Binding of (acetyl-14C)trichodermin to the peptidyl transferase centre of eukaryotic ribosomes.
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Acetates↗
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Trichodermin, a new sesquiterpene antibiotic.
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Anti-Bacterial Agents↗
Peptidyltransferase inhibition by trichodermin.
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Acyltransferases↗
Total synthesis of (+)-trichodermin.
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Anti-Bacterial Agents↗
Quantitative binding of antibiotics to ribosomes from a yeast mutant altered on the peptidyl-transferase center.
Quantitative binding studies of [G-3H]anisomycin and [acetyl-14C]trichodermin to sensitive and resistant 80-S ribosomes from yeasts are described in this work. A single mutation, most probably affecting the ribosome peptidyl transferase centre, appears to have pleiotropic effects on the ribosome leading to resistance to trichodermin and anisomycin and to an increased sensitivity to sparsomycin. Resistance to trichodermin is due to a reduced affinity of ribosomes from the mutant for the antibiotic. Ribosomes from the sensitive strain (Y 1661 bind [acetyl-14C]trichodermin with a dissociation constant of 0.99 muM while those from the resistant one (TR1) bind [acetyl-14C]trichodermin with a dissociation constant of 15.4 muM. Similar results are obtained when the binding of [acetyl-14C]trichodermin to Y 166 and TR1 60-S subunits is studied. The mutant TR1 is also resistant to anisomycin. Although trichodermin and anisomycin bind to the ribosome at mutually exclusive sites, the higher affinity binding of [G-3H]anisomycin that is responsible for the inhibition of the peptidyl transferase center is practically identical for Y 166 and TR1 ribosomes. Therefore, the mutation in the ribosome leading to resistance to trichodermin and anisomycin decreases the affinity for trichodermin but not for anisomycin. Trichodermin, trichothecin and fusarenon X inhibit the binding of [G-3H]anisomycin to TR1 ribosomes to a lower extent than to Y 166 ribosomes, suggesting that the resistance of TR1 ribosomes to the effects of trichothecin and fusarenon X is caused by a decrease in the affinity of the ribosomes for these drugs, as was seen with trichodermin. On the other hand, verrucarin A inhibits [G-3H]anisomycin binding to Y 166 and TR1 ribosomes to a similar extent and therefore its affinity for the ribosome does not appear to be affected by the mutation leading to resistance. Trichothecin, trichodermin and fusarenon X appear to have a common binding site on the 60-S ribosomal subunits, which overlaps or is closely linked to the binding sites of anisomycin and verrucarin A.
Acyltransferases↗
Mechanism of action of the 12,13-epoxytrichothecene, anguidine, an inhibitor of protein synthesis.
Anguidine, muconomycin A, T-2 toxin, crotocin and trichodermin, a group of 12,13-epoxytrichothecenes, inhibit protein synthesis in HeLa cells and in rabbit reticulocyte lysates. These five mycotoxins can be divided into two groups on the basis of the reversibility of their effects in HeLa cells, and kinetics of inhibition and effects on polyribosome structure in rabbit reticulocyte lysates. Anguidine, muconomycin A and T-2 toxin are irreversible inhibitors of protein synthesis; crotocin and trichodermin are reversible inhibitors of protein synthesis. After addition of low concentrations (1 muM) of anguidine, muconomycin A or T-2 toxin to rabbit reticulocyte lysates, polyribosomes are broken down to monosomes. At higher concentrations, 1 mM, these drugs begin to freeze the polyribosomes. Crotocin and trichodermin freeze the polyribosomes at a concentration of 10 muM. We conclude that anguidine, muconomycin A and T-2 toxin act primarily as inhibitors of initiation of protein synthesis, whereas crotocin and trichodermin inhibit the process of chain elongation.
Cell-Free System↗
Prevention, by ribosome-bound nascent polyphenylalanine chains, of the functional interaction of t-2 toxin with its receptor site.
