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The nomenclature and taxonomy of Acarospora molybdina is revised using morphological and molecular data. The new combination Myriospora molybdina is proposed and Acarospora hysgina is recognized as a distinct species while A. brunneola is reduced to synonymy with A. hysgina. In its new circumscription M. molybdina is an arctic species, in Scandinavia only occurring in northernmost Norway. Further localities are reported from Greenland, Russia, Svalbard and the USA (Alaska). Acarospora hysgina is the correct name for a species distributed along the west coast of Sweden and Norway, formerly thought to belong to A. molybdina. Localities are also reported from Canada (New Brunswick), Greenland and the USA (Maine). The following names are lectotypified: Acarospora brunneola, A. molybdina var. confusa, Lecanora ereutica beta microcyclos, Parmelia ereutica, P. hysgina and P. molybdina.

Using next-generation sequencing (NGS), we developed and validated a whole-genome sequencing (WGS)-based clinical test for fungal species identification on clinical isolates. The identification is mainly based on the fungal ribosomal internal transcribed spacer (ITS) region as the primary marker, and additional marker and genomic analysis applied for species within the Mucorales family (using the 28S rRNA gene) and Aspergillus genus (using the beta-tubulin gene and k-mer tree-based phylogenetic clustering). The validation study involving 74 unique fungal isolates (22 yeasts, 51 molds, and 1 mushroom-forming fungus) showed high accuracy, with 100% (74/74) concordance on the genus-level identifications and 89.2% (66/74) concordance on the species level. The 8 discrepant results were due to either the limitation of conventional morphology-based methodology or taxonomic changes. After one year of implementation in our clinical laboratory, this fungal NGS test was utilized in 29 cases; the majority of them were transplant and cancer patients. We demonstrated the utility of this test by detailing five case studies, in which accurate fungal species identification led to correct diagnosis, treatment adjustment or was ruled out for hospital acquired infection. This study provides a model for validation and implementation of WGS for fungal identification in a complex health system that serves a large immunocompromised patient population.

“Pébrine” is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the β-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.

Few ophiostomatoid fungi have been reported from the margin of the Tibetan Plateau and none have been found in the central portion of the region. In a survey of ophiostomatoid fungi associated with spruce bark beetles in Tibetan subalpine forests, numerous strains of Leptographium s. l. (Ophiostomataceae) were isolated from Orthotomicus sp. (Coleoptera: Scolytinae) and its galleries infesting Picea likiangensis var. balfouriana. Morphological characters and phylogenetic analysis based on multiple DNA sequence data (ITS2-partial LSU rDNA region, beta-tubulin and transcription elongation factor-1a genes) revealed a new species in the “Grosmannia penicillata complex”, which is proposed as G. tibetensis. The species is characterized by both Leptographium and Pesotum asexual states, which is unique in the “G. penicillata complex”. Additionally, sequences of the tubC paralogue gene were found combining with tub2 sequences in many species of the “G. penicillata complex”, resulting in incongruent trees. This is the first report of tubulin paralogue genes in ophiostomatoid fungi. Gene duplication and losses make betatubulin a potentially challenging locus for use as a molecular marker for tracing speciation.

Plocabulin (PM060184) is a microtubule depolymerizing agent with potent antiproliferative activity undergoing phase II clinical trials for the treatment of solid tumors. Plocabulin shows antifungal activity virtually abolishing growth of the filamentous fungus Aspergillus nidulans. A. nidulans hyphae depend both on mitotic and interphase microtubules, as human cells. Here, we exploited the A. nidulans genetic amenability to gain insight into the mechanism of action of plocabulin. By combining mutations in the two A. nidulans β-tubulin isotypes we obtained a plocabulin-insensitive strain, showing that β-tubulin is the only molecular target of plocabulin in fungal cells. From a genetic screen, we recovered five mutants that show plocabulin resistance but do not carry mutations in β-tubulin. Resistance mutations resulted in amino acid substitutions in (1) two subunits of the eukaryotic translation initiation factor eIF2B activating the General Amino Acid Control, (2) TIM44, an essential component of the inner mitochondrial membrane translocase, (3) two transcription factors of the binuclear zinc cluster family potentially interfering with the uptake or efflux of plocabulin. Given the conservation of some of the identified proteins and their respective cellular functions in the tumor environment, our results pinpoint candidates to be tested as potential biomarkers for determination of drug efficiency.

