Metabolism and excretion of nitrogen during metamorphosis and egg production in the sawfly, Neodiprion sertifer

Measurements of whole-body dry matter, total nitrogen, water-soluble protein, amino acids, and uric acid were determined at successive stages during metamorphosis in Neodiprion sertifer. The major change was in the uric acid fraction: in females, it increased up to the non-pharate pupa and then decreased during the subsequent stages of adult development and egg production; in males, it continued to increase during adult development. The decline of uric acid could not be explained by the accumulation of allantoin, allantoic acid, urea, or uric acid riboside. Examination of amino acid levels in the gut revealed an accumulation in the pupa followed by a depletion at the onset of adult development. This was followed by an excretory phase marked by the progressive accumulation of large quantities of uric acid and small quantities of urea, ammonia, and amino acids during the formation of the meconium. Amino acid analysis of the meconium revealed the presence of large proportions of proline, hydroxyproline, and histidine in comparison with the other amino acids.     

The Assimilation of Ureides in Shoot Tissues of Soybeans : 1. CHANGES IN ALLANTOINASE ACTIVITY AND UREIDE CONTENTS OF LEAVES AND FRUITS

The ureides, allantoin and allantoic acid, are major forms of N transported from nodules to shoots in soybeans (Merr.). Little is known about the occurrence, localization, or properties of the enzymes involved in the assimilation of ureides in shoot tissues. We have examined the capacity of the shoot tissues to assimilate allantoin via allantoinase (EC 3.5.2.5) during leaf and fruit development in nodulated soybeans. Specific activity of allantoinase in leaves peaked during pod formation and early seed filling. In developing fruits allantoinase activity in the seeds was 2 to 4 times that in the pods when expressed on a fresh weight or organ basis. In seeds, the embryos contained the highest specific allantoinase activity. Stems and petioles also had appreciable allantoinase activity. With development, peaks in the amounts of allantoic acid, but not allantoin, were measured in both leaves and fruits suggesting that the assimilation of allantoic acid may be a limiting factor in ureide assimilation. Highest amounts of ureides were measured in the pith and xylem of stem tissues and in developing pod walls.     

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.     

Genetic and phenotypic studies of the dark-like mutant mouse

The dark-like (dal) mutant mouse has a pleiotropic phenotype that includes dark dorsal hairs and reproductive degeneration. Their pigmentation phenotype is similar to Attractin (Atrn) mutants, which also develop vacuoles throughout the brain. In further characterizing the testicular degeneration of dal mutant males, we found that they had reduced serum testosterone and developed vacuoles in their testes. Genetic crosses placed dal upstream of the melanocortin 1 receptor (Mc1r) and downstream of agouti, although dal suppressed the effect of agouti on pigmentation but not body weight. Atrn(mg-3J) and dal showed additive effects on pigmentation, testicular vacuolation, and spongiform neurodegeneration, but transgenic overexpression of Attractin-like-1 (Atrnl1), which compensates for loss of ATRN, did not rescue dal mutant phenotypes. Our results suggest dal and Atrn function in the same pathway and that identification of the dal gene will provide insight into molecular mechanisms of vacuolation in multiple cell types.