Application of the disk method to cultured plant cells. I. Inhibition zones

When paper disks carrying small volumes of highly concentrated drugs were placed on the suface of the medium in plant cell culture plates, diffusion of the drugs led to a circular area of non-dividing cells (an inhibition zone) around the disks. Out of 63 drugs tested 37 were inhibitory and 15 of these produced a clear inhibition zone.Drug concentrations could be estimated by measuring the inhibition zone diameter. Cell growth and drug diffusion were analysed and the influence of several variables on inhibition zone formation studied. Inhibition zones occured with cultures of Zea mays, Acer pseudoplatanus, Daucus carota and Hyoscyamus muticus and protoplast-derived cells of Rosa. Possible applications of the method in plant cell genetics and physiology are discussed.Abbreviations ETH L-ethionine - MMC methylmercury chloride - NAA l-naphthaleneacetic acid - BAP 6-benzylaminopurine - PCV Packed cell volume - PE Plating efficiency     

Aspartokinase of a lysine producing mutant ofMicrococcus glutamicus

Aspartokinase fromMicrococcus glutamicus AEC RN-13-6/1 [a homoserine requiring, S-(2-aminoethyl)-L-cysteine resistant, lysine producing strain] was purified 71 fold. The partially purified enzyme was inhibited by L-lysine. L-threonine, L-methionine, L-isoleucine, L-valine and L-phenylalanine activated the enzyme and reversed the inhibition by L-lysine. Aspartokinase activity was not derepressed by growth-limiting concentrations of L-threonine and/or L-methionine. It was not repressed by an excess of L-lysine (20 mM) and/or L-isoleucine (15.3 mM). The degree of activation or inhibition by amino acids was dependant on the composition of the growth medium. This observation is in contrast with the enzyme from the original (non-lysine-producing) strain which was inhibited by lysine or threonine and in a concerted manner by threonine plus lysine.     

Mechanism of inhibition of Chromatium D growth by L-methionine regulation of L-threonine biosynthesis by the intracellular level of S-adenosylmethionine

1. (1) An unusual accumulation of S-adenosyl-L-methionine in Chromatium D was associated with a marked growth inhibition by L-methionine. The inhibition was overcome by L-isoleucine, L-leucine, L-phenylalanine, L-threonine, L-valine and putrescine. Based on their effects, these compounds are classified into 3 types.

2. (2) L-Isoleucine, L-leucine, L-phenylalanine and L-valine (Type I) inhibited the L-methionine uptake and consequently prevented the bacterium from the unusual accumulation of S-adenosyl-L-methionine even in the presence of L-methionine in the medium. Putrescine (Type II) stimulated the consumption of S-adenosyl-L-methionine, but did not influence the L-methionine uptake. Hence, the effect of putrescine would be explained by the action to diminish the intracellular level of S-adenosyl-L-methionine. L-Threonine (Type III) neither inhibited the L-methionine uptake nor affected the content of S-adenosyl-L-methionine due to the addition of L-methionine.

3. (3) The specific activity of homoserine kinase (EC 2.7.1.39) was greatly lowered by the addition of L-methionine under conditions in which Chromatium D unusually accumulates S-adenosyl-L-methionine. Homoserine dehydrogenase (EC 1.1.1.3) activity was inhibited by S-adenosyl-L-methionine (50% inhibition index, 3.5 mM). These facts strongly suggest that the growth inhibition by L-methionine is associated with the L-threonine deficiency caused by the unusual accumulation of S-adenosyl-L-methionine.

Disruption of thymidylate synthesis and glycine-serine interconversion by L-methionine and L-homocystine in Raji cells

Excessive concentrations of L-methionine inhibited the folate-dependent de novo synthesis of thymidylic acid (TMP) in Raji cells, demonstrating the usefulness of this cell line for the study of methionine-folate antagonism. The effect was also produced by L-homocystine but not by other amino acids including D-methionine and L-ethionine, suggesting that this effect is exerted by a common intermediate of methionine and homocystine metabolism. L-Methionine, L-homocysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) are not inhibitors of thymidylate synthase activity. On the other hand the capacity of the cells to incorporate serine 3-carbon and glycine 2-carbon into DNA is impaired by the presence of L-methionine or L-homocystine. Studies with cell-free extracts demonstrated that the glycine cleavage enzyme is inhibited by 45% by L-methionine, L-homocysteine, SAM or SAH. Serine hydroxymethylase on the other hand was slightly stimulated by these sulfur-containing compounds and this stimulation was shown to occur in the intact cell as well. These findings suggest that when levels of L-methionine metabolites are elevated, there is an increase in the use of glycine to maintain the intracellular concentration of serine, which is required for homocysteine detoxification by conversion to cystathionine. The reduction in TMP synthesis caused by excess L-methionine or L-homocystine may result from increased utilization of one-carbon units for serine synthesis.     

