Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

Screening of substrate analogs as potential enzyme inhibitors for the arginine kinase of Trypanosoma cruzi

Arginine kinase catalyzes the transphosphorylation between phosphoarginine and ADP. Phosphoarginine is involved in temporal ATP buffering and inorganic phosphate regulation. Trypanosoma cruzi arginine kinase phosphorylates only L-arginine (specific activity 398.9 x mUE-min(-1) x mg(-1)), and is inhibited by the arginine analogs, agmatine, canavanine, nitroarginine, and homoarginine. Canavanine and homoarginine also produce a significant inhibition of the epimastigote culture growth (79.7% and 55.8%, respectively). Inhibition constants were calculated for canavanine and homoarginine (7.55 and 6.02 mM, respectively). In addition, two novel guanidino kinase activities were detected in the epimastigote soluble extract. The development of the arginine kinase inhibitors of T. cruzi could be an important feature because the phosphagens biosynthetic pathway in trypanosomatids is different from the one in their mammalian hosts.     

Relative argininosuccinate synthetase mRNA levels and gene copy number in canavanine-resistant lymphoblasts

Mutants resistant to the arginine analogue, canavanine, have been isolated from two normal lymphoblast lines, MGL8B2 and MGL33. These mutants constitutively express up to 200-fold higher amounts of structurally normal argininosuccinate synthetase, the urea cycle enzyme that converts citrulline to argininosuccinate. Relative levels of argininosuccinate synthetase mRNA were compared among normal and canavanine-resistant lines using in vitro translation of poly(adenylic acid) RNA and blot hybridization of total cytoplasmic RNA to an argininosuccinate synthetase cDNA. Both of these approaches indicated that the canavanine-resistant lines contain increased steady-state levels of synthetase-specifc mRNA relative to their sensitive parents and that these were roughly correlated with levels of enzyme activity. Blot hybridization of Eco RI-digested genomic DNA preparations revealed no detectable differences in argininosuccinate synthetase structural gene copy number between normal and canavanine-resistant lymphoblasts, demonstrating that the canavanine-resistant phenotype is not caused by gene amplification.     

Canavanine-lnduced inhibition of growth and heterocyst differentiation inAnabaena doliolum and isolation of a canavanine-resistant mutant

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen-fixing algaAnabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μM. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. A mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.     

Canavanine derivatives useful in peptide synthesis

Summary The objective of this work is to investigate the possibilities for introducing the currently used N-, N_G- and C-protective groups into the canavanine molecule and the preparation of canavanines selectively blocked at the guanidino function. These novel compounds will find application in the synthesis of canavanine derivatives expected to be amino acid antimetabolites and of canavanine modified biologically active peptides.     

Allelochemical induced stress: Effects of l-canavanine on the pathogenicity of Bacillus thuringiensis in Manduca sexta

Increased susceptibility of Manduca sexta to commercial formulations of the microbial insecticide, Bacillus thuringiensis, as evidenced by lower LD₅₀ and LT₅₀ values, was observed when M. sexta were reared on an artificial diet supplemented with a sublethal concentration (2.5 mm) of l-canavanine. At several dosages of B. thuringiensis, which were administered either by diet contamination or by per os forced feeding, a greater than 70% reduction (P < 0.05) occurred in the LT₅₀ response with canavanine-treated larvae. The LD₅₀ values also were lowered by canavanine treatment. This constitutes the first report of a plant allelochemical enhancing the effect of B. thuringiensis in vivo. It is suggested that canavanine enhances the effect of B. thuringiensis on gut permeability and active transport.     