Cercospora leaf spot (CLS) caused by Cercospora beticola occurs annually in Serbia causing severe yield losses of sugar beet, which requires intensive use of fungicides. In recent years we have observed unsatisfactory control of CLS originating from northwestern Serbia. Frequency of C. beticola populations resistant to Quinone outside inhibitors (QoI) was 81% (51/63 isolates), 98% (62/63) to sterol-demethylation inbibitors (DMI) and 54% (34/63) to methyl-2-benzimidazole carbamate fungicides (MBC). The genetic basis underlying the resistance was tested by characterizing the cob, CYP51 and -tubulin genes, associated with resistance to QoI, DMI and MBC fungicides, respectively. Isolates that were resistant to QoI fungicides had the G143A mutation in the cob gene. Characterization of the CYP51 gene revealed seven diverse haplotypes; however, no correlation with sensitivity or resistance to DMI fungicides could be identified. Resistance to MBC fungicides was associated with the presence of the E198A mutation in the -tubulin gene of all resistant isolates. From a total of 63 isolates originating from sugar beet fields of northwestern Serbia, 62 isolates showed resistance to multiple modes of action. Three multi-resistant phenotypes were identified: MR1 (N = 29) - resistant to QoI and DMI fungicides (QoI-R and DMI-R) but sensitive to MBC fungicides (MBC-S); MR2 (N = 11, QoI-S, DMI-R and MBC-R); and MR3 (N = 22), resistant to all three groups of fungicides (QoI-R, DMI-R and MBC-R). This is the first report of C. beticola resistance to QoI fungicides in Serbia. This study revealed development of multi-resistance of C. beticola isolates to MBC, DMI and QoI fungicides, which represents the first record of this phenomenon in C. beticola populations.

In autumn 2015, hazelnut plants with leaf blight symptoms were noticed in a commercial plantation in the Province of Vojvodina, Serbia. Symptomatic samples were collected and submitted to laboratory analysis. Based on morphological characterization, the fungus isolated from the material was initially identified as Pestalotiopsis sp. Pathogenicity tests showed that two selected isolates infected hazelnut leaves and fruits that developed symptoms after artificial inoculation. The pathogen was re-isolated from diseased leaves and fruits, confirming Koch's postulates. Molecular identification was performed with sequence and phylogenetic analysis of ITS, EF1-alpha, and TUB genomic regions. Phylogenetic analysis confirmed the results of the morphological identification. The detection of Pestalotiopsis sp., a causal agent of leaf blight on hazelnut in Serbia, is one of a few reports of these pathogenic fungi on hazelnut.

The efficacy of microtubule targeting agents in cancer treatment has been compromised by the development of drug resistance that may involve both, P-glycoprotein overexpression and the changes in beta-tubulin isoforms' expression. The anti-Topoisomerase II activity of methyl 4-((E)-2-(methoxycarbonyl)vinyloxy)oct-2-ynoate (DTA0100) was recently reported. Herein, we further evaluated this propargylic enol ether derivative and found that it exerts inhibitory effect on tubulin polymerization by binding to colchicine binding site. DTA0100 mitotic arrest properties were investigated in two multi-drug resistant cancer cell lines with P-glycoprotein overexpression (colorectal carcinoma and glioblastoma). The sensitivity of multi-drug resistant cancer cell lines to DTA0100 was not significantly changed in contrast to microtubule targeting agents such as paclitaxel, vinblastine and colchicine. DTA0100 clearly induced microtubule depolymerization, leading to disturbance of cell cycle kinetics and subsequent apoptosis. The fine-tuning in beta-tubulin isoforms expression observed in multi drug resistant cancer cells may influence the efficacy of DTA0100. Importantly, DTA0100 blocked the Pglycoprotein function in both multi-drug resistant cancer cell lines without inducing the increase in Pglycoprotein expression. Therefore, DTA0100 acting as dual inhibitor of Topoisomerase II and microtubule formation could be considered as multi-potent anticancer agent. Besides, it is able to overcome the problem of drug resistance that emerges in the therapeutic approaches with either Topoisomerase II or microtubule targeting agents.