L-tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris Speg. et Comes

The biosynthetic route to L-tyrosine was identified in isogenic suspension-cultured cells of N. silvestris. Arogenate (NADP⁺) dehydrogenase, the essential enzyme responsible for the conversion of L-arogenato L-tyrosine, was readily observed in crude extracts. In contrast, prephenate dehydrogenase (EC 1.3.1.13) activity with either NAD⁺ or NADP⁺ was absent altogether. Therefore, it seems likely that this tobacco species utilizes the arogenate pathway as the exclusive metabolic route to L-tyrosine. L-Tyrosine (but not L-phenylalanine) was a very effective endproduct inhibitor of arogenate dehydrogenase. In addition, analogs of L-tyrosine (m-fluoro-DL-tyrosine [MFT], D-tyrosine and N-acetyl-DL-tyrosine), but not of L-phenylalanine (o-fluoro-DL-phenylalanine and p-fluoro-DL-phenylalanine), were able to cause inhibition of arogenate dehydrogenase. The potent antimetabolite of L-tryptophan, 6-fluoro-DL-tryptophan, had no effect upon arogenate dehydrogenase activity. Of the compounds tested, MFT was actually more effective as an inhibitor of arogenate dehydrogenase than was L-tyrosine. Since MFT was found to be a potent antimetabolite inhibitor of growth in N. silvestris and since inhibition was specifically and effectively reversed by L-tyrosine, arogenate dehydrogenase is an outstanding candidate as the in vivo target of analog action. Although chorismate mutase (EC 5.4.99.5) cannot be the prime target of MFT action, MFT can mimick L-tyrosine in partially inhibiting this enzyme activity. The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was insensitive to L-phenylalanine or L-tyrosine. The overall features of this system indicate that MFT should be a very effective analog mimick for selection of feedback-insensitive regulatory mutants L-tyrosine biosynthesis.Abbreviations DAHP synthase 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase - 6FT 6-fluoro-DL-tryptophan - MFT m-fluoro-DL-tyrosine - OFP o-fluoro-DL-phenylalanine - PFP p-fluoro-DL-phenylalanine     

Effect of L-ethionine the expression of the SOS system in Escherichia coli

The effect of L-ethionine, the ethyl analog of the essential amino acid methionine the SOS system of Escherichia coli was studied. This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA⁺ Met⁺ RelA⁺ strain, nor in RecA⁺ Met⁻ RelA⁺ or RecA⁺ Met⁻ RelA⁻ mutants. Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42°C. Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift. Moreover, cultures of the recA441 mutant incubated at 42°C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid. On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA⁺ strain even if this compound is added to the cells several hours before irradiation.     

Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway

Methionine gamma-lyase (MGL) catalyzes the degradation of L-methionine to alpha-ketobutyrate, methanethiol and ammonia. The Arabidopsis (Arabidopsis thaliana) genome includes a single gene (At1g64660) encoding a protein (AtMGL) with approximately 35% identity to bacterial and protozoan MGLs. When overexpressed in Escherichia coli, AtMGL allowed growth on L-methionine as sole nitrogen source and conferred a high rate of methanethiol emission. The purified recombinant protein exhibited a spectrum typical of pyridoxal 5'-phosphate enzymes, and had high activity toward l-methionine, L-ethionine, L-homocysteine and seleno-L-methionine, but not L-cysteine. Quantitation of mRNA showed that the AtMGL gene is expressed in aerial organs and roots, and that its expression in leaves was increased 2.5-fold by growth on low sulfate medium. Emission of methanethiol from Arabidopsis plants supplied with 10 mM L-methionine was undetectable (<0.5 nmol min(-1) g(-1) FW), suggesting that AtMGL is not an important source of volatile methanethiol. Knocking out the AtMGL gene significantly increased leaf methionine content (9.2-fold) and leaf and root S-methylmethionine content (4.7- and 7-fold, respectively) under conditions of sulfate starvation, indicating that AtMGL carries a significant flux in vivo. In Arabidopsis plantlets fed L-[(35)S]methionine on a low sulfate medium, label was incorporated into protein-bound cysteine as well as methionine, but incorporation into cysteine was significantly (30%) less in the knockout mutant. These data indicate that plants possess an alternative to the reverse trans-sulfuration pathway (methionine-->homocysteine-->cystathionine-->cysteine) in which methanethiol is an intermediate.     