1. The inhibitory effects of T-2 toxin and trichodermin on poly(U)-directed polyphenylalanine synthesis were studied by using cell-free systems from reticulocytes. Conditions for amino acid incorporation were carefully chosen in an attempt to ensure that the large majority of poly(U) chains bound only one ribosome engaged in protein synthesis and that all such ribosomes carried nascent polyphenylalanine chains containing approximately the same number of residues. 2. Cell-free systems were allowd to synthesize polyphenylalanine, and T-2 toxin and trichodermin were added to the incorporation mixtures at various times. Irrespective of the time of addition, trichodermin (50 mug/ml) inhibited polyphenylalanine synthesis by approx. 70%. In contrast, although T-2 toxin (40 mug/ml), when added at early incubation times, could inhibit polyphenylalanine synthesis with a maximum of 50%, the drug had no effect on the system when added after a critical time-period. 3. It is concluded that although both T-2 toxin and trichodermin can inhibit peptide-bond formation on ribosomes at the level of the peptidyl transferase catalytic centre the presence, on ribosomes, of nascent polyphenylalanine chains above a certain critical chain length excludes T-2 toxin from functional interaction with its receptor site.
Animals↗
Ribosomal protein L3 is involved in replication or maintenance of the killer double-stranded RNA genome of Saccharomyces cerevisiae.
Ability to secrete the K1 (or K2) toxin protein and immunity to that toxin [the K1 (or K2) killer trait] are determined by a double-stranded (ds) RNA, called M1 (or M2), whose replication and maintenance depend on at least one of the larger (L) ds RNAs and 29 chromosomal genes, called MAK genes (maintenance of killer). The location of the MAK8 gene near TCM1 (trichodermin resistance) on the yeast map suggested the possible identity of these two genes. Of six independently isolated tcm1 mutants, five were clearly mak-, and the sixth was weakly mak-. In each case, the mak- phenotype and the trichodermin-resistant phenotypes cosegregated in meiosis and showed the expected tight linkage to pet17. The mak- mutations in the trichodermin-resistant strains did not complement mak8-1, indicating that MAK8 and TCM1 are the same gene. The mak8-1 mutation does not make strains resistant to trichodermin, and one tcm1 mutation is only slightly mak-. Whereas tcm1 mutants lose M1 or M2 ds RNA, they do not lose L ds RNA. Because TCM1 codes for ribosomal protein L3 [Fried, H. M. & Warner, J. R. (1981) Proc. Natl. Acad. Sci, USA 78, 238--242], we conclude that ribosomal protein L3 is involved in the replication and maintenance of M ds RNA. Mutations in cyh2 or cry1, producing resistance to cycloheximide and crytopleurine due to mutant ribosomal proteins, do not produce a mak- phenotype. In analogy with bacterial ribosome assembly mutants, yeast low-temperature-sensitive (lts) mutants may have defective ribosomes. We thus examined mutants for an effect on the killer system. An lts5 mutant, unable to grow at 5 degrees C, also has a mak- phenotype (at 30 degrees C) that cosegregates in meiosis with the lts- phenotype. Mutations in seven other lts genes do not result in the mak- phenotype.
Chromosome Mapping↗
Mechanism of inhibition of eukaryotic protein synthesis by trichothecene fungal toxins.
The 12,13-epoxytrichothecenes, a group of sesquiterpenoid fungal antibiotics, inhibit protein synthesis in eukaryotic cells but do not share a common mode of action. Trichodermin stabilizes polyribosomes, prevents their disaggregation by puromycin, and also prevents the release of nascent peptides from ribosomes by puromycin. Nivalenol, T-2 toxin, and verrucarin A cause rapid and almost quantitative breakdown of polyribosomes in H-HeLa cells, a process which is inhibited by anisomycin, cycloheximide, or trichodermin. Similar effects of trichodermin, nivalenol, and verrucarin A are also observed in yeast spheroplasts. We conclude that nivalenol, T-2 toxin, and verrucarin A are potent and highly selective inhibitors of polypeptide chain initiation in eukaryotes, whereas trichodermin inhibits chain elongation and (or) termination. We have compared the structural formulae of various trichothecenes and suggest that the presence of substituents on carbon-15 of the common trichothecene ring may be important in determining the precise modes of action of this group of compounds.
