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Trichodermin (TCD), a trichothecene first isolated from marine Trichoderma viride, is an inhibitor of eukaryotic protein synthesis. However, the potential effects of TCD on human oral squamous cell carcinoma (OSCC) cells and the underlying molecular mechanisms remain unknown. In this study, the exposure of OSCC cells (Ca922 and HSC-3 cells) to TCD suppressed cell proliferation assessed using MTT assays and colony formation assays. TCD inhibited the migration and invasion of OSCC cells (Ca922 and HSC-3 cells) through the downregulation of matrix metalloproteinase 9. After treatment of OSCC cells with TCD, the G2/M phase was arrested, caspase-related apoptosis (cleaved caspase-3 and PARP expression) was induced, and the protein level of x-linked inhibitor of apoptosis was reduced. Meanwhile, the TCD-induced cell death was reversed by the pan-caspase inhibitor Z-VAD-FMK. Furthermore, TCD diminished mitochondrial membrane potential, mitochondrial oxidative phosphorylation and glycolytic function in OSCC cells. In addition, TCD decreased the levels of histone deacetylase 2 (HDAC-2) and downstream signaling proteins, including phosphorylated STAT3 and NF-κB. Finally, TCD significantly suppressed tumor growth in a zebrafish OSCC xenotransplantation model. Overall, this evidence demonstrates that TCD is a novel promising strategy for the treatment of OSCCs., Competing Interests: Conflict of interest statement The authors declare that they have no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Ovarian cancer is a fatal gynecological cancer because of a lack of early diagnosis, which often relapses as chemoresistant. Trichodermin, a trichothecene first isolated from Trichoderma viride , is an inhibitor of eukaryotic protein synthesis. However, whether trichodermin is able to suppress ovarian cancer or not was unclear. In this study, trichodermin (0.5 µM or greater) significantly decreased the proliferation of two ovarian cancer cell lines A2780/CP70 and OVCAR-3. Normal ovarian IOSE 346 cells were much less susceptible to trichodermin than the cancer cell lines. Trichodermin predominantly inhibited ovarian cancer cells by inducing G0/G1 cell cycle arrest rather than apoptosis. Trichodermin decreased the expression of cyclin D1, CDK4, CDK2, retinoblastoma protein, Cdc25A, and c-Myc but showed little effect on the expression of p21 Waf1/Cip1 , p27 Kip1 , or p16 Ink4a . c-Myc was a key target of trichodermin. Trichodermin regulated the expression of Cdc25A and its downstream proteins via c-Myc. Overexpression of c-Myc attenuated trichodermin's anti-ovarian cancer activity. In addition, trichodermin decelerated tumor growth in BALB/c nude mice, proving its effectiveness in vivo. These findings suggested that trichodermin has the potential to contribute to the treatment of ovarian cancer.

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum , the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds 9 , 13 , and 15 , which presented average IC 50 values of 2 μg/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes).

The silkworm infection assay is a useful method for directly evaluating the in vivo therapeutic effects of drug candidates. In the present study, 3 known trichothecenes, trichodermin, epiisororidin E, and verrucarin A, were evaluated as antifungal agents in the silkworm-Candida albicans assay. Trichodermin and epiisororidin E yielded effective therapeutic effects, while verrucarin A exhibited no efficacy in this assay system. These results strongly suggest that trichodermin and epiisororidin E are the lead compounds for developing a new antifungal agent.

In an attempt to discover more potential antifungal agents, in this study, 21 novel trichodermin derivatives containing conjugated oxime ester (5a-5u) were designed and synthesized and were screened for in vitro antifungal activity. The bioassay tests showed that some of them exhibited good inhibitory activity against the tested pathogenic fungi. Compound 5a exhibited better activity against Pyricularia oryzae and Sclerotonia sclerotiorum than trichodermin, and compound 5j showed particular activity against P.oryzae and Botrytis cinerea. The quantitative structure-activity relationship (QSAR) indicated that log P and hardness were two critical parameters for the biological activities. The result suggested that these would be potential lead compounds for the development of fungicides with further structure modification.

