Screening saikosaponin d (SSd)-producing endophytic fungi from Bupleurum scorzonerifolium Willd.

Saikosaponin d (SSd) is an important bioactive compound of traditional Chinese medicinal plant Bupleurum scorzonerifolium Willd. and exhibits many effects, such as anti-tumor, anti-inflammation and immunomodulatory. Since endophytic fungi possess the natural capacity to produce the similar secondary metabolite to that of their host plants, they are promising as alternative sources of plant bioactive natural products. In this study, in order to search for SSd-producing strains, endophytes were isolated from B. scorzonerifolium and were authenticated by the ITS sequence and the translation elongation factor-1alpha gene (TEF-1α) sequence analysis. The profile of metabolites present in the crude exacts was carried out by ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis. The results showed that two strains, CHS2 and CHS3 from B. scorzonerifolium could produce SSd by UPLC/Q-TOF-MS analysis, and the amount of SSd produced by strain CHS2 and CHS3 were about 2.17 and 2.40 µg/mL, respectively. CHS2 and CHS3 showed a close phylogenetic relationship to Fusarium oxysporum and Fusarium acuminatum, respectively. According to our concern, no endophytic fungi capable of producing SSd from B. scorzonerifolium have been found before. Our clear intention was to isolate and identify these endophytic fungi that produce important active secondary metabolites, and then study the strains that produce this compound on a large scale through fermentation or even genetic study, to provide a feasible and more convenient way for the production of SSd.

eEF-2K knockdown synergizes with STS treatment to inhibit cell proliferation, migration, and invasion via the TG2/ERK pathway in A549 cells.

Emerging research has suggested the anticancer potential of tanshinone IIA, the bioactive ingredient isolated from the traditional Chinese herb Salvia miltiorrhiza. However, the molecular mechanism of sodium tanshinone IIA sulfonate (STS) antilung cancer effect is not very clear. In this study, our purpose is to investigate the roles of STS and elongation factor-2 kinase (eEF-2K) in regulating the proliferation, migration, and invasion of A549 cells and explore the implicated pathways. We found that STS suppressed A549 cell survival and proliferation in a time- and xdose-dependent manner. Knockdown of eEF-2K and treatment with STS synergistically exerted antiproliferative, -migratory, and -invasive effects on A549 cells. These effects were caused by attenuation of the extracellular signal-regulated kinase (ERK) pathway via inhibition of tissue transglutaminase (TG2). In summary, the inhibition of eEF-2K synergizes with STS treatment, exerting anticancer effects on lung adenocarcinoma cells through the TG2/ERK signaling pathway, which provides a potential therapeutic target for treating lung adenocarcinoma.

Cladosporium cladosporioides and C. tenuissimum Cause Blossom Blight in Strawberry in Korea

Blossom blight in strawberry was first observed in a green house in Nonsan, Damyang, and Geochang areas of Korea, between early January to April of 2012. Disease symptoms started as a grey fungus formed on the stigma, which led to the blossom blight and eventually to black rot and necrosis of the entire flower. We isolated the fungi purely from the infected pistils and maintained them on potato dextrose agar (PDA) slants. To test Koch's postulates, we inoculated the fungi and found that all of the isolates caused disease symptoms in the flower of strawberry cultivars (Seolhyang, Maehyang, and Kumhyang). The isolates on PDA had a velvet-like appearance, and their color ranged between olivaceous-brown and smoky-grey to olive and almost black. The intercalary conidia of the isolates were elliptical to limoniform, with sizes ranging from 5.0~10.5 x 2.5~3.0 microm to 4.0~7.5 x 2.0~3.0 microm, respectively. The secondary ramoconidia of these isolates were 0- or 1-septate, with sizes ranging betweem 10.0~15.0 x 2.5~3.7 microm and 8.7~11.2 x 2.5~3.2 microm, respectively. A combined sequence analysis of the internal transcribed spacer regions, partial actin (ACT), and translation elongation factor 1-alpha (TEF) genes revealed that the strawberry isolates belonged to two groups of authentic strains, Cladosporium cladosporioides and C. tenuissimum. Based on these results, we identified the pathogens causing blossom blight in strawberries in Korea as being C. cladosporioides and C. tenuissimum.

Homologous overexpression of RfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide.

