High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

Bacteria do not incorporate ß-N-methylamino-L-alanine into their proteins.

ß-N-methylamino-l-alanine (BMAA), is commonly found in both a free and proteinassociated form in various organisms exposed to the toxin. The long latency of development of neurodegeneration attributed to BMAA, is hypothesized to be the result of excitotoxicity following slow release of the toxin from protein reservoirs. It was recently suggested that these BMAA-protein associations may reflect misincorporation of BMAA in place of serine, as occurs, for example, when canavanine misincorporates in place of arginine. We therefore compared BMAA and canavanine toxicty in various bacterial species, and misincorporation of these amino acids into proteins in a bacterial protein expression system. None of the bacterial species showed any physiological stress responses to BMAA in contrast to the growth reduction observed when cultures were incubated in media containing canavanine. LC-MS analysis confirmed uptake of BMAA from growth media. However, after immobilized metal affinity chromatography and SDS-PAGE purification of proteins produced in an E scherichia coli expression system, no BMAA was detected by either LC-MS or LC-MS/MS analysis using two derivatization methods, or by orbitrap MS of trypsin digests of the protein. We therefore conclude that BMAA is not misincorporated into proteins in bacteria and that the observed BMAA-protein association in bacteria is superficial.

Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.

The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress.

Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.

Amino acid discrimination by the nuclear encoded mitochondrial arginyl-tRNA synthetase of the larva of a bruchid beetle (Caryedes brasiliensis) from northwestern Costa Rica.

L-canavanine, the toxic guanidinooxy analogue of L-arginine, is the product of plant secondary metabolism. The need for a detoxifying mechanism for the producer plant is self-evident but the larvae of the bruchid beetle Caryedes brasiliensis, that is itself a non-producer, have specialized in feeding on the Lcanavanine-containing seeds of Dioclea megacarpa. The evolution of a seed predator that can imitate the enzymatic abilities of the host permits us to address the question of whether the same problem of amino acid recognition in two different kingdoms has been solved by the same mechanism. A discriminating arginyl-tRNA synthetase, detected in a crude C. brasiliensis larval extract, was proposed to be responsible for insect's ability to survive the diet of L-canavanine (Rosenthal, G. A., Dahlman, D. L., and Janzen, D. H. (1976) A novel means for dealing with L-canavanine, a toxic metabolite. Science 192, 256e258). Since the arginyl-tRNA synthetase of at least three genetic compartments (insect cytoplasmic, insect mitochondrial and insect gut microflora) may participate in conferring L-canavanine resistance, we investigated whether the nuclear-encoded C. brasiliensis mitochondrial arginyl-tRNA synthetase plays a role in this discrimination. Steady state kinetics of the cloned, recombinant enzyme have revealed and quantified an amino acid discriminating potential of the mitochondrial enzyme that is sufficient to account for the overall L-canavanine misincorporation rate observed in vivo. As in the cytoplasmic enzyme of the L-canavanine producer plant, the mitochondrial arginyl-tRNA synthetases from a specialist seed predator relies on a kinetic discrimination that prevents L-canavanine misincorporation into proteins.

Quantification of canavanine, 2-aminoethanol, and cyanamide in Aphis craccivora and its host plants, Robinia pseudoacacia and Vicia angustifolia: effects of these compounds on larval survivorship of Harmonia axyridis.

The cowpea aphid Aphis craccivora that infests the black locust Robinia pseudoacacia shows toxicity to its predator, the multicolored Asian ladybird beetle, Harmonia axyridis. In contrast, the same aphid species that infests the common vetch, Vicia angustifolia, is suitable prey for H. axyridis larvae. Previously, it was reported that the toxicity of A. craccivora infesting R. pseudoacacia was due to canavanine and 2-aminoethanol, but there was some doubt about the toxicity of these compounds and their concentrations in the aphids. In the present study, we determined the concentrations of cyanamide, canavanine, and 2-aminoethanol in A. craccivora infesting the two host plants. In the extracts of A. craccivora that infested either of the host plants, canavanine was undetectable, and 2-aminoethanol was detected at the concentration of 3.0-4.0 µg/g fresh weight. Cyanamide was detected in the extract of A. craccivora that infested R. pseudoacacia (7.7 µg/g fresh weight) but not in that infesting V. angustifolia. The toxicity of canavanine, 2-aminoethanol, and cyanamide was evaluated against H. axyridis larvae in a bioassay by using an artificial diet containing these compounds at various concentrations. Cyanamide exhibited 10-100 times stronger toxicity than canavanine and 2-aminoethanol. These results indicate that the toxicity is at least partly due to cyanamide, which is present in the toxic A. craccivora that infests R. pseudoacacia but absent from the non-toxic A. craccivora that infests V. angustifolia.