To discover more potential antifungal agents, 17 novel trichodermin derivatives were designed and synthesized by modification of 3 and 4a. The structures of all the synthesized compounds were confirmed by (1)H NMR, ESI-MS and HRMS. Their antifungal activities against Ustilaginoidea oryzae and Pyricularia oryzae were evaluated. Most of the target compounds showed potent inhibitory activity, in which 4g showed superior inhibitory effects than 4a and commercial fungicide prochloraz. Furthermore, 4h demonstrated comparable inhibitory activity to 4a. Moreover, 4i and 4l exhibited excellent inhibitory activity for Pyricularia oryzae. Additionally, compound 9 was found to be more active against all tested fungal strains than 3, with EC50 values of 0.47 and 3.71 mg L(-1), respectively., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8S- ITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4β-acetoxy-12,13- epoxy-Δ(9)-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 μg mL(-1). Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 μg mL(-1). However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 μg mL(-1)). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin.

Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress., (Copyright © 2013. Published by Elsevier Inc.)

Twenty new trichodermin derivatives, 2a-5, containing alkoxy, acyloxy, and Br groups in 4-, 8-, 9-, 10- and 16-positions were synthesized and characterized. The antifungal activities of the new compounds against rice false smut (Ustilaginoidea virens), rice sheath blight (Rhizoctonia solani), and rice blast (Magnaporthe grisea) were evaluated. The results of bioassays indicated that the antifungal activities were particularly susceptible to changes at 4-, 8-, and 16-positions, but low to changes at 9- and 10-positions. Most of these target compounds exhibited good antifungal activities at the concentration of 50 mg l(-1) . Compound 4 (9-formyltrichodermin; EC50 0.80 mg l(-1) ) with an CHO group at 9-position displayed nearly the same level of antifungal activity against Ustilaginoidea virens as the commercial fungicide prochloraz (EC50 0.82 mg l(-1) ), while compound 3f ((8R)-8-{[(E)-3-phenylprop-2-enoyl]oxy}trichodermin; EC50 3.58 and 0.74 mg l(-1) ) with a cinnamyloxy group at C(8) exhibited much higher antifungal activities against Rhizoctonia solani and Magnaporthe grisea than the commercial fungicides prochloraz (EC50 0.96 mg l(-1) ) and propiconazole (EC50 5.92 mg l(-1) ), respectively. These data reveal that compounds 3f and 4 possess high antifungal activities and may serve as lead compounds for the development of fungicides in the future., (Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.)

Through bioassay-guided fractionation, the EtOAc extract of a culture broth of the endophytic fungus Phoma species ZJWCF006 in Arisaema erubescens afforded a new α-tetralone derivative, (3S)-3,6,7-trihydroxy-α-tetralone (1), together with cercosporamide (2), β-sitosterol (3), and trichodermin (4). The structures of compounds were established on the basis of spectroscopic analyses. Compounds 1, 2, and 3 were obtained from Phoma species for the first time. Additionally, the compounds were subjected to bioactivity assays, including antimicrobial activity, against four plant pathogenic fungi (Fusarium oxysporium, Rhizoctonia solani, Colletotrichum gloeosporioides, and Magnaporthe oryzae) and two plant pathogenic bacteria (Xanthomonas campestris and Xanthomonas oryzae), as well as in vitro antitumor activities against HT-29, SMMC-772, MCF-7, HL-60, MGC80-3, and P388 cell lines. Compound 1 showed growth inhibition against F. oxysporium and R. solani with EC₅₀ values of 413.22 and 48.5 μg/mL, respectively. Additionally, compound 1 showed no cytotoxicity, whereas compound 2 exhibited cytotoxic activity against the six tumor cell lines tested, with IC₅₀ values of 9.3 ± 2.8, 27.87 ± 1.78, 48.79 ± 2.56, 37.57 ± 1.65, 27.83 ± 0.48, and 30.37 ± 0.28 μM, respectively. We conclude that endophytic Phoma are promising sources of natural bioactive and novel metabolites.