BACKGROUND: Glycosaminoglycans, such as hyaluronic acid, heparin, and chondroitin sulfate, are among the top ranked products in industrial biotechnology for biomedical applications, with a growing world market of billion dollars per year. Recently a remarkable progress has been made in the development of tailor-made strains as sources for the manufacturing of such products. The genetic modification of E. coli K4, a natural producer of chondroitin sulfate precursor, is challenging considering the lack of detailed information on its genome, as well as its mobilome. Chondroitin sulfate is currently used as nutraceutical for the treatment of osteoarthritis, and several new therapeutic applications, spanning from the development of skin substitutes to live attenuated vaccines, are under evaluation. RESULTS: E. coli K4 was used as host for the overexpression of RfaH, a positive regulator that controls expression of the polysaccharide biosynthesis genes and other genes necessary for the virulence of E. coli K4. Various engineering strategies were compared to investigate different types of expression systems (plasmid vs integrative cassettes) and integration sites (genome vs endogenous mobile element). All strains analysed in shake flasks on different media showed a capsular polysaccharide production improved by 40 to 140%, compared to the wild type, with respect to the final product titer. A DO-stat fed-batch process on the 2L scale was also developed for the best performing integrative strain, EcK4r3, yielding 5.3 g ∙ L(-1) of K4 polysaccharide. The effect of rfaH overexpression in EcK4r3 affected the production of lipopolysaccharide and the expression of genes involved in the polysaccharide biosynthesis pathway (kfoC and kfoA), as expected. An alteration of cellular metabolism was revealed by changes of intracellular pools of UDP-sugars which are used as precursors for polysaccharide biosynthesis. CONCLUSIONS: The present study describes the identification of a gene target and the application of a successful metabolic engineering strategy to the unconventional host E. coli K4 demonstrating the feasibility of using the recombinant strain as stable cell factory for further process implementations.

The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.

Selenocysteine insertion into selenoproteins involves the translational recoding of UGA stop codons. In mammals, selenoprotein expression further depends on selenium availability, which has been particularly described for glutathione peroxidase 1 and 4 (Gpx1 and Gpx4). The SECIS element located in the 3'UTR of the selenoprotein mRNAs is a modulator of UGA recoding efficiency in adequate selenium conditions. One of the current models for the UGA recoding mechanism proposes that the SECIS binds SECIS-binding protein 2 (SBP2), which then recruits a selenocysteine-specific elongation factor (EFsec) and tRNA (Sec) to the ribosome, where L30 acts as an anchor. The involvement of the SECIS in modulation of UGA recoding activity was investigated, together with SBP2 and EFsec, in Hek293 cells cultured with various selenium levels. Luciferase reporter constructs, in transiently or stably expressing cell lines, were used to analyze the differential expression of Gpx1 and Gpx4. We showed that, upon selenium fluctuation, the modulation of UGA recoding efficiency depends on the nature of the SECIS, with Gpx1 being more sensitive than Gpx4. Attenuation of SBP2 and EFsec levels by shRNAs confirmed that both factors are essential for efficient selenocysteine insertion. Strikingly, in a context of either EFsec or SBP2 attenuation, the decrease in UGA recoding efficiency is dependent on the nature of the SECIS, GPx1 being more sensitive. Finally, the profusion of selenium of the culture medium exacerbates the lack of factors involved in selenocysteine insertion.

Increased E4 activity in mice leads to ubiquitin-containing aggregates and degeneration of hypothalamic neurons resulting in obesity.

Obesity has become a serious worldwide public health problem. Although neural degeneration in specific brain regions has been suggested to contribute to obesity phenotype in humans, a causal relationship between these two conditions has not been demonstrated experimentally. We now show that E4B (also known as UFD2a), a mammalian ubiquitin chain elongation factor (E4), induces the formation of intracellular aggregates positive for ubiquitin and the adaptor protein p62 when overexpressed in cultured cells or the brain. Mice transgenic for E4B manifested neural degeneration in association with aggregate formation, and they exhibited functional impairment specifically in a subset of hypothalamic neurons that regulate food intake and energy expenditure, resulting in development of hyperphagic obesity and related metabolic abnormalities. The neural pathology of E4B transgenic mice was similar to that of human neurodegenerative diseases associated with the formation of intracellular ubiquitin-positive deposits, indicating the existence of a link between such diseases and obesity and related metabolic disorders. Our findings thus provide experimental evidence for a role of hypothalamic neurodegeneration in obesity, and the E4B transgenic mouse should prove to be a useful animal model for studies of the relationship between neurodegenerative diseases and obesity.