Acremonium↗
Synergistic interaction in simultaneous exposure to Streptomyces californicus and Stachybotrys chartarum.
The microbial exposure associated with health complaints in moldy houses consists of a heterogeneous group of components, including both living and dead bacteria, fungi, and their metabolites and active compounds. However, little is known about the interactions between different microbes and their metabolites, although the cytotoxicity and inflammatory potential of certain individual microbes have been reported. In this study, we investigated the inflammatory responses of mouse RAW264.7 macrophages after exposure to six indoor air microbes (Aspergillus versicolor, Penicillium spinulosum, Stachybotrys chartarum, Bacillus cereus, Mycobacterium terrae, and Pseudomonas fluorescens) alone and together with the actinomycete Streptomyces californicus. The production of nitric oxide, levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), and cytotoxicity were measured. The coexposure to Sta. chartarum and Str. californicus caused a synergistic increase in the production of IL-6 but not other cytokines. In further experiments, the metabolites from Sta. chartarum or from closely related fungi (atranones B and E, satratoxin G, trichodermin, 7-alpha-hydroxytrichodermol, staplabin, and SMTP-7) and the known fungal toxins sterigmatocystin, citrinin, and ochratoxin A were each tested with Str. californicus. The testing revealed a synergistic response in TNF-alpha and IL-6 production after coexposure to Str. californicus with both trichodermin and 7-alpha-hydroxytrichodermol. Finally, the synergistic inflammatory response caused by Str. californicus and trichodermin together was studied by analyzing for the presence of nuclear factor-kappa-B (NF-kappa-B) in nuclear extracts of the exposed cells. The exposure to Str. californicus induced the binding of NF-kappa-B proteins to the NF-kappa-B consensus sequence as well as to the natural NF-kappa-B site of the IL-6 promoter. Adding trichodermin to the exposure did not increase the DNA binding.
Animals↗
Characterisation of ribosomes from drug resistant strains of Schizosaccharomyces pombe in a poly U directed cell free protein synthesising system.
Mutants of Schizosaccharomyces pombe were isolated as resistant either to trichodermin or to anisomycin. Growth tests showed that the majority of mutants isolated were cross resistant to both drugs and also to cycloheximide. A limited genetic analysis showed that mutants at least four loci, tri3, tri4, ani1 and ani2, had this phenotype as was also the case for mutants at three cycloheximide resistant loci, cyh2, cyh3 and cyh4 reported previously (Ibrahim and Coddington, 1976). Allelism tests showed that the tri3, ani2 and cyh4 strains were allelic. A mutant at another trichodermin resistant locus, tri5, was cross resistant to anisomycin but sensitive to cycloheximide. Ribosomes from wild type and selected strains were analysed in a poly U directed cell free protein synthesising system. Three strains, cyh1-C7, ani1-F1 and tri-N15 (probably a tri5 allele) possessed ribosomes which were more resistant than the wild type to the drugs used in their isolation. In each case the site of the resistance was in the 60S subunit. Ribosomes from the cyh2, cyh3 and cyh4 strains were as sensitive to cycloheximide as those from wild type.
Anisomycin↗
Phenotypic resistance to amphotericin B in Candida albicans: relationship to glucan metabolism.
