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Journal of Cell Science May 2009In general, coronins play an important role in actin-based processes, and are expressed in a variety of eukaryotic cells, including Leishmania. Here, we show that...
In general, coronins play an important role in actin-based processes, and are expressed in a variety of eukaryotic cells, including Leishmania. Here, we show that Leishmania coronin preferentially distributes to the distal tip during cytokinesis, and interacts with microtubules through a microtubule-based motor, kinesin K39. We further show that reduction in coronin levels by 40-50% in heterozygous coronin mutants results in generation of bipolar cells (25-30%), specifically in the log phase, owing to unregulated growth of the corset microtubules. Further analysis of bipolar cells revealed that the main cause of generation of bipolar cell morphology is the intrusion of the persistently growing corset microtubules into the other daughter cell corset from the opposite direction. This defect in cytokinesis, however, disappears upon episomal gene complementation. Additionally, our attempts to prepare homozygous mutants were unsuccessful, as only the aneuploid cells survive the selection process. These results indicate that coronin regulates microtubule remodeling during Leishmania cytokinesis and is essentially required for survival of these parasites in culture.
Topics: 4-Butyrolactone; Aneuploidy; Animals; Antigens, Protozoan; Cytokinesis; Leishmania donovani; Macrolides; Microtubules; Mutation; Protozoan Proteins; Time Factors; Tubulin Modulators
PubMed: 19401334
DOI: 10.1242/jcs.044651 -
Angewandte Chemie (International Ed. in... 2009A new "branch" for polyketide synthases was discovered in the biosynthesis of the antimitotic rhizoxin complex in the endofungal bacterium Burkholderia rhizoxinica....
A new "branch" for polyketide synthases was discovered in the biosynthesis of the antimitotic rhizoxin complex in the endofungal bacterium Burkholderia rhizoxinica. Genetic engineering and the structural elucidation of pathway intermediates revealed that a complex polyketide chain is branched at the beta position by an unprecedented conjugate addition of an acetyl building block to an acryloyl precursor (see scheme).
Topics: Antineoplastic Agents; Biocatalysis; Burkholderia; Gene Knockout Techniques; Macrolides; Multigene Family; Polyketide Synthases; Protein Structure, Tertiary
PubMed: 19266509
DOI: 10.1002/anie.200900277 -
Journal of Natural Products Mar 2009This review provides an overview of the discovery, structures, and biological activities of anticancer natural products that act by inhibiting or promoting the assembly... (Review)
Review
This review provides an overview of the discovery, structures, and biological activities of anticancer natural products that act by inhibiting or promoting the assembly of tubulin to microtubules. The emphasis is on providing recent information on those compounds in clinical use or in advanced clinical trials. The vinca alkaloids, the combretastatins, NPI-2358, the halichondrin B analogue eribulin, dolastatin 10, noscapine, hemiasterlin, and rhizoxin are discussed as tubulin polymerization inhibitors, while the taxanes and the epothilones are the major classes of tubulin polymerization promoters presented, with brief treatments of discodermolide, eleutherobin, and laulimalide. The challenges and future directions of tubulin-interactive natural products-based drug discovery programs are also discussed briefly.
Topics: Antineoplastic Agents; Biological Products; Diketopiperazines; Imidazoles; Microtubules; Molecular Structure; Piperazines; Tubulin; Vinca Alkaloids
PubMed: 19125622
DOI: 10.1021/np800568j -
The Journal of Infectious Diseases Oct 2008Environmental isolates of the fungus Rhizopus have been shown to harbor a bacterial endosymbiont (Burkholderia) that produces rhixozin, a plant mycotoxin. We sought to...