Effects of cysteine and structurally related compounds on ochratoxin production by Aspergillus ochraceus

Addition of 10(-2) M L-cysteine, L-cystine, or S-ethyl-L-cysteine to a synthetic medium containing xylose as the sole carbon source did not decrease ochratoxin production by Aspergillus ochraceus. At that concentration, DL-homocysteine thiolactone HC1, DL-cysteine HC1, L-ethionine, S-methyl-L-cysteine, and glutathione (reduced) strongly inhibited ochratoxin production. DL-Homocysteine thiolactone HC1, DL-cysteine HC1, and L-ethionine also strongly inhibited fungal growth. At lower concentrations (10(-3) and 10(-4) M) only L-ethionine decreased the toxin production. Ochratoxin inhibition caused by DL-homocysteine thiolactone HC1, DL-cysteine HC1, and glutathione was observed only in cases where the pH of the media was below 5.0. The inhibition caused by 10(-3) M ethionine was partially prevented by the addition of 10(-3) M methionine but this was not the case after the addition of S-methyl-L-cysteine to the medium.     

Effect of L-ethionine on the expression of the SOS system in Escherichia coli

The effect of L-ethionine, the ethyl analog of the essential amino acid methionine, on the SOS system of Escherichia coli was studied. This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA+ Met+ RelA+ strain, nor in RecA+ Met- RelA+ or RecA+ Met- RelA- mutants. Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42 degrees C. Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift. Moreover, cultures of the recA441 mutant incubated at 42 degrees C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid. On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA+ strain even if this compound is added to the cells several hours before irradiation.     

Interaction of odorous lactones with phospholipids: implications in toxicity towards producing yeast cells

Some lactonic aroma compounds, that can be produced industrially by microorganisms, become toxic towards the producing cells as these compounds reach high concentrations in the culture medium. To determine the manner by which these metabolites may influence yeast physiology, the effects of four lactones (concentration range of 100 to 300 mg l^–1) on the growth of Yarrowia lipolytica and on the phase behaviour of deuterated dimyristoylphosphatidylcholine (DMPC-d27) were studied. The results showed that the hydrophobic lactones decrease the phase transition temperature (Tm) of DMPC-d27 bilayers and that Tm decrease (Tm) was related to the inhibitory action of the lactones on yeast growth (evaluated by the lag time). These results suggest that whatever the lactone, a Tm of at least 2.5 °C resulted in a total growth inhibition: this implicates the lactone-phospholipids interaction in the mechanism of yeast growth inhibition. The test used in the present study may be a predicting method to assess the in vivo action of potential membrane active compounds.     

Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates

A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solaniAG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicilliumstrains with R. solani prepared in sterilized soil and when the Penicilliumstrains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicilliumspecies against R. solani is discussed.     

In vitro cultures of Solanum malacoxylon Sendt.: nutritional requirements and sterol production

Callus cultures were established from hypocotyl, root and leaf explants of Solanum malacoxylon. The growth rate of calli was evaluated on Murashige and Skoog medium as well as Gamborg's B5 medium. Sterols were isolated from calli grown on both media however the B5 proved to be more effective in promoting the production of these metabolites. No calcitriol was found in the cultures. Feeding experiments with vitamin D₃ were scarcely effective in promoting the production of vitamin D₃ hydroxylated metabolites.Abbreviations GC/MS gas-liquid chromatographic-mass spectrometric analysis - CHCL₃ chloroform - GLC gas liquid chromatography - PCV packed cell volume - TLC thin layer chromatography     

Effects on winter wheat seedling growth by toxin-producing rhizobacteria

Summary Root-colonizing pseudomonads capable of inhibiting seedling winter wheat (Triticum aestivum L.) root growth in an agar seedling bioassay also significantly inhibited wheat root growth in vermiculite; however, the inhibitory trait is quite labile in laboratory culturing. The extent of inhibition in both the agar and vermiculite medium depended on inoculum level. These pseudomonads were found to produce a toxin capable of inhibiting growth ofEscherichia coli C-la andBacillus subtilis. Field isolates that strongly inhibit growth of indicator bacteria also inhibited root growth. Toxin production by the bacteria appeared necessary for inhibition of root growth and indicator bacteria as toxin-negative (TOX^–) mutants no longer inhibited either. Antibiosis towardsE. coli as well as wheat seedling root inhibition in agar was reversed by L-methionine, providing further evidence that a toxin, produced by these organisms, is involved in growth retardation.Contribution in cooperation with the College of Agric. Res. Center, Washington State Univ., Pullman, WA 99164. Scientific Paper No. 6837.     

The pathophysiology of neurofibromatosis. I. Resistance in vitro to 3-nitrotyrosine as an expression of the mutation

The in vitro expression of the autosomal dominant mutation responsible for neurofibromatosis was probed using the amino acid analogue 3-nitrotyrosine as a cell culture selective agent. The presence of 3-nitrotyrosine in culture medium led to inhibition of growth and cell death among normal skin fibroblasts in log phase growth, whereas cell strains derived from six different patients' neurofibromas or skin cells, or both, exhibited a consistently enhanced ability to survive under the same conditions. At 0.8 mM 3-nitrotyrosine, four patient-derived skin fibroblast strains could be differentiated from five strains of control skin fibroblasts with a high level of confidence (P < 0.0000). In the same way four neurofibroma-derived fibroblast strains were differentiated from control skin fibroblasts (P < 0.0022). Neurofibroma-derived cells were not different from control cells when treated with 5-fluorotryptophan or p-fluorophenylalanine.     