Amino acid analog toxicity in primary rat neuronal and astrocyte cultures: implications for protein misfolding and TDP-43 regulation.

Amino acid analogs promote translational errors that result in aberrant protein synthesis and have been used to understand the effects of protein misfolding in a variety of physiological and pathological settings. TDP-43 is a protein that is linked to protein aggregation and toxicity in a variety of neurodegenerative diseases. This study exposed primary rat neurons and astrocyte cultures to established amino acid analogs (canavanine and azetidine-2-carboxylic acid) and showed that both cell types undergo a dose-dependent increase in toxicity, with neurons exhibiting a greater degree of toxicity compared with astrocytes. Neurons and astrocytes exhibited similar increases in ubiquitinated and oxidized protein following analog treatment. Analog treatment increased heat shock protein (Hsp) levels in both neurons and astrocytes. In neurons, and to a lesser extent astrocytes, the levels of TDP-43 increased in response to analog treatment. Taken together, these data indicate that neurons exhibit preferential toxicity and alterations in TDP-43 in response to increased protein misfolding compared with astrocytes.

Misincorporation of amino acid analogues into proteins by biosynthesis.

Despite astounding diversity in their structure and function, proteins are constructed from 22 protein or 'canonical' amino acids. Hundreds of amino acid analogues exist; many occur naturally in plants, some are synthetically produced or can be produced in vivo by oxidation of amino acid side-chains. Certain structural analogues of the protein amino acids can escape detection by the cellular machinery for protein synthesis and become misincorporated into the growing polypeptide chain of proteins to generate non-native proteins. In this review we seek to provide a comprehensive overview of the current knowledge on the biosynthetic incorporation of amino acid analogues into proteins by mammalian cells. We highlight factors influencing their incorporation and how the non-native proteins generated can alter cell function. We examine the ability of amino acid analogues, representing those commonly found in damaged proteins in pathological tissues, to be misincorporated into proteins by cells in vitro, providing us with a useful tool in the laboratory to generate modified proteins representing those present in a wide-range of pathologies. We also discuss the evidence for amino acid analogue incorporation in vivo and its association with autoimmune symptoms. We confine the review to studies in which the synthetic machinery of cell has not been modified to accept non-protein amino acids.

African herbal medicines in the treatment of HIV: Hypoxis and Sutherlandia. An overview of evidence and pharmacology.

In Africa, herbal medicines are often used as primary treatment for HIV/AIDS and for HIV-related problems. In general, traditional medicines are not well researched, and are poorly regulated. We review the evidence and safety concerns related to the use of two specific African herbals, which are currently recommended by the Ministry of Health in South Africa and member states for use in HIV: African Potato and Sutherlandia. We review the pharmacology, toxicology and pharmacokinetics of these herbal medicines. Despite the popularity of their use and the support of Ministries of Health and NGOs in some African countries, no clinical trials of efficacy exist, and low-level evidence of harm identifies the potential for drug interactions with antiretroviral drugs. Efforts should be made by mainstream health professionals to provide validated information to traditional healers and patients on the judicious use of herbal remedies. This may reduce harm through failed expectations, pharmacologic adverse events including possible drug/herb interactions and unnecessary added therapeutic costs. Efforts should also be directed at evaluating the possible benefits of natural products in HIV/AIDS treatment.

The mechanism of L-canavanine cytotoxicity: arginyl tRNA synthetase as a novel target for anticancer drug discovery.

There is a clear need for agents with novel mechanisms of action to provide new therapeutic approaches for the treatment of pancreatic cancer. Owing to its structural similarity to L-arginine, L-canavanine, the beta-oxa-analog of L-arginine, is a substrate for arginyl tRNA synthetase and is incorporated into nascent proteins in place of L-arginine. Although L-arginine and L-canavanine are structurally similar, the oxyguanidino group of L-canavanine is significantly less basic than the guanidino group of L-arginine. Consequently, L-canavanyl proteins lack the capacity to form crucial ionic interactions, resulting in altered protein structure and function, which leads to cellular death. Since L-canavanine is selectively sequestered by the pancreas, it may be especially useful as an adjuvant therapy in the treatment of pancreatic cancer. This novel mechanism of cytotoxicity forms the basis for the anticancer activity of L-canavanine and thus, arginyl tRNA synthetase may represent a novel target for the development of such therapeutic agents.