The genus Torulomyces was characterised by species that typically have conidiophores consisting of solitary phialides that produce long chains of conidia connected by disjunctors. Based on the phylogenetic position of P. lagena (generic ex-neotype), the genus and its seven species were transferred to Penicillium and classified in sect. Torulomyces along with P. cryptum and P. lassenii. The aim of this study was to review the species currently classified in sect. Torulomyces using morphology and phylogenies of the ITS, BenA, CaM and RPB2 regions. Based on our results, we accept 16 species in sect. Torulomyces, including 12 new species described as P. aeris, P. austricola, P. cantabricum, P. catalonicum, P. oregonense, P. marthae-christenseniae, P. riverlandense, P. tubakianum, P. variratense, P. williamettense, P. wisconsinense and P. wollemiicola. In addition, we reclassify P. laeve and P. ovatum in sect. Exilicaulis and correct the typification of P. lagena. We provide descriptions and notes on the identification of the species.

We introduce 15 new species of Penicillium isolated from a diverse range of locations, including Canada, Costa Rica, Germany, Italy, New Zealand, Tanzania, USA and the Dry Valleys of Antarctica, from a variety of habitats, including leaf surfaces in tropical rain forests, soil eaten by chimpanzees, infrabuccal pockets of carpenter ants, intestinal contents of caterpillars and soil. The new species are classified in sections Aspergilloides (1), Canescentia (2), Charlesia (1), Exilicaulis (3), Lanata-Divaricata (7) and Stolkia (1). Each is characterised and described using classical morphology, LC-MS based extrolite analyses and multigene phylogenies based on ITS, BenA and CaM. Significant extrolites detected include andrastin, pulvilloric acid, penitrem A and citrinin amongst many others.

Phaeoacremonium spp. are commonly isolated from stems and branches of diseased woody hosts, and humans with phaeohyphomycosis. The genus Phaeoacremonium (Togniniaceae, Togniniales) has recently been monographed, and presently contains 46 species, while its sexual morph, Togninia, contains 26 epithets, of which 13 are insufficiently known. In this review we summarise information pertaining to the global distribution, pathology, ecology, and detection of these species, and present a case for retaining the genus Phaeoacremonium over that of Togninia. Furthermore, to obtain a single nomenclature, the following new combinations are also proposed: Phaeoacremonium africanum, P. aquaticum, P. fraxinopennsylvanicum, P. griseo-olivaceum, P. inconspicuum, P. leptorrhynchum, P. minimum, and P. vibratile.

Background: The development of anthelmintic resistance (AR) to macrocyclic lactones in the equine roundworm Parascaris equorum has resulted in benzimidazoles now being the most widely used substance to control Parascaris infections. However, over-reliance on one drug class is a risk factor for the development of AR. Consequently, benzimidazole resistance is widespread in several veterinary parasites, where it is associated with single nucleotide polymorphisms (SNPs) in drug targets encoded by the beta-tubulin genes. The importance of these SNPs varies between different parasitic nematodes, but it has been hypothesised that they occur, at low allele frequencies, even in unselected populations. This study investigated whether these SNPs exist in the P. equorum population and tested the hypothesis that BZ resistance can develop from pre-existing SNPs in codons 167, 198 and 200 of the beta-tubulin isotype 1 and 2 genes, reported to be associated with AR in strongylids. The efficacy of the oral paste formula fenbendazole on 11 farms in Sweden was also assessed. Methods: Two isotype-specific primer pairs were designed, one on either side of the codon 167 and one on either side of codons 198 and 200. A pool of 100 000 larvae was sequenced using deep amplicon sequencing by Illumina HiSeq. Faecal egg count reduction test was used to assess the efficacy of fenbendazole. Results: No SNPs were observed in codons 167, 198 or 200 of the beta-tubulin isotype 1 or 2 genes of P. equorum, even though 100 000 larvae were sequenced. Faecal egg count reduction testing of fenbendazole showed that this anthelmintic was still 100% effective, meaning that the likelihood of finding high allele frequency of SNPs associated with benzimidazoles resistance in P. equorum was low. Unexpectedly, the allele frequencies observed in single worms were comparable to those in pooled samples. Conclusions: We concluded that fenbendazole does not exert selection pressure on the beta-tubulin genes of isotype