Mycological analysis throughout the vegetation period of potato (Solanum tuberosum) made it possible to study in detail the structure of micromycete community, to determine typical dominant (frequency, more than 60%), typical common (frequency, 30 to 60%), typical rare (frequency, 10 to 30%), and casual (frequency, less than 10%) species and to estimate changes in the microorganism community caused by plant protection preparations with different mechanisms of action. It was shown that, as a result of occurrence of resistant forms, synthetic preparations against fungal pathogens of potato (such as TMTD, Ridomil gold MC, and Cupricol) were only slightly more effective than biological preparations (Trichodermin and AgroChit), with the former considerably changing the natural saprophytic mycological community. An increase in the soil pool of Trichoderma harzianum as a result of application of a biological preparation based on this antagonistic fungus correlated with its effectiveness against the soil pathogen Fusarium sp., which causes root rots. A chitosan-based elicitor preparation more effectively suppressed the development of early (Alternaria sp. and Macrosporium sp.) and late (Phytophthora sp.) blights of leaves and had a weaker effect on the soil microflora.

Bacillus intermedius RNAase (with specific activity of 1,000,000 units per one mg of protein) at concentration of 1 x 10(-3) mg/ml was shown to increase antagonistic and growth-stimulating properties of Trichoderma harzianum. An application of trichodermin which was treated with an enzyme enhanced cucumber crop capacity by 15-18% in industrial conditions.

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.

The role of eukaryotic 5'-untranslated messenger RNA leaders is not entirely clear, since they share little sequence similarity among each other. The importance of the leader in determining the efficiency of translation initiation was addressed here by examining the polyribosome distribution of several leader-deletion alleles of the yeast tcm1 gene (coding for ribosomal protein L3). Shortening of this 22-nucleotide leader, or complete removal of it (the first nucleotide of the mRNA becoming the A of the translation initiation codon AUG) permitted translation, albeit reduced. Further deletion of as few as the first two nucleotides of the initiation codon leads to a substantial reduction in ribosome loading, which is compatible with inefficient initiation at the next downstream, out-of-frame, AUG triplet. A second measure of translation initiation was obtained by assaying qualitatively for the production of biologically active L3 protein using growth-resistance to trichodermin. This experiment indicates that ribosomes can recognize the correct initiation codon even in the complete absence of a leader. We conclude that the 5'-untranslated leader of the yeast tcm1 gene is not essential for accurate translation initiation, but enhances its efficiency.

Thirteen strains of viridans group streptococci and two strains of other streptococci were tested for coaggregation with Candida albicans. Streptococcus sanguis strains generally exhibited low levels of adherence to 28 degrees C-grown exponential-phase yeast cells, but starvation of yeast cells for glucose at 37 degrees C (or at 28 degrees C) increased their coaggregating activity with these streptococci by at least tenfold. This was a property common to four C. albicans strains tested, two of which were able to form mycelia (6406 and MEN) and two of which were not (MM2002 and CA2). The expression of the coaggregation adhesin during yeast cell starvation was inhibited by addition of trichodermin or amphotericin B. The strains of S. sanguis, Streptococcus gordonii, and Streptococcus oralis tested for coaggregating activity encompassed a diverse range of physiological and morphological types, yet all exhibited saturable coaggregation with starved C. albicans cells. There was no correlation of cell surface hydrophobicity, of either yeast or streptococcal cells, with their abilities to coaggregate. Strains of Streptococcus anginosus also coaggregated with starved yeast cells; Streptococcus salivarius and Streptococcus pyogenes coaggregated to a lesser degree with C. albicans, and the coaggregation with S. pyogenes was not promoted by yeast cell starvation; Streptococcus mutans and Enterococcus faecalis did not coaggregate with yeast. The coaggregation reactions of S. sanguis and S. gordonii with C. albicans were inhibited by EDTA and by heat or protease treatment of the yeast cells and were not reversible by the addition of lactose or other simple sugars. These observations extend the range of intergeneric coaggregations that are known to occur between oral microbes and suggest that coaggregations of C. albicans with viridans group streptococci may be important for colonization of oral surfaces by the yeast.