The antiproliferative and immunotoxic effects of L-canavanine and L-canaline.

L-Canavanine and its arginase-catalyzed metabolite, L-canaline, are two novel anticancer agents in development. Since the immunotoxic evaluation of agents in development is a critical component of the drug development process, the antiproliferative effects of L-canavanine and L-canaline were evaluated in vitro. Both L-canavanine and L-canaline were cytotoxic to peripheral blood mononucleocytes (PBMCs) in culture. Additionally, the mononucleocytes were concurrently exposed to either L-canavanine or L-canaline and each one of a series of compounds that may act as metabolic inhibitors of the action of L-canavanine and L-canaline (L-arginine, L-ornithine, D-arginine, L-lysine, L-homoarginine, putrescine, L-omega-nitro arginine methyl ester and L-citrulline). The capacity of these compounds to overcome the cytotoxic effects of L-canavanine or L-canaline was assessed in order to provide insight into the biochemical mechanisms that may underlie the toxicity of these two novel anticancer agents. The results of these studies suggest that the mechanism of L-canavanine toxicity is mediated through L-arginine-utilizing mechanisms and that the L-canavanine metabolite, L-canaline, is toxic to human PBMCs by disrupting polyamine biosynthesis. The elucidation of the biochemical mechanisms associated with the effects of L-canavanine and L-canaline on lymphoproliferation may be useful for maximizing the therapeutic effectiveness and minimizing the toxicity of these novel anticancer agents.

Synthesis and biological activity of canavanine hydrazide derivatives.

The canavanine derivatives L-canavanine hydrazide (CH), L-canavanine-bis-(2-chloroethyl)hydrazide (CBCH) and L-canavanine phenylhydrazide (CPH) were synthesized and evaluated for biological activity in microorganisms, plants and tumor cells using canavanine as a positive control. (1) In microbial systems, the compounds exerted activity, as assessed in 14 bacterial strains. The effect of canavanine was easily removed by equimolar concentrations of arginine or ornithine, while the effect of CBCH or CPH was abolished by 10-fold excess of arginine or 10- to 100-fold excess of ornithine. (2) In plants, the activity of CH and CBCH were relatively low, whereas the inhibitory potential of CPH was comparable or even superior to that of canavanine, resulting at 1 mM concentration in a nearly complete block of tomato cell growth, and reducing by up to 80% the length of radicles of cress, amaranth, cabbage and pumpkin. (3) In pumpkin seeds, CPH or canavanine induced the synthesis of four small heat shock proteins of hsp-17 family in the pH range of 6 to 7.5. The proteins exhibited in both cases a similar profile, but differed in the timing of their expression and/or accumulation. With canavanine, the highest hsp-17 expression was found after 48 h of drug treatment, while with CPH this maximum was shifted to 24 h. (4) CPH proved to be highly cytotoxic against Friend leukemia cells in culture, exceeding by one order of magnitude the cytotoxicity of canavanine. The effect of canavanine was completely removed in the presence of equimolar amounts of arginine, while a 20-fold excess of arginine failed to abolish the cytotoxicity of CPH. Thus, a proper hydrazide modification of canavanine may lead to a significant increase in its growth-inhibitory activity and to a change in the mode of action of the parent compound.

Growth inhibition of A549 human lung adenocarcinoma cells by L-canavanine is associated with p21/WAF1 induction.

L-Canavanine (CAV) is a higher plant nonprotein amino acid and a potent L-arginine antimetabolite. CAV can inhibit the proliferation of tumor cells in vitro and in vivo, but little is known regarding the molecular mechanisms mediating these effects. We demonstrated that the treatment of human lung adenocarcinoma A549 cells with CAV caused growth inhibition; G1 phase arrest is accompanied by accumulation of an incompletely phosphorylated form of the retinoblastoma protein, whose phosphorylation is necessary for cell cycle progression from G1 to S phase. In addition, CAV induces the expression of p53 and subsequent expression of a cyclin-dependent kinase inhibitor, p21/WAF1. The p53-dependent induction of p21/WAF1 and the following dephosphorylation of the retinoblastoma protein by CAV could account for the observed CAV-mediated G1 phase arrest.