Environmental isolates of the fungus Rhizopus have been shown to harbor a bacterial endosymbiont (Burkholderia) that produces rhixozin, a plant mycotoxin. We sought to define the role of rhizoxin production by endosymbionts in the pathogenesis of mucormycosis. Endosymbiotic bacteria were identified by polymerase chain reaction in 15 (54%) of 28 clinical isolates of Zygomycetes, with 33% of the bacterial strains showing 87% identity to Burkholderia 16S rDNA. The presence of rhizoxin in myclial extracts from fungi harboring bacteria was confirmed by high-performance liquid chromatography analysis. However, fungal strains with or without endosymbionts did not differ in their ability to cause endothelial cell injury in vitro, nor did antibiotic-mediated eradication of endosymbionts and rhizoxin production decrease the virulence of fungal strains in mice or flies. In summary, although bacterial endosymbiosis is widely detected in clinical isolates of Zygomycetes, including Rhizopus oryzae strains, we found no evidence that bacterial endosymbionts and rhizoxin contribute to the pathogenesis of mucormycosis in the models studied.
Topics: Animals; Burkholderia; Endothelial Cells; Humans; Macrolides; Male; Mice; Mice, Inbred BALB C; Mucor; Mucormycosis; Rhizopus; Symbiosis
PubMed: 18694335
DOI: 10.1086/591461 -
Mycological Research May 2008The fungus Rhizopus oligosporus (R. microsporus var. oligosporus) is traditionally used to make tempe, a fermented food based on soybeans. Interest in the fungus has... (Comparative Study)
Comparative Study
The fungus Rhizopus oligosporus (R. microsporus var. oligosporus) is traditionally used to make tempe, a fermented food based on soybeans. Interest in the fungus has steadily increased, as it can also ferment other substrates, produce enzymes, and treat waste material. R. oligosporus belongs to the R. microsporus group consisting of morphologically similar taxa, which are associated with food fermentation, pathogenesis, or unwanted metabolite production (rhizonins and rhizoxins). The ornamentation pattern, shape, and size of sporangiospores of 26 R. microsporus group strains and two R. oryzae strains were studied using low-temperature SEM (LT-SEM) and LM. This study has shown that: (1) LT-SEM generates images from well-conserved sporangiophores, sporangia, and spores. (2) Robust spore ornamentation patterns can be linked to all different taxa of the R. microsporus group, some previously incorrectly characterized as smooth. Ornamentation included valleys and ridges running in parallel, granular plateaus, or smooth polar areas. Distribution of ornamentation patterns was related to spore shape, which either was regular, ranging from globose to ellipsoidal, or irregular. Specific differences in spore shape, size, and ornamentation were observed between Rhizopus taxa, and sometimes between strains. (3) R. oligosporus has a defect in the spore formation process, which may be related to the domesticated nature of this taxon. It had a high proportion, 10-31%, of large and irregular spores, and was significantly differentiated from other, natural Rhizopus taxa as evaluated with partial least squares discriminant analysis. It is remarkable that the vehicle of distribution, the sporangiospore, is affected in the strains that are distributed by human activity. This provides information about the specificity and speed of changes that occur in fungal strains because of their use in (food) industry.
Topics: Microscopy; Microscopy, Electron, Scanning; Multivariate Analysis; Rhizopus; Spores, Fungal; Temperature
PubMed: 18400482
DOI: 10.1016/j.mycres.2007.11.006 -
Current Medicinal Chemistry 2008Despite the conventional and high-dose chemotherapy with hematopoietic stem cell transplantation, multiple myeloma eventually relapses, resulting in an incurable... (Review)
Review
Despite the conventional and high-dose chemotherapy with hematopoietic stem cell transplantation, multiple myeloma eventually relapses, resulting in an incurable hematological malignancy. Therefore, novel therapeutic approaches in clinical settings are desired. Recently, thalidomide was introduced for the treatment of myeloma, and many clinical trials have since confirmed its efficacy in patients with relapsed/refractory or newly diagnosed multiple myeloma. Multiple mechanisms have been proposed to explain thalidomide's anti-myeloma activity. However, the precise mechanism underlying this activity remains unclear, because thalidomide rapidly undergoes spontaneous, nonenzymatic, hydrolytic cleavage to numerous metabolites in vivo. To elucidate the exact anti-myeloma mechanism of thalidomide in vivo, we have performed structural development studies of thalidomide, and obtained various analogs with specific molecular properties. Among these derivatives, we found that a new thalidomide analog, 2-(2,6-diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33), has the most potent anti-myeloma effect with tubulin polymerization inhibiting activity. 5HPP-33 directly inhibited the growth and survival of various myeloma cells in a dose-dependent manner with IC(50) of 1-10 microM. In contrast, thalidomide itself did not inhibit RPMI8226 cell growth. A tubulin polymerization assay using microtubule protein from porcine brain revealed that 5HPP-33 had potent tubulin polymerization inhibiting activity with IC(50) of 8.1 microM, comparable to that of rhizoxin, a known tubulin polymerization inhibitor. Moreover, its activity was more potent than that of a known thalidomide metabolite, 5-hydroxythalidomide. Our data suggest that 5HPP-33 is a promising candidate as a therapeutic agent for multiple myeloma. In addition, the results suggest that thalidomide's tubulin polymerization inhibiting activity might be the mechanism underlying the induction of apoptosis in myeloma cells.