1. A yeast mutant (CLP-8), resistant at the ribosome level to the trichothecene antibiotic trichodermin, differs from its parent in having an unusual distribution of native ribosomal subunits. Sucrose gradient analysis of cytoplasmic extracts from this mutant revealed a large excess of material sedimenting at 60 S with little or no material sedimenting at 40 S. 2. The excess 609-S material consists predominantly of functionally active 60-S ribosomal subunits, as indicated by both analysis of ribosomal RNA and studies in vitro using a poly(U)-directed protein-synthesizing system. 3. Using the poly(U) system it was found that high-salt-washed particles derived from either the excess 60-S peak or 80-S ribosomes of CLP-8 exhibited very similar levels of resistance to the antibiotic fusarenon-X, a drug closely related chemically to trichodermin. The same level of resistance to fusarenon-X was also shown by high-salt-washed 60-S ribosomal particles obtained from a further trichodermin-resistant yeast strain (TR-1), although this strain has a normal distribution of native ribosomal subunits. In addition, both CLP-8 and TR-1 are equally resistant to inhibition of protein synthesis by trichothecene antibiotics, as assayed in vivo. 4. Genetic analysis of CLP-8 indicates that the trichodermin-resistant trait can be segregated from the lesion responsible for the inbalance of native ribosomal subunits. However, the latter defect is only expressed phenotypically in cells that retain the trichodermin-resistant character. 5. CLP-8 has a further defect in that both in vivo and in vitro it fails to generate native 40-S ribosomal subunits from 80-S particles. There may be a lesion in the protein factor normally required for this process.

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.

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.

Yeast cells sensitive to the eukaryotic protein synthesis inhibitor trichodermin have been transformed with autonomously replicating recombinant plasmids carrying DNA fragments of the genome of a trichodermin-resistant yeast strain. After selection for trichodermin-resistant cells, several transformants yielded a plasmid containing a 13.5-kilobase (kb) DNA fragment that encodes the trichodermin resistance gene, tcm1, and the gene for ribosomal protein L3, the largest of the yeast ribosomal proteins. Cells carrying this plasmid are resistant to trichodermin and to the related drug verrucarin A as well as to the unrelated drug anisomycin. This pattern of resistance is similar to that exhibited by strains carrying a chromosomal copy of tcm1. Moreover, polyribosomes prepared from transformed cells are resistant to trichodermin when tested in an in vitro protein synthesis assay. Subcloning of the 13.5-kb DNA fragment revealed that the gene for tcm1 and the gene for protein L3 are contained within a 3.2-kb segment. These results suggest that the gene for trichodermin resistance in yeast specifies ribosomal protein L3.

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.

Cycloheximide-resistant mutants of Schizosaccharomyces pombe were isolated either as spontaneous mutants or after mutagenic treatment with nitrous acid, UV and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Twenty-three spontaneous mutants and 64 induced mutants were analysed genetically. Crosses revealed that at least four loci, designated cyh1, cyh2, cyh3 and cyh4 are responsible for resistance. Alleles of cyh1 show good growth on either high (100 mug/ml) or low (40 mug/ml) concentrations of cycloheximide whereas alleles at the cyh2, cyh3 and cyh4 loci gorw well on 40 mug/ml but poorly on 100 mug/ml. Some alleles at the cyh2 and cyh3 loci are also temperature sensitive (ts), the ts phenotype being conferred by the same gene as the resistance. In diploids, cyh1 and cyh4 are re-essive to wild type whereas cyh2 and cyh3 are semi-dominant. There was no intragenic complementation between three cyh1 alleles. Cross-resistance to trichodermin and anisomycin was shown by cyh2, cyh3 and cyh4 but not cyh1. Most cyh1 alleles, of spontaneous and UV origin only, were cold sensitive (cs) at 14 degrees and some of these were also cycloheximide dependent at the same temperature. It is suggested that the cyh1 and cyh4 genes are involved in ribosome formation or function and the other loci probably affect the uptake of cycloheximide by the cells.