The biochemical basis for L-canavanine tolerance by the tobacco budworm Heliothis virescens (Noctuidae).

The tobacco budworm, Heliothis virescens (Noctuidae), a destructive insect pest, is remarkably resistant to L-canavanine, L-2-amino-4-(guanidinooxy)butyric acid, an arginine antimetabolite that is a potent insecticide for nonadapted species. H. virescens employs a constitutive enzyme of the larval gut, known trivially as canavanine hydrolase (CH), to catalyze an irreversible hydrolysis of L-canavanine to L-homoserine and hydroxyguanidine. As such, it represents a new type of hydrolase, one acting on oxygen-nitrogen bonds (EC 3.13.1.1). This enzyme has been isolated from the excised gut of H. virescens and purified to homogeneity; it exhibits an apparent Km value for L-canavanine of 1.1 mM and a turnover number of 21.1 micromol x min(-1)x micromol(-1). This enzyme has a mass of 285 kDa and is composed of two subunits with a mass of 50 kDa or 47.5 kDa. CH has a high degree of specificity for L-canavanine as it cannot function effectively with either L-2-amino-5-(guanidinooxy)pentanoate or L-2-amino-3-(guanidinooxy)propionate, the higher or lower homolog of L-canavanine, respectively. L-Canavanine derivatives such as methyl-L-canavanine, or L-canaline and O-ureido-L-homoserine, are not metabolized significantly by CH.

Effects of the naturally occurring arginine analogues indospicine and canavanine on nitric oxide mediated functions in aortic endothelium and peritoneal macrophages.

The ability of the naturally occurring non-protein toxic amino acids indospicine and canavanine to inhibit nitric oxide synthesis was tested in isolated rat aorta and cultured rat peritoneal macrophages. Both compounds inhibited acetylcholine induced relaxation of rat aorta contracted with noradrenaline, a process mediated by nitric oxide generated in vascular endothelium. Nitric oxide is generated in vascular endothelium from arginine by a constitutive nitric oxide synthase. Indospicine and canavanine also increased superoxide mediated reduction of cytochrome c by phorbol myristate acetate stimulated rat peritoneal macrophages. The increase in superoxide under these conditions was due to decreased nitric oxide synthesis. Macrophage synthesis of nitric oxide is mediated by an inducible form of nitric oxide synthase. It is concluded that indospicine and canavanine are inhibitors of constitutive and inducible nitric oxide synthases and it is suggested that the toxicity associated with these compounds could be related to this activity.

The effect of food chemicals on cell aging of human diploid cells in in vitro culture.

The potency of food chemicals to induce cell aging was evaluated in human diploid fibroblast cells HAIN-55 having a finite replicative potential by using in vitro aging markers, i.e., decreases of maximum proliferative potential (lifespan) of cells, saturation density in monolayer culture (SD), plating efficiency (PE) and mitotic index (MI), and an increase of cells with polyploid karyotypes. By treatment twice with low concentration of genotoxic chemicals aflatoxin B1, allylisothiocyanate or trans-cinnamaldehyde (severe clastogenic flavoring agent; Kasamaki et al., 1982), lifespan (expressed by the number of cumulative cell population doubling (CPD)) of the treated cells was reduced by 8-12 CPDs accompanied by change of the other aging markers. By successive treatment (29 or 25 times) with non-genotoxic chemical aspartame (N-L-aspartyl-L-phenylalanine) or L-canavanine (structural analogue of L-arginine), lifespan of the treated cells was also slightly shortened (by 2-6 CPDs) compared with the untreated control cells. In the process of cell aging, Mitochondrial activity (MTT activity) decreased almost in parallel with the decrease of SD and MI. On the basis of these results, a variety of genotoxic and non-genotoxic chemicals were examined by using MTT activity as the aging marker for their effects on the aging of HAIN-55 cells and bovine artery endothelial cells which also had a finite replicative potential. The results showed that seven genotoxic and nine non-genotoxic chemicals promoted cell aging.

[Isolation and characteristics of new mutants of Saccharomyces cerevisiae with increased spontaneous mutability]. / Vydelenie i kharakteristika novykh mutantov drozhzhei Saccharomyces cerevisiae s povyshennoi spontannoi mutabil'nost'iu.