Thirty-eight Phaeoacremonium isolates collected from pruning wounds of tropical sandalwood in Western Australia were studied with morphological and cultural characteristics as well as phylogenetic analyses of combined DNA sequences of the actin and beta-tubulin genes. Three known Phaeoacremonium species were found, namely P. alvesii, P. parasiticum, and P. venezuelense. Phaeoacremonium venezuelense represents a new record for Australia. Two new species are described: P. luteum sp. nov. can be identified by the ability to produce yellow pigment on MEA, PDA, and OA, the predominance of subcylindrical to subulate type II phialides, and the mycelium showing prominent exudate droplets observed as warts; and P. santali sp. nov. which can be separated from other species producing pink colonies on MEA by the predominance of type I and II phialides, the distinct brownish olive colonies in OA, and slow growth.

Benzimidazole (BZ) resistance in gastrointestinal nematodes has been associated with single nucleotide polymorphisms (SNPs) at codons 200,167 and 198 in the beta-tubulin isotype 1 gene and, recently, these SNPs have also been found in macrocyclic lactone (ML) resistant strains of Haemonchus contortus. On this basis, we have studied the same putative SNPs in Spanish Teladorsagia circumcincta field isolates by pyrosequencing. Single L3 (infective 3rd stage larvae) from five sheep flocks were tested after confirming their BZ susceptibility and degree of ivermectin (IVM) resistance. According to the Faecal Egg Count Reduction Test (FECRT) one flock was classified as IVM susceptible, another one was resistant, and the rest had a suspicion of resistance to IVM. DNA extraction was carried out on 598 single L3 and 56% of these were identified as T. circumcincta after the amplification of a species-specific ITS2 fragment. The number of L3 analyzed for the SNPs 198/200 was 255 and for the SNP 167 was 187. Results clearly indicate no resistance-associated SNPs were present at any codon, before or after treatment. Therefore, all T. cicumcincta L3 were designated as susceptible homozygous genotypes for all SNPs. The absence of the mutations in these populations would argue against resistance haplotypes being present in the parasite population prior to drug treatment, at least in Spanish T. circumcincta.

Species of Calonectria are important plant pathogens, several of which have a worldwide distribution. Contemporary taxonomic studies on these fungi have chiefly relied on DNA sequence comparisons of the ß-tubulin gene region. Despite many new species being described, there has been no phylogenetic synthesis for the group since the last monographic study almost a decade ago. In the present study, the identity of a large collection of Calonectria isolates from various geographic regions was determined using morphological and DNA sequence comparisons. This resulted in the discovery of seven new species; Ca. densa, Ca. eucalypti, Ca. humicola, Ca. orientalis, Ca. pini, Ca. pseudoscoparia and Ca. sulawesiensis, bringing the total number of currently accepted Calonectria species to 68. A multigene phylogeny was subsequently constructed for all available Calonectria spp., employing seven gene regions, namely actin, ß-tubulin, calmodulin, histone H3, the internal transcribed spacer regions 1 and 2 and the 5.8S gene of the ribosomal RNA, 28S large subunit RNA gene and translation elongation 1-alpha. Based on these data 13 phylogenetic groups could be distinguished within the genus Calonectria that correlated with morphological features. Dichotomous and synoptic keys to all Calonectria spp. currently recognised are also provided.