毛萼田菁茎瘤菌被种子浸提物诱导表达基因的筛选及其功能

摘要：【目的】选择毛萼田菁茎瘤菌ORS571为研究对象,筛选毛萼田菁茎瘤菌在菌植互作早期被宿主植物种子浸提物诱导表达的基因。【方法】采用新颖的基于抗性的活体表达筛选技术,以种子浸提物为诱导物,筛选获得能够稳定被种子浸提物诱导的突变株。通过中间片断融合报告基因的方法研究信号分子对相关基因的诱导情况。【结果】随机引物PCR产物测序及网上BLAST结果表明,被诱导表达的基因为LysE家族的成员。而且,对另外3个属根瘤菌中lysE基因的研究发现,它们都可以被相应的宿主植物种子浸提物诱导表达。此外,这四株根瘤菌的lysE突变株都表现对刀豆氨酸极为敏感。本文首次报道了lysE家族在根瘤菌中的生理功能。【结论】我们推测：LysE家族蛋白可能广泛存在于根瘤菌中,并在根瘤菌与宿主植物的早期相互作用中发挥重要作用。     

Selective cytotoxicity of L-canavanine in tumorigenic Madin-Darby canine kidney T1 cells

L-Canavanine, an analog of L-arginine, was examined for toxicity in a normal canine kidney epithelial cell line, Madin-Darby canine kidney (MDCK), and in its chemically transformed derivative MDCK-T₁. Under conditions where the analog reversibly arrested growth of MDCK cells, more than 90% of the tumorigenic cells were killed. This selective cytotoxicity was not due to any difference in growth rate (both lines doubled every 24 h). Nor was it caused by the inhibition of protein synthesis or DNA replication, since amino acid deprivation and drugs which inhibited replication directly and indirectly did not kill the transformed cells preferentially. To the contrary, cycloheximide killed MDCK cells preferentially.

Although the mechanism for the selective cytotoxicity of canavanine in the tumorigenic cells remains obscure, one clue was afforded by the observation that the analog caused a loss in plating efficiency of the T₁ cells prior to their detachment from plates. Selective sensitivity to canavanine may therefore reside in cell surface proteins, which are known to differ both qualitatively and quantitatively between normal and transformed cells. The present results support our previous findings suggesting the possible value of using canavanine as an agent for cancer chemotherapy.     

The effect of canavanine on protein synthesis and protein degradation in IMR-90 fibroblasts

Proteins of IMR-90 fibroblasts incorporating [³⁵S]methionine during a 1 h labelling period in the presence of the arginine analogue canavanine were degraded twice as rapidly in the cells as were proteins similarly made in the presence of arginine. Using both isoelectric focusing and SDS-polyacrylamide gel electrophoretic analyses, the banding patterns of proteins labelled in the presence of canavanine and arginine were found to differ. This banding difference was detected as early as 15 min after canavanine treatment. With the exception of one minor band in isoelectric focusing gel, the relative intensity of labelled protein bands for the control samples remained unchanged during the 2 h period of protein degradation being investigated. This was also true for the proteins labelled in the presence of canavanine, despite the increase in their rate of degradation. Banding difference between canavanine and arginine treatment was also detected in an in vitro reticulocyte lysate translation system dependent on fibroblast mRNA. Proteins labelled in the presence of a different analogue, p-fluorophenylalanine instead of phenylalanine, however, had similar banding patterns as the control both in the lysate system and in intact cells.     

Non-protein amino acids in plant defense against insect herbivores: representative cases and opportunities for further functional analysis

Chemical defense against herbivores is of utmost importance for plants. Primary and secondary metabolites, including non-protein amino acids, have been implicated in plant defense against insect pests. High levels of non-protein amino acids have been identified in certain plant families, including legumes and grasses, where they have been associated with resistance to insect herbivory. Non-protein amino acids can have direct toxic effects via several mechanisms, including misincorporation into proteins, obstruction of primary metabolism, and mimicking and interfering with insect neurological processes. Additionally, certain non-protein amino acids allow nitrogen to be stored in a form that is metabolically inaccessible to herbivores and, in some cases, may act as signals for further plant defense responses. Specialized insect herbivores often possess specific mechanisms to avoid or detoxify non-protein amino acids from their host plants. Although hundreds of non-protein amino acids have been found in nature, biosynthetic pathways and defensive functions have been elucidated in only a few cases. Next-generation sequencing technologies and the development of additional plant and insect model species will facilitate further research on the production of non-protein amino acids, a widespread but relatively uninvestigated plant defense mechanism.