Arginase, the enzyme responsible for arginine degradation in Saccharomyces cerevisiae, is an inducible protein whose inhibition of ornithine carbamoyl-transferase has been studied extensively. Mutant strains defective in the normal regulation of arginase production have also been isolated. However, in spite of these studies, the macromolecular biosynthetic events involved in production of arginase remain obscure. We have, therefore, studied the requirements of arginase induction. We observed that: (i) 4 min elapsed between the addition of inducer (homoarginine) and the appearance of arginase activity at 30 degrees C; (ii) induction required ribonucleic acid synthesis and a functional rna1 gene product; and (iii) production of arginase-specific synthetic capacity occurred in the absence of protein synthesis but could be expressed only when protein synthesis was not inhibited. Termination of induction by inducer removal, addition of the ribonucleic acid synthesis inhibitor lomofungin, or resuspension of a culture of organisms containing temperature-sensitive rna1 gene products in a medium at 35 degrees C resulted in loss of ability for continued arginase synthesis with half-lives of 5.5, 3.8, and 4.5 min, respectively. These and other recently published data suggest that a variety of inducible or repressible proteins responding rapidly to the environment may be derived from labile synthetic capacities, whereas constitutively produced proteins needed continuously throughout the cell cycle may be derived from synthetic capacities that are significantly more stable.

A new h.p.l.c. cation-exchange method has been used to separate proteins from 60S ribosomal subunits prepared from strains of Saccharomyces cerevisiae sensitive or resistant to trichodermin. Ribosomal protein L3 was identified in column eluates by one-dimensional and two-dimensional gel electrophoresis and purified further by reverse-phase h.p.l.c. The protein was cleaved with CNBr and the products were analysed, again by reverse-phase h.p.l.c. A marked difference was observed in the peptide profiles between preparations from trichodermin-sensitive and trichodermin-resistant yeast strains. These results provide the first direct demonstration that, in yeast, mutationally induced resistance to trichodermin can alter the covalent structure of ribosomal protein L3. They convincingly demonstrate the potential of the experimental technique for the rapid and preparative separation of a selected yeast ribosomal protein and its subsequent characterization.

Centromeres are the structural elements of eukaryotic chromosomes that hold sister chromatids together and to which spindle tubules connect during cell division. Centromeres have been shown to suppress meiotic recombination in some systems. In this study yeast strains genetically marked within and flanking a centromere, were used to demonstrate that gene conversion (nonreciprocal recombination) tracts in mitosis can enter into and extend through the centromere.

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

Trichodermin, a eukaryotic-specific antibiotic, inhibits protein synthesis in Drosophila cells. We have synthesized a 14C-labeled bromoacetyl derivative of trichodermin that binds to Drosophila 80S ribosomes and once bound reacts covalently with ribosomal proteins. It does not react with rRNA. Three large-subunit proteins (L1, L3, and L24) and three small-subunit proteins (S3/S5, 2/3S, and S8) are labeled by [14C] (bromoacetyl)trichodermin. Reaction with each of these proteins can be competed by an excess of unmodified trichodermin, indicating that the labeling has occurred from the native binding site of the parent drug. One of the (bromoacetyl)trichodermin-labeled proteins (S8) is also labeled by photoactivated puromycin in the A site. A second protein (S3/S5) is found to be labeled by a P-site affinity reagent. The results suggest that the trichodermin binding site spans both the small and large subunits and portions of both the A and P sites. These data combined with previous studies on the A and P sites of Drosophila ribosomes have allowed us to construct a model of the protein locations in this important active site.