Topics: Humans; Multiple Myeloma; Thalidomide; Tubulin Modulators
PubMed: 18393844
DOI: 10.2174/092986708783955473 -
Applied and Environmental Microbiology May 2008The products synthesized from a hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster in the genome of Pseudomonas fluorescens Pf-5 were identified...
The products synthesized from a hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster in the genome of Pseudomonas fluorescens Pf-5 were identified using a genomics-guided strategy involving insertional mutagenesis and subsequent metabolite profiling. Five analogs of rhizoxin, a 16-member macrolide with antifungal, phytotoxic, and antitumor activities, were produced by Pf-5, but not by a mutant with an insertion in the gene cluster. The five rhizoxin analogs, one of which had not been described previously, were differentially toxic to two agriculturally important plant pathogens, Botrytis cinerea and Phytophthora ramorum. The rhizoxin analogs also caused swelling of rice roots, a symptom characteristic of rhizoxin itself, but were less toxic to pea and cucumber roots. Of the rhizoxin analogs produced by Pf-5, the predominant compound, WF-1360 F, and the newly described compound 22Z-WF-1360 F were most toxic against the two plant pathogens and three plant species. These rhizoxin analogs were tested against a panel of human cancer lines, and they exhibited potent but nonselective cytotoxicity. This study highlights the value of the genomic sequence of the soil bacterium P. fluorescens Pf-5 in providing leads for the discovery of novel metabolites with significant biological properties.
Topics: Antifungal Agents; Antineoplastic Agents; Botrytis; Cell Line, Tumor; Cucumis; Genomics; Humans; Inhibitory Concentration 50; Macrolides; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Molecular Structure; Multigene Family; Mutagenesis, Insertional; Oryza; Pisum sativum; Peptide Synthases; Phytophthora; Plant Roots; Polyketide Synthases; Pseudomonas fluorescens
PubMed: 18344330
DOI: 10.1128/AEM.02848-07 -
The ISME Journal Jun 2008The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin....
The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of beta-tubulin sequences in the kingdom Fungi. A total of 49 new beta-tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism--mutualism shift in insensitive fungi.
Topics: Amino Acid Sequence; Bacterial Toxins; Burkholderia; Drug Resistance, Fungal; Evolution, Molecular; Fungal Proteins; Fungi; Macrolides; Models, Molecular; Molecular Sequence Data; Phylogeny; Protein Binding; Rhizopus; Symbiosis; Tubulin
PubMed: 18309361
DOI: 10.1038/ismej.2008.19 -
International Journal of Molecular... Feb 2008To ascertain the exact anti-myeloma mechanism of thalidomide in vivo, we performed structural development studies of thalidomide, and obtained various analogues with...
Novel tubulin-polymerization inhibitor derived from thalidomide directly induces apoptosis in human multiple myeloma cells: possible anti-myeloma mechanism of thalidomide.