The phenotypic resistance to amphotericin methyl ester (AME) of stationary phase cultures of Candida albicans was decreased by alkaline pH values and by treatment with 2-mercaptoethanol or glucanase preparations, and was increased by acid pH values, increased aeration, treatment with N-ethylmaleimide, or the presence of inhibitors of protein synthesis such as trichodermin. The effects of such treatments on endogenous glucanase activity and on the incorporation of glucose residues into the 'glucan fraction' of the organism were studied. The changes in the endogenous levels of lytic activities on laminarin [as a measure of the total (1 leads to 3)-beta-D-glucanase] and on p-nitrophenyl-beta-D-glucoside [reflecting the exo-(1 leads to 3)-beta-D-glucanase] were followed in C. albicans cells under a variety of conditions. Treatments which increased AME sensitivity stimulated both total and exo-(1 leads to 3)-beta-D-glucanase activities, while treatments which promoted resistance decreased the levels of both (1 leads to 3)-beta-D-glucanases. Changes in the 'glucan fraction' were followed by incubating suspensions of organisms in the presence of trace amounts of [U-14C]glucose. The rate of incorporation of radioactivity fell during the first 2-3 d of stationary phase culture and then rose to high values by 7-8 d; AME resistance increased throughout this period. The rate of incorporation was markedly stimulated by prior treatment of the organisms with 2-mercaptoethanol or glucanase and inhibited by trichodermin or treatment with N-ethylmaleimide. The addition in the concentration range 0.3-3 mM of the glucose analogues beta-D-allose, 3-O-methyl-D-glucose, 2-deoxy-D-glucose or 5-thio-D-glucose to cultures 24 h after inoculation prevented any further increase in AME resistance for the next 2-3 d and resulted in a decrease in the level of resistance established at the time of addition. Radioactivity from 14C- or 3H-labelled analogues added, 24 h after inoculation, to stationary phase cultures was incorporated into the 'glucan fraction' of the organisms. The incorporation of glucose residues into the 'glucan fraction' is controlled by the activity of glucanases in producing glucose acceptor sites. The results reported confirm that there is a correlation between glucan metabolism, glucanase activity and resistance to AME, in that any factor leading to increased glucanase action also results in decreased resistance and vice versa, while incorporation of certain glucose analogues into the 'glucan fraction' delays the further increase in resistance.
Amphotericin B↗
The effect of aeration and metabolic inhibitors on resistance to amphotericin in starved cultures of Candida albicans.
The development of resistance to amphotericin methyl ester, measured in terms of the amount of drug required to induce a standard rate of release of K+ from suspensions of washed organisms, has been followed in Candida albicans in starved cultures under controlled conditions of aeration, stirring and temperature. Resistance develops at a rate which increases with the rate of aeration, limited by the onset of damage due to turbulence. Resistance decreases rapidly if gassing with N2 is substituted for aeration, but sensitivity does not reach that of exponentially growing cells. Resumption of aeration is followed by a slow recovery of resistance. The addition of inhibitors of protein synthesis (trichodermin, verrucarin) or uncoupling agents (2,4-dinitrophenol, sodium azide) at the beginning of starvation results in an increased rate of development of resistance. Adding inhibitors at a later stage, when resistance has developed after 72 h aeration, does not affect the decrease in resistance produced by gassing with N2 but the presence of trichodermin or verrucarin delays the recovery of resistance o
Amphotericin B↗
[Cycloheximide-dependent mutants of the yeast Saccharomyces cerevisiae].
Selection of sup1 and sup2 mutants in the yeast Saccharomyces cerevisiae on cycloheximide containing media revealed classes of mutants that either are completely unable to grow on YAPD without cycloheximide or need this drug under high temperature incubation (30 or 36 degrees C). Some of these mutants also exhibit the growth dependence on another antibiotic--trichodermin, and, at the same time, the osmotic dependence. A hypothesis claiming that sup1 and sup2 mutations cause conformational lability fo yeast cytoplasmic ribosomes has been put forward. It is also proposed that binding of cycloheximide and trichodermin to the mutant ribosomes cause their conformational shift, which compensates the functional defects.
Culture Media↗
Trichothecene production by Trichoderma brevicompactum.
Trichoderma brevicompactum, T. viride, T. harzianum, T. atroviride, T. longibrachiatum, T. erinaceum, T. citrinoviride, and Hypocrea lutea were screened for production of trichothecenes after growth on one or several solid and liquid media. Trichothecenes were detected by liquid chromatography combined with online UV/vis spectroscopy and electrospray high-resolution mass spectrometry. T. brevicompactum produced trichodermin and/or harzianum A on all media investigated, with liquid media yielding the largest amounts. Detection of octa-2Z,4E,6E-trienedioic acid in the harzianum-A-producing strains indicated that harzianum A was synthesized directly by esterification of trichodermol with octa-2Z,4E,6E-trienedioic acid. Both the T. viride strain from which trichodermin was originally isolated and the T. harzianum strain from which harzianum A was originally isolated were shown to belong to T. brevicompactum based on four independent criteria: metabolite profiles, micromorphology, macromorphology on yeast extract sucrose agar and potato dextrose agar, and DNA sequences of the ITS1/ITS2 regions of the nuclear ribosomal DNA.