Addition of putrescine of spermidine prevents the increase in ornithine decarboxylase activity in cultures of 3T3 cells brought about by pituitary growth factors and results in a rapid, specific, and reversible reduction of enzyme activity in cultures previously stimulated by the growth factors. These effects are not due to polyamine toxicity and do not require other organic medium components. The amines apparently share a single carrier-mediated transport system in 3T3 cells. Methylglyoxal bis(guanylhydrazone), an inhibitor of spermidine synthesis from putrescine was found to also inhibit uptake of each amine. Studies with this drug indicate that each amine is effective without further metabolism. Since ornithine decarboxylase activity decays more rapidly in the presence of each polyamine after addition of camptothecin, the major locus of amine action appears to be in the cytoplasm. However, direct inhibition of the enzyme in vivo by assimilated amines appears to account for at most a small part of the reduction in activity, a conclusion supported by the inability to recover activity in vitro. Also, neither amine seems to act by accelerating enzyme inactivation. When amines are removed from the medium, the subsequent recovery of enzyme activity is totally prevented by trichodermin, an inhibitor of protein synthesis, but is only slightly reduced by camptothecin. It is suggested that both putrescine and spermidine reduce ornithine decarboxylase activity by selectively inhibiting translation.

Resistance to the protein synthesis inhibitor trichodermin in Saccharomyces cerevisiae has been studied. A single recessive nuclear gene was responsible for resistance. The resistance locus, tcm1 was found to be closely linked (1 centi-morgan) to the locus pet 17 on the right arm of chromosome XV. The mutation to trichodermin resistance conferred resistance to other 12,13-epoxytrichothecenes and to the structurally unrelated antibiotic anisomycin.

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.

1. The effect of trichodermin as an inhibitor of eukaryotic protein synthesis was studied in a reticulocyte cell-free system. 2. Trichodermin at a concentration of 25 mug/ml inhibits total protein synthesis instantaneously and stabilizes polyribosome profiles. Conversely, at a concentration of 0.25 mug/ml the drug inhibits total protein synthesis by only 70-75% and allows 30-35% breakdown of the polyribosomes in the system. These effects were compared with those produced by two other drugs (pactamycin and anisomycin) examined under conditions identical with those used for trichodermin.

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.

The dimorphic fungus Mucor racemosus exhibited a single-step, inducible resistance to cycloheximide, trichodermin, and amphotericin B. Cells adapted to inhibitory levels of the antibiotics after 12 to 40 h. The adaptation involved all the cells in the population and was not the result of the selection of resistant mutants. Adaptation to one drug provided cross resistance to other, dissimilar drugs. Resistance was lost within several generations of growth in the absence of the inhibitors.

In the petite positive yeast, Saccharomyces cerevisiae, cycloheximide selectively inhibits protein synthesis on cytoplasmic ribosomes, and, as a consequence, nuclear DNA synthesis. Mitochondrial DNA, however, is synthesized for 4-6 h after cessation of protein synthesis. In this paper we show that in contrast to Saccharomyces cerevisiae, synthesis of mitochondrial and nuclear DNA is tightly coordinated in the petite negative yeast Schizosaccharomyces pombe, since inhibition of cytoplasmic protein synthesis leads immediately to cessation of both nuclear and mitochondrial DNA synthesis.

Mucor racemosus exhibited inducible phenotypic resistance toward the protein synthesis inhibitor trichodermin. Induction of resistance was elicited by exposure to trichodermin or to cycloheximide. Both adapted and nonadapted cells took up [14C]trichodermin from the medium. Trichodermin was found to be rapidly deacetylated to trichodermol upon entering the cell. Adapted cells deacetylated the drug more rapidly than nonadapted cells both in vivo and in vitro. The trichodermol resulting from deacetylation appeared in the medium, but the growth of adapting cells began well before the total conversion of trichodermin to trichodermol. Based on these data and the observation that trichodermol was a poor inhibitor of Mucor, adaptation appears to result from deacylation of the active antibiotic.