Purification and properties of gamma-glutamyltranspeptidase from Proteus mirabilis.

gamma-Glutamyltranspeptidase was purified ca. 15,200-fold from cell-free extracts of Proteus mirabilis to electrophoretic homogeneity and then crystallized. The enzyme has an estimated molecular weight of 80,000 and consists of two different subunits with molecular weights of ca. 47,000 and 28,000. The purified enzyme catalyzed hydrolysis and transpeptidation of various gamma-glutamyl compounds, including the oxidized and reduced forms of glutathione, gamma-glutamyl compounds of L-phenylalanine, L-tyrosine, L-histidine, L-alpha-aminobutyrate, L-leucine, and p-nitroaniline. Glycylglycine, L-phenylalanine, L-methionine, L-histidine, L-tryptophan, and L-isoleucine were good acceptors of the gamma-glutamyl moiety in the transpeptidation reaction. Km values for gamma-glutamyl compounds were on the order of 10(-4) to 10(-5) M, and those for acceptor peptides and amino acids were on the order of 10(-2) to 10(-3) M. The enzyme was inhibited by L-serine plus borate and 6-diazo-5-oxo-L-norleucine, which are inhibitors of gamma-glutamyltranspeptidases isolated from mammals. Various amino acids alone were found to inhibit the transpeptidation competitively with a gamma-glutamyl donor. Kinetic analysis suggested that the reaction sequence of substrate binding and product release proceeds according to a ping pong bi bi mechanism.     

Biological evaluation of 10-(diphenylmethylene)- 4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione derivatives

Antibacterial and antifungal activity of 10-(diphenylmethylene)-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione derivatives were examined by the disc-diffusion method (growth inhibition zone diameter in agar medium). The MIC’s for the most active agents were determined. Title compounds were also evaluated in vitro against representatives of different virus classes. Most of the tested compounds exhibit activity against CVB-2 virus.     

Cytotoxic effects of butyrate and other ‘differentiation inducers’ on immature lymphoid cells

A connection between the processes of cell death and differentiation is suggested by observations which show that chemical inducers of differentiation are cytotoxic to CCRF-CEM human leukaemic lymphoblasts, cells which have properties typical of immature lymphoid cells. Sodium $\text{n}$-butyrate, salts of other short-chain fatty acids, 5-azacytidine, hypoxanthine, L-ethionine and dimethyl sulphoxide were all cytotoxic to these cells at concentrations similar to those reported to produce reversible growth inhibition in more mature lymphocytes or growth inhibition and differentiation in other cell types. Only actively cycling cells were susceptible to killing by $\text{n}$-butyrate. Inhibitory effects of these compounds on DNA methylation are postulated to be responsible for their cytotoxic actions.     

Bacteriological tests as an aid in the management of dental caries in Iceland

The activities of three natural coumarins, xanthotoxin and bergapten (fromAmmi majus, Umbelliferae) and psoralene (fromFicus cycomorus, Moraceae), were tested against mycelial growth and aflatoxin production of a toxigenic strain ofAspergillus flavus grown in a rice/corn steep liquor medium. Two other natural chromones, khellin and visnagin (fromAmmi visnaga) were also compared. Complete inhibition of aflatoxin release occurred with either xanthotoxin or khellin at 5 mM. The other three compounds also at 5 mM reduced aflatoxin to 12 to 16% of its original concentration. The mould growth was only slightly inhibited by all the compounds used.     

Ethylene production by cress roots and excised cress root segments and its inhibition by 3,5-diiodo-4-hydroxybenzoic acid

Summary 3,5-Diiodo-4-hydroxybenzoic acid (DIHB) has been shown to exert an inhibitory effect on the formation of ethylene by the roots of intact cress Lepidium sativum seedlings in light, and by excised cress root segments. Adding IAA to the culture solution greatly promoted ethylene production, which was suppressed by DIHB. The findings together with results obtained with dinitrophenol (DNP), L-methionine and L-ethionine and also the horseradish peroxidase/methional system of Yang are discussed.The results indicate that the effect of DIHB in promoting the root growth of cress seedlings in nutrient solution in the light operates, at least in part, by suppressing the formation of the root growth inhibitor ethylene.Abbreviation GLC gas-liquid chromatography - dIHB 3,5-diiodo-4-hydroxybenzoic acid This study forms part of research to be submitted for PhD degree and supported by a grant from Consejo Nacional de Ciencia y Tecnología (México).