Species of Calonectria and their Cylindrocladium anamorphs are important plant pathogens worldwide. At present 52 Cylindrocladium spp. and 37 Calonectria spp. are recognised based on sexual compatibility, morphology and phylogenetic inference. The polyphasic approach of integrating Biological, Morphological and Phylogenetic Species Concepts has revolutionised the taxonomy of fungi. This review aims to present an overview of published research on the genera Calonectria and Cylindrocladium as they pertain to their taxonomic history. The nomenclature as well as future research necessary for this group of fungi are also briefly discussed

The family Sporormiaceae (Pleosporales, Dothideomycetes, Ascomycota) occur worldwide and a majority of the species are coprophilous. The taxonomy and classification of the family are based on a small number of morphological and ecological characters. Several taxa are easily confused by their shared morphological features, and the relationships between genera are poorly known and in need of critical study. The aims of this study were to resolve the phylogenetic relationships within the Sporormiaceae, test the current generic classification, and study the utility of traditional characters for the taxonomy in the group. To resolve these questions, we analysed combined data sets of ITS-nLSU rDNA, mtSSU rDNA and beta-tubulin sequences with parsimony and Bayesian methods. The results showed that many characters, which previously have been used in the taxonomy and classification of the Sporormiaceae, such as the substrate choice, presence or absence of an ostiole, and presence or absence of germ slits, were all homoplastic and not useful for circumscribing monophyletic groups. A number of characters may be useful for circumscribing smaller clades if used in combination with other features, such as the shape of the ascus with the shape of the ascospores. Our phylogenetic analyses show that Preussia and Sporormiella are non-monophyletic, and a constrained analysis forcing these genera into monophyly resulted in significantly worse trees. Spororminula is nested in Preussia s. lat., and Eremodothis and Pycnidiophora are nested within Westerdykella. Finally, we suggest a new generic classification for the family Sporormiaceae, including Sporormia, Preussia (including Sporomiella and Spororminula) and Westerdykella (including Eremodothis and Pycnidiophora). We also propose 14 new combinations: Preussia alloiomera (S.I. Ahmed & Cain) Kruys, Preussia antarctica (Speg.) Kruys, Preussia bipartis (Cain) Kruys, Preussia borealis (I.Egeland) Kruys, Preussia dubia (S.I. Ahmed &

Dendroctonus valens is an invasive pest in coniferous forests of northern China. It was suspected of being responsible for the death of more than three million Pinus tabuliformis trees. The present study sought to identify the ophiostomatoid fungi associated with D. valens in northern China and understand the possible role of these fungi in the pine decline. On the basis of morphology, physiology, mating compatibility and phylogenetic analyses of multiple DNA sequences, seven species of ophiostomatoid fungi were isolated from and around D. valens galleries: Leptographium alethinum, Grosmannia koreana (teleomorph of L. koreanum), L. procerum, L. sinoprocerum, L. truncatum, Pesotum aureum and P. pini. All have been recorded for the first time in China. Among them, the occurrence of the dominant species L. procerum is positively linked to attack intensities of D. valens. The pathogenicity of four species (L. koreanum, L. procerum, L. sinoprocerum and L. truncatum) was tested on mature P. tabuliformis trees by stem inoculation. All inoculated strains caused significant necrotic lesions on the inner bark. However, L. koreanum and L. truncatum induced more extensive lesions than L. procerum and L. sinoprocerum. Their association with D. valens and the P. tabuliformis decline is discussed.

Calonectria (Ca.) species and their Cylindrocladium (Cy.) anamorphs are well-known pathogens of forest nursery plants in subtropical and tropical areas of the world. An investigation of the mortality of rooted Pinus cuttings in a commercial forest nursery in Colombia led to the isolation of two Cylindrocladium anamorphs of Calonectria species. The aim of this study was to identify these species using DNA sequence data and morphological comparisons. Two species were identified, namely one undescribed species, and Cy. gracile, which is allocated to Calonectria as Ca. brassicae. The new species, Ca. brachiatica, resides in the Ca. brassicae species complex. Pathogenicity tests with Ca. brachiatica and Ca. brassicae showed that both are able to cause disease on Pinus maximinoi and P. tecunumanii. An emended key is provided to distinguish between Calonectria species with clavate vesicles and 1-septate macroconidia