During the yeast-to-hyphae transition of the dimorphic phycomycete Mucor racemosus, there was a 30- to 50-fold increase in the activity of ornithine decarboxylase. Increased enzyme activity preceded the emergence of germ tubes and reached a maximum before conversion was completed. Subsequently, enzyme levels rapidly declined, despite the continuation of mycelial growth. Both putrescine and spermidine blocked the enzyme activity response. Protein synthesis was required for the increase in enzyme activity during morphogenesis. A combination of actinomycin D and netropsin inhibited ribonucleic acid synthesis but failed to inhibit the increase in ornithine decarboxylase activity. There was a twofold increase in the enzyme half-life during morphogenesis with either trichodermin or verrucarin to inhibit protein synthesis.

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.

Yeast polysomes are very active for amino acid incorporation when supplemented with elongation factors and the different components required for elongation of the polypeptide chain. This polysomal system is suitable for the study of the individual streps of the elongation cycle and to test the effect of different inhibitors. Anisomycin, trichodermin, trichodermol, trichothecin, fusarenon X, sparsomycin and blasticidin S inhibit peptide bond formation on these polysomes, whereas diphtheria toxin, pederine, cycloheximide and cryptopleurine block translocation.

We have previously determined the amounts of time required to complete various macromolecular synthetic processes needed for induction of allophanate hydrolase in Saccharomyces cerevisiae. This information provided a means of testing, in vivo, an early hypothesis suggesting that cycloheximide inhibited the initiation as well as elongation steps of protein synthesis. Our data suggest that initiation of protein synthesis in yeast may be inhibited by low concentrations of cycloheximide which do not significantly affect polypeptide chain elongation.

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.

The effect of drugs such as puromycin and cycloheximide, which inhibit protein synthesis, on the accumulation of La Crosse virus S genome RNAs in vivo has been examined. We have found that if these drugs are added to the cultures before infection, minuscule amounts of S-mRNA can be detected late in infection. Genome replication, on the other hand, cannot be detected at any time. When these drugs are added later in infection when RNA synthesis is well established, S-mRNA accumulation decreases in a dose-dependent manner proportional to the effect of these drugs on protein synthesis. This decrease cannot be accounted for by increased turnover of the mRNA in the presence of the drug. S genome replication, curiously, was found to be hypersensitive to the effects of these drugs. Our results confirm those of Abraham and Pattnaik (1983) that ongoing protein synthesis is required for the accumulation of complete bunyavirus S-mRNA.

Mutants resistant to the protein synthesis inhibitor trichodermin have been selected in Chinese hamster ovary (CHO) cells. The mutants vary in their stability from those which rapidly lose their resistance to others which are relatively stable after prolonged growth in nonselective medium. Protein synthesis in extracts from the latter class of mutants (Trir) is resistant to the inhibitory action of trichodermin as compared to similar extracts from wild-type cells. After dissociation into subunits, the ability of the 60S ribosomal subunits from Trir cells to function in a protein-synthesizing system is greatly diminished. This subunit also shows reduced binding of [acetyl-14C]TRICHODERMIN. The lesion in Trir mutants therefore seems to have affected this ribosomal subunit. Trir X Tris hybrids are sensitive to trichodermin indicating that the Trir mutation behaves recessively to Tris in hybrids. The Emtr and Trir markers segregate independently from hybrid cells showing that the Trir mutation is probably not linked to the Emtr locus, which as we have shown earlier affects the 40S ribosomal subunit.

Inhibition of protein synthesis by trichodermol, diacetoxyscirpenol and verrucarin A in cells and spheroplasts of Saccharomyces cerevisiae was investigated. Inhibition was reversible for trichodermol and diacetoxyscirpenol, both drugs being removed from their target site(s) by washing, but was reversible for verrucarin A. These results are interpreted in relation to variations in chemical structure between these trichothecenes.