Unusual antimicrotubule activity of the antifungal agent spongistatin 1.
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Spongistatin 1, a macrocyclic lactone from the marine sponge Hyrtios erecta, has broad-spectrum antifungal activity. Since this compound is a potent antimicrotubule agent in mammalian cells, we examined its effects on the filamentous fungus Aspergillus nidulans to determine if its antifungal effects are due to antimicrotubule activity. At 25 microg/ml (twice the MIC), spongistatin 1 caused a greater-than-twofold elevation of the chromosome and spindle mitotic indices. Immunofluorescence microscopy revealed that mitotic spindles were smaller and shorter than in control germlings. However, late-anaphase and telophase nuclei were seen occasionally, and this suggests that the spindles are capable of segregating chromosomes. Spongistatin 1 had more dramatic effects on cytoplasmic microtubules. At 30 min after initiation of treatment, 83% of germlings contained fragmented microtubules and after 2 h of treatment, microtubules had disappeared completely from 82% of germlings. In contrast, microtubules disappeared rapidly and completely from germlings treated with benomyl. We conclude that spongistatin 1 has antimicrotubule activity in A. nidulans and that its mechanism of action may involve a novel microtubule-severing activity. (+info)
The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins.
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The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to the spindle. When spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability. We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle. Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another spindle checkpoint component, Mad1p. This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins. In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE. Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p. (+info)
Rhodamine 6G efflux for the detection of CDR1-overexpressing azole-resistant Candida albicans strains.
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We investigated the drug efflux mechanism in azole-resistant strains of Candida albicans using rhodamine 6G (R6G). No significant differences in R6G uptake were observed between azole-sensitive B2630 (9.02 +/- 0.02 nmol/10(8) cells) and azole-resistant B67081 (8.86 +/- 0.03 nmol/10(8) cells) strains incubated in glucose-free phosphate buffered saline. A significantly higher R6G efflux (2.0 +/- 0.21 nmol/10(8) cells) was noted in the azole-resistant strain (B67081) when glucose was added, compared with that in the sensitive strain B2630 (0.23 < or = 0.14 nmol/10(8) cells). A fluconazole-resistant strain C40 that expressed the benomyl resistance gene (CaMDR) also showed a low R6G efflux (0.16 +/- 0.06 nmol/10(8) cells) as did the sensitive strains. Accumulation of R6G in growing C. albicans cells was inversely correlated with the level of CDR1 mRNA expression. Our data also suggest that measurement of intracellular accumulation of R6G is a useful method for identification of azole-resistant strains due to CDR1-expressed drug efflux pump. (+info)
Translational regulation of ribonucleotide reductase by eukaryotic initiation factor 4E links protein synthesis to the control of DNA replication.
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Ribonucleotide reductase synthesizes dNDPs, a specific and limiting step in DNA synthesis, and can participate in neoplastic transformation when overexpressed. The small subunit (ribonucleotide reductase 2 (RNR2)) was cloned as a major product in a subtraction library from eukaryotic initiation factor 4E (eIF4E)-transformed cells (Chinese hamster ovary-4E (CHO-4E)). CHO-4E cells have 20-40-fold elevated RNR2 protein, reflecting an increased distribution of RNR2 mRNA to the heavy polysomes. CHO-4E cells display an altered cell cycle with shortened S phase, similar to cells selected for RNR2 overexpression with hydroxyurea. The function of ribonucleotide reductase as a checkpoint component of S progression was studied in yeast in which elevated eIF4E rescued S-arrested rnr2-68(ts) cells, by increasing recruitment of its mRNA to polysomes. Crosses between rnr2-68(ts) and mutant eIF4E (cdc33-1(ts)) engendered conditional synthetic lethality, with extreme sensitivity to hydroxyurea and the microtubule depolymerizing agent, benomyl. The double mutant (cdc33-1 rnr2-68) also identified a unique terminal phenotype, arrested with small bud and a randomly distributed single nucleus, which is distinct from those of both parental single mutants. This phenotype defines eIF4E and RNR2 as determinants in an important cell cycle checkpoint, in early/mid-S phase. These results also provide a link between protein and DNA synthesis and provide an explanation for cell cycle alterations induced by elevated eIF4E. (+info)
Structure-function relationships in yeast tubulins.
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A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major alpha-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitive tub1 alleles were synthetically lethal in combination with tub3Delta, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of alpha-tubulin. The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast alpha-beta-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of beta-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of alpha-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the alpha-beta interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201 allele. (+info)
Hybridization and breeding of the benomyl resistant mutant, Trichoderma harziantum antagonized to phytopathogenic fungi by protoplast fusion.
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A diploid strain obtained from heterokaryons of Trichoderma harzianum by protoplast fusion grew on minimal medium containing 100ppm benomyl. This strain inhibited the growth of the phytopathogenic fungus Fusarium oxysporum f. sp. raphani on paired cultures and also protected against radish yellows and a drop in germination induced by F. oxysporum f. sp. raphani. (+info)
Effect of enzyme inhibitors on protein quaternary structure determined by on-line size exclusion chromatography-microelectrospray ionization mass spectrometry.
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Aldehyde dehydrogenases (ALDH) are a family of enzymes primarily involved in the oxidation of various aldehydes. Most ALDH enzymes derived from mammalian sources have been shown to exist as homotetramers, consisting of four identical subunits of approximately 54 kDa. The presence of the homotetramer appears to be necessary for enzyme activity. In this study, recombinant rat liver mitochondrial ALDH (rmALDH) was inhibited in vitro with four different inhibitors, namely, disulfiram (MW, 296.5), prunetin (MW, 284.3), benomyl (MW, 290.3), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (MW, 351.8). Subsequently, inhibited rmALDH was analyzed by a novel approach of on-line size exclusion chromatography-microelectrospray ionization-mass spectrometry (SEC-muESI-MS) to examine the noncovalent quaternary structural stability of the inhibited enzyme. Analysis of native rmALDH by SEC-muESI-MS revealed predominantly the homotetramer (Mr = approximately 217,457 Da, +/- 0.01%) with some in-source, skimmer-induced dissociation to afford monomer (Mr = approximately 54,360 Da, +/- 0.01%). Both disulfiram and prunetin inhibited rmALDH by >70% and >90%, respectively, but did not disrupt the quaternary structure of rmALDH. Furthermore, there was no detectable change within experimental error (+/- 0.01%) of the disulfiram or the prunetin homotetramers (Mr = approximately 217,448 Da and Mr = approximately 217,446 Da). This may possibly indicate that inhibition occurred via formation of intramolecular disulfide bond at the enzyme active site, or weak affinity noncovalent binding. In contrast, benomyl-inhibited rmALDH homotetramer (>90% inhibition) exhibited a Mr = approximately 217,650 Da (+/- 0.01%) corresponding to two butylcarbamoyl adducts on two of the four enzyme subunits. The skimmer-induced monomer afforded a mixture of unmodified rmALDH (Mr = approximately 54,365 Da, +/- 0.01%) and butylcarbamoylated enzyme (Mr = approximately 54,459 Da, +/- 0.01%). Finally, TPCK (>90% inhibition) modified all four subunits of rmALDH to give Mr = approximately 218,646 Da (+/- 0.01%). In all four cases while significant enzyme inhibition occurred, no destabilization of the quaternary complex was detected. (+info)
Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii.
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We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network. (+info)