During a study of ophiostomatoid fungi associated with the invasive pest Dendroctonus valens in the Pinus tabuliformis ecosystem in northern China, a multigenic (ITS2-LSU, beta-tubulin and EF1-alpha) phylogenetic analysis and examination of morphological features revealed in addition to Leptographium procerum the occurrence of an undescribed species. The new species, Leptographium sinoprocerum, belongs to the L. procerum-L. profanum clade. Both L. procerum and L. sinoprocerum are similar to each other and occur sympatrically in the ecosystem studied. Nevertheless L. sinoprocerum can be distinguished from L. procerum by shorter conidiophore stipes arising from both submerged and aerial hyphae, slightly more oblong conidia, a granular ornamentation on the submerged hyphae and dark olivaceous colonies on MEA.

During a survey of Prunus wood from South Africa, isolations were made of three presumably Calosphaerialean fungi that formed hyphomycetous, phialidic anamorphs in culture. In order to reveal the phylogenetic relationship of these fungi, they were characterised on a morphological and molecular (LSU and ITS rDNA) basis. Two isolates that formed a teleomorph in culture are newly described as Calosphaeria africana sp. nov. Although asci of Calosphaeria are characterised by having non-amyloid apical rings, two functional wall layers were observed in asci of C. africana, which has hitherto not been observed in any member of the Calosphaeriaceae. However, Calosphaeriaceae (Calosphaeriales, Sordariomycetes) are not closely related to other bitunicate fungi like Dothideomycetes, Chaetothyriales and bitunicate lichens. Possession of two separating wall layers is considered to be a result of both inherited abilities and convergent evolution under a strong selection pressure of the environmental conditions that favour an extension of the ascus. The other two species represented a separate lineage within Calosphaeriaceae, and formed phialophora-like anamorphs. By obtaining the teleomorph in culture, one of them could be identified as a species of Jattaea, described here as Jattaea prunicola sp. nov., while the second, which only produced the anamorph, is named as Jattaea mookgoponga sp. nov. These findings suggest that some species of Jattaea are true members of the Calosphaeriaceae, though the phylogenetic relation of the type, J. algeriensis, remains unknown. Furthermore, it also represents the first report of Jattaea on Prunus wood, and from South Africa.

The present study compares all known species of Cylindrocladium that have clavate vesicles. Several isolates were obtained from baited soils collected in various parts of the world, while others were associated with leaf litter or symptomatic plant hosts. Isolates were compared based on morphology, as well as DNA sequence data from their ß-tubulin and histone gene H3 regions. Cylindrocladium australiense and Cy. ecuadoriae, are described as new species, a decision based on morphology and molecular data. A group of isolates associated with toppling disease of banana in the West Indies is identified as Cy. flexuosum. An epitype is designated for Cy. ilicicola, and a new name, Curvicladiella, proposed to replace the anamorphic genus Curvicladium, which is a homonym.

Cancer chemotherapy with taxol often fails due to acquired resistance of cancer cells, which is frequently associated with an overexpression of P-gp and alterations of beta -tubulin. A taxol-resistant cell line, QGY-TR50, derived from a human hepatocellular carcinoma (HCC) QGY-7703 cell line was used to investigate the mechanisms of taxol-resistance, QGY-TR50 cells showed more than 250-fold resistance to taxol and exhibited cross-resistance to other drugs including actinomycin D, doxorubicin, vinblastine, and vincristine, P-gp was highly expressed in QGY-TR50 cells. Expression levels of five human beta -tubulin isotypes (beta (I), beta (II)-, beta (III)-, beta (IVa), and beta (IVb)-tubulin) were examined by real-time semi-quantitative PCR. Comparing with QGY-7703 cells, QGY-TR50 cells did not show any significant change in the expression levels of beta (I)-, beta (IVa), and beta (IVb)-tubulin, While a 1.2-fold increased in beta (II)-tubulin and a 0.5-fold decreased in beta (III)-tubulin levels were observed. All results suggest that the P-glycoprotein could be one key factor involved in enhancing drug resistance in QGY-TR50 cells. (C) 2001 Academic Press.