Chromatography, Liquid↗
Control of transfer RNA synthesis in the presence of inhibitors of protein synthesis.
The rate of synthesis of transfer RNA in suspension cultures of Chang's liver cells, has been examined in the presence of various inhibitors of protein synthesis with different modes of action. Inhibitors of polypeptide chain elongation such as cycloheximide and emetine stimulated the rate of synthesis of transfer RNA at concentrations that inhibited protein synthesis by 60-90%. Trichodermin, an inhibitor of the elongation and termination steps in protein synthesis, had as effect similar to that of cycloheximide and emetine. On the other hand verrucarin, an inhibitor of initiation, and puromycin, an analogue of the aminoacyl terminus of tRNA, had little effect of the synthesis of transfer RNA at low concentrations. At high concentrations these compounds inhibited transfer RNA synthesis. Inhibitors of protein synthesis can be divided in two groups based on their effect on the polysom pattern. The first group increased the number of large polysomes, while verrucarin and puromycin lead to a break down of large polysomes and to an accumulation of monosomes and small polysomes. Thus, there appears to be a correlation between the effect of these inhibitors of protein synthesis on the aggregational state of the polysomes and their effect on the rate of transfer RNA synthesis.
Anti-Bacterial Agents↗
Assessment of potential therapies for acute T-2 toxicosis in the rat.
The efficacy of a variety of approaches for the treatment of animals with acute T-2 toxicosis was assessed utilizing young female rats. A single large dose of the water soluble salt of methylprednisolone significantly prolonged survival times in T-2 toxin treated animals. The use of diltiazem hydrochloride, dazemgrel, N-acetylcysteine, dimethyl sulfoxide, adenosine triphosphate (ATP), ATP combined with magnesium chloride, ascorbic acid, and aprotinin did not prolong survival times at the dosages administered. Trichodermin, a trichothecene similar in structure and biochemical activity to T-2 toxin but much less acutely toxic, had a detrimental effect on survival times whether given 1 hr prior to or after T-2 toxin.
Adenosine Triphosphate↗
Mutants of CHO cells resistant to the protein synthesis inhibitors, cryptopleurine and tylocrebrine: genetic and biochemical evidence for common site of action of emetine, cryptopleurine, tylocrebine, and tubulosine.
Stable mutants resistant to the protein synthesis inhibitors cryptopleurine and tylocrebine can be isolated in Chinese hamster ovary (CHO) cells, in a single step. The frequency of occurrence of cryptopleurine (CryR) and tylocrebrine (TylR) resistant mutants in normal and mutagenized cell populations is similar to that observed for emetine resistant (EmtR) mutants. The CryR, TylR, and EmtR mutants exhibit strikingly similar cross-resistance to the three drugs used for selection, to tubulosine and also to two emetine derivatives cephaeline and dehydroemetine, based on assays of in vivo cytotoxicity and on assays of protein synthesis in cell-free extracts. The identity of cross-resistance patterns of the CryR, TylR, and EmtR mutants indicates that the resistance to all these compounds results from the same primary lesion, which in the case of EmtR cells has been shown to affect the 40S ribosomal subunit. This conclusion is strongly supported by the failure of EmtR, TylR, and CryR mutants to complement each other in somatic cell hybrids. Based on these results it is suggested that the above group of compounds possesses common structural determinants which are responsible for their activity. The above mutants, however, do not show any cross-resistance to other inhibitors of protein synthesis such as cycloheximide, trichodermin, anisomycin, pactamycin, and sparsomycin, either in vivo or in vitro, indicating that the site of action of these inhibitors is different from that of the emetine-like compounds.
Alkaloids↗