To isolate some new genes controlling the process of spontaneous mutagenesis, a collection of 16 yeast strains with enhanced rate of spontaneous canavanine resistant mutations was obtained. Genetical analysis allowed to define that the mutator phenotype of these strains is due to a single nuclear mutation. Such mutations were called hsm (high spontaneous mutagenesis). Recombinational test showed that 5 mutants under study carried 5 nonallelic mutations. It was revealed that the mutation hsm3-1 is a nonspecific mutator elevating the rate of both spontaneous canavanine resistant mutations and the frequency of reversions in mutations lys1-1 and his1-7. Genetical analysis revealed that mutation hsm3-1 is recessive. The study of cross sensitivity of mutator strains to physical and chemical mutagens demonstrated that 12 of 16 hsm mutants were resistant to the lethal action of UV, gamma rays and methylmethanesulfonate, and 4 mutants were only sensitive to these factors. Possible nature of hsm mutations is discussed.

Toxicity and pharmacokinetics of the nonprotein amino acid L-canavanine in the rat.

The toxicity of L-canavanine was investigated because of its demonstrated potential as an antitumor drug. This natural product was only slightly toxic to Sprague-Dawley rats following a single sc injection: the LD50 was 5.9 +/- 1 8 g/kg in adult rats and 5.0 +/- 1.0 g/kg in 10-day-old rats. Following a single dose of 2.0 g/kg, the systemic clearance value for canavanine in adult rats was 0.114 liter/hr, the volume of distribution at steady state was 0.154 liter, and the half-life was 1.56 hr. Forty-eight percent of the dose was excreted unaltered in the urine following an iv injection, and 16% of a sc dose was recovered in the urine. Bioavailability of a 2.0 g/kg sc dose was 72%. Single oral doses of canavanine were less toxic to adult rats than sc injections. Bioavailability of a 2.0 g/kg po dose was 43%, and only 1% of the administered canavanine was recovered in the urine. Twenty-one percent of the administered canavanine remained in the gastrointestinal tract 24 hr after an oral dose. Less than 1% of a 2.0 g/kg dose of L-[guanidinooxy-14C]canavanine was incorporated into the proteins of adult and neonatal rats 4 or 24 hr following administration. Repeated sc administration of canavanine resulted in more severe toxicity. Weight loss and alopecia were observed in rats given daily sc canavanine injections for 7 days. Food intake was decreased by 80% in adult rats subjected to this dosing regimen, but returned to normal after canavanine injections were terminated. Histological studies of tissues from adult rats treated with 3.0 g/kg canavanine daily for 6 days revealed pancreatic acinar cell atrophy and fibrosis. Serum amylase and lipase levels were elevated following one sc injection of 2.0 g/kg canavanine; after three daily injections both serum enzymes were depleted. Elevations in serum glucose and urea nitrogen, and depletion of cholesterol, were observed. The most significant changes were severe attenuations of serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activity.

Metabolism of L-[guanidinooxy-14C]canavanine in the rat.

The metabolism of L-canavanine, a nonprotein amino acid with significant antitumor effects, was investigated. L-Canavanine, provided at 2.0 g/kg, was supplemented with 5 microCi of L-[guanidinooxy-14C]canavanine (58 microCi/mumol) and administered iv, sc, or orally to female Sprague-Dawley rats weighing approximately 200 g. 14C recovery in the urine at 24 hr was 83, 68, or 61%, respectively, of the administered dose. Another 5-8% of the 14C was expired as 14CO2. The gastrointestinal tract contained 21% of orally administered 14C. Serum, feces, tissues, and de novo synthesized proteins only accounted for a few percent of the original dose by any administrative route. Analysis of the 14C-containing urinary metabolites revealed that [14C] urea accounted for 88% of the urinary radioactivity for an iv injection, 75% for sc administration, and 50% following an oral dose. By all routes of administration, [14C]guanidine represented 5% of the radioactivity in the urine and [14C]guanidinoacetic acid accounted for 2%. Serum and urine amino acid analysis showed a markedly elevated ornithine level. Basic amino acids such as histidine, lysine, and arginine were also higher in the urine. Plasma ammonia levels were determined following oral canavanine doses of 1.0, 2.0, and 4.0 g/kg. A rapid but transient elevation in plasma ammonia was observed only at the 4.0 g/kg dose. This indicates that elevated plasma ammonia is not a likely cause of canavanine toxicity at the drug concentrations used in this study.