The effect of bound nucleotide on the conformation of cell division protein FtsZ from Methanococcus jannaschii has been investigated using molecular dynamics and site-directed mutagenesis. The molecular dynamics indicate that the gamma-phosphate of GTP induces a conformational perturbation in loop T3 (Gly88-Gly99 segment), in a position structurally equivalent to switch II of Ha-ras-p21. In the simulated GTP-bound state, loop T3 is pulled by the gamma-phosphate into a more compact conformation than with GDP, related to that observed in the homologous proteins alpha- and beta-tubulin. The existence of a nucleotide-induced structural change in loop T3 has been confirmed by mutating Thr92 into Trp (T92W-W319Y FtsZ). This tryptophan (12 A away from gamma-phosphate) shows large differences in fluorescence emission, depending on which nucleotide is bound to FtsZ monomers. Loop T3 is located at a side of the contact interface between two FtsZ monomers in the current model of FtsZ filament. Such a structural change may bend the GDP filament upon hydrolysis by pushing against helix H8 of next monomer, thus, generating force on the membrane during cell division. A related curvature mechanism may operate in tubulin activation.

We have determined the kinetic scheme and the reaction rates of binding to microtubules of two fluorescent taxoids, 7-O-[N-(4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-1) and 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-2). Flutax-1 and Flutax-2 bind to microtubules with high affinity (K(a) approximately 10(7) m(-1), 37 degrees C). The binding mechanism consists of a fast bimolecular reaction followed by at least two monomolecular rearrangements, which were characterized with stopped-flow techniques. The kinetic constants of the bimolecular reaction were 6.10 +/- 0.22 x 10(5) m(-1) s(-1) and 13.8 +/- 1.8 x 10(5) m(-1) s(-1) at 37 degrees C, respectively. A second slow binding step has been measured employing the change of fluorescence anisotropy of the probe. The reversal of this reaction is the rate-limiting step of dissociation. A third step has been detected using small angle x-ray scattering and involves a 2-nm increase in the diameter of microtubules. It is suggested that the first step entails the binding of the Taxol moiety and the second a relative immobilization of the fluorescent probe. The equilibrium and some kinetic measurements required the use of stabilized cross-linked microtubules, which preserved taxoid binding. The results indicate that the Taxol binding site is directly accessible, in contrast with its location at lumen in the current model of microtubules. An alternative structural model is considered in which the binding site is located between protofilaments, accessible from the microtubule surface.

We have investigated the accessibility of the Taxol-binding site and the effects of Taxol binding on the structures of assembled microtubules. Taxol and docetaxel readily bind to and dissociate from microtubules, reaching 95% ligand exchange equilibrium in less than 3 min under our solution conditions (microtubules were previously assembled from GTP-tubulin, GTP-tubulin and microtubule-associated proteins, or GDP-tubulin and taxoid). Microtubules assembled from purified tubulin with Taxol are known to have typically one protofilament less than with the analogue docetaxel and control microtubules. Surprisingly, Taxol binding and exchange induce changes in the structure of preformed microtubules in a relatively short time scale. Cryoelectron microscopy shows changes toward the protofilament number distribution characteristic of Taxol or docetaxel, with a half-time of approximately 0.5 min, employing GDP-tubulin-taxoid microtubules. Correspondingly, synchrotron x-ray solution scattering shows a reduction in the mean microtubule diameter upon Taxol binding to microtubules assembled from GTP-tubulin in glycerol-containing buffer, with a structural relaxation half-time of approximately 1 min. These results imply that microtubules can exchange protofilaments upon Taxol binding, due to internal dynamics along the microtubule wall. The simplest interpretation of the relatively fast taxoid exchange observed and labeling of cellular microtubules with fluorescent taxoids, is that the Taxol-binding site is at the outer microtubule surface. On the contrary, if Taxol binds at the microtubule lumen in agreement with the electron crystallographic structure of tubulin dimers, our results suggest that the inside of microtubules is easily accessible from the outer solution. Large pores or moving lattice defects in microtubules might facilitate the binding of taxoids, as well as of possible endogenous cellular ligands of the inner microtubule wall.