To ascertain the exact anti-myeloma mechanism of thalidomide in vivo, we performed structural development studies of thalidomide, and obtained various analogues with specific molecular properties. Among these derivatives, we found that a new thalidomide analogue, 2-(2,6-diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33) had the most potent anti-myeloma effect and tubulin-polymerization-inhibiting activity. 5HPP-33 directly inhibited the growth and survival of various myeloma cell lines (RPMI8226, U266, and IM9) in a dose-dependent manner with IC50 of 1-10 microM. In contrast, thalidomide itself did not inhibit cellular growth of RPMI8226 cells. Cultivation with 10 microM 5HPP-33 induced G2/M phase cell cycle arrest, followed by apoptosis of myeloma cells. Treatment with 5HPP-33 induced caspase-3 activity and PARP cleavage. A tubulin polymerization assay using microtubule protein from porcine brain revealed that 5HPP-33 showed potent tubulin-polymerization-inhibiting activity with IC50 of 8.1 microM, comparable to that of the known tubulin-polymerization inhibitor, rhizoxin. Moreover, its activity was more potent than that of a known thalidomide metabolite, 5-hydroxythalidomide. Notably, the structural requirement for its activity was critical, as other analogues and derivatives of 5HPP-33 showed only slight tubulin-polymerization-inhibiting activity. Our data suggest that 5HPP-33 is a promising candidates for a therapeutic agent of multiple myeloma. In addition, these results suggest that the tubulin-polymerization inhibiting activity of thalidomide might be a possible mechanism for inducing the apoptosis of myeloma cells by thalidomide.
Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Division; Cell Line, Tumor; Drug Screening Assays, Antitumor; G2 Phase; Humans; Isoindoles; Multiple Myeloma; Thalidomide; Time Factors; Tubulin Modulators
PubMed: 18204782
DOI: No ID Found -
International Journal of Systematic and... Nov 2007Several strains of the fungus Rhizopus microsporus harbour endosymbiotic bacteria for the production of the causal agent of rice seedling blight, rhizoxin, and the toxic...
Several strains of the fungus Rhizopus microsporus harbour endosymbiotic bacteria for the production of the causal agent of rice seedling blight, rhizoxin, and the toxic cyclopeptide rhizonin. R. microsporus and isolated endobacteria were selected for freeze-fracture electron microscopy, which allowed visualization of bacterial cells within the fungal cytosol by their two parallel-running envelope membranes and by the fine structure of the lipopolysaccharide layer of the outer membrane. Two representatives of bacterial endosymbionts were chosen for phylogenetic analyses on the basis of full 16S rRNA gene sequences, which revealed that the novel fungal endosymbionts formed a monophyletic group within the genus Burkholderia. Inter-sequence similarities ranged from 98.94 to 100%, and sequence similarities to members of the Burkholderia pseudomallei group, the closest neighbours, were 96.74-97.38%. In addition, the bacterial strains were distinguished from their phylogenetic neighbours by their fatty acid profiles and other biochemical characteristics. The phylogenetic studies based on 16S rRNA gene sequence data, together with conclusive DNA-DNA reassociation experiments, strongly support the proposal that these strains represent two novel species within the genus Burkholderia, for which the names Burkholderia rhizoxinica sp. nov. (type strain, HKI 454T=DSM 19002T=CIP 109453T) and Burkholderia endofungorum sp. nov. (type strain, HKI 456T=DSM 19003T=CIP 109454T) are proposed.
Topics: Bacterial Typing Techniques; Burkholderia; DNA, Bacterial; DNA, Ribosomal; Fatty Acids; Freeze Fracturing; Genes, rRNA; Microscopy, Electron; Molecular Sequence Data; Nucleic Acid Hybridization; Oryza; Phylogeny; Plant Diseases; RNA, Ribosomal, 16S; Rhizopus; Sequence Analysis, DNA; Species Specificity; Symbiosis
PubMed: 17978222
DOI: 10.1099/ijs.0.64660-0