Eukaryotic selenoprotein synthesis: mechanistic insight incorporating new factors and new functions for old factors.

Selenium is an essential micronutrient that has been linked to various aspects of human health. Selenium exerts its biological activity through the incorporation of the amino acid, selenocysteine (Sec), into a unique class of proteins termed selenoproteins. Sec incorporation occurs cotranslationally at UGA codons in archaea, prokaryotes, and eukaryotes. UGA codons specify Sec coding rather than termination by the presence of specific secondary structures in mRNAs termed selenocysteine insertion (SECIS) elements, and trans-acting factors that associate with SECIS elements. Herein, we discuss the various proteins known to function in eukaryotic selenoprotein biosynthesis, including several players whose roles have only been elucidated very recently.

A análise comparativa entre alongamento muscular sob eletroestimulação e inibição ativa em atletas amadores de futebol / Analysis comparative between muscles strenghtening electric stimulation and active inhibition in soccer athlethes

Na pesquisa foram utilizados duas técnicas de alongamento, a eletroestimulação e a inibição ativa, onde através dos resultados obtidos procurou verificar a eficácia das mesmas. Foram estudados 15 atletas da seleção universitária de futebol, divididos igualmente em 3 grupos, com idade entre 18 e 25 anos. O estudo foi aplicado em 3 etapas: grupo I, eletroestimulação com 10 Hz de freqüência, 250us de largura de pulso, durante 10 minutos, através de dois eletrodos instalados sobre o ventre muscular dos ísquios-tibiais; grupo II, inibição ativa com contração muscular dos ísquios-tibiais por 10s seguido por um alongamento mantido por 30s, durante 10 minutos; grupo III, para controle, no qual os voluntários permaneceram em decúbito dorsal durante 10 minutos. Os atletas foram avaliados através da goniometria inicial e final do quadril com joelho em extensão total. A análise dos resultados mostrou que nas comparações entre os instantes inicial e final, houve variação significativa nos grupos submetidos aos protocolos I e II; no grupo controle não houve variação, e ambos os grupos de forma superior ao grupo controle. na comparação entre grupos, a técnica de inibição ativa mostrou-se mais eficaz em relação ao grupo de eletroestimulação e ambos ao grupo controle. Conclui-se que as técnicas aplicadas foram eficientes no aumento do grau de amplitude articular dos atletas; contudo a técnica de inibição ativa foi mais eficaz.

Selenium-dependent pre- and posttranscriptional mechanisms are responsible for sexual dimorphic expression of selenoproteins in murine tissues.

Important enzymes for thyroid hormone metabolism, antioxidative defense, and intracellular redox control contain selenocysteine (Sec) in their active centers. Expression of these selenoproteins is tightly controlled, and a sex-specific phenotype is observed on disturbance of selenium (Se) transport in mice. Therefore, we analyzed Se concentrations and expression levels of several selenoproteins including type I iodothyronine deiodinase (Dio1) and glutathione peroxidase (GPx) isozymes in male and female mice. On regular lab chow, serum Se levels were comparable, but serum GPx3 activity was higher in females than males (1.3-fold). Selenoprotein P (SePP) mRNA levels were higher in livers (1.3-fold) and lower in kidneys (to 31%) in female compared with male mice. Orchidectomy alleviated the sex-specific differences in SePP mRNA amounts, indicating modulatory effects of androgens on SePP expression. Female mice expressed higher levels of Dio1 mRNA in kidney (2.6-fold) and liver (1.4-fold) in comparison with male mice. This sexual dimorphic expression of Dio1 mRNA was paralleled by increased Dio1 activity in female kidney (1.8-fold) but not in liver in which males expressed higher Dio1 activity (2.8-fold). Interestingly, Se deficiency decreased Dio1 activity more effectively in males than females, and resulting hepatic enzyme levels were then comparable between the sexes. At the same time, the sex-specific difference of Dio1 activity widened in kidney. Orchidectomy or estradiol treatment of ovariectomized females impacted stronger on renal than hepatic Dio1 expression. Thus, we conclude that Se-dependent posttranscriptional mechanisms are operational that affect either translational efficiency or Dio1 stability in a sex- and tissue-specific manner.

Characterization of a high-throughput screening assay for inhibitors of elongation factor p and ribosomal peptidyl transferase activity.

Elongation Factor P (EF-P) is an essential component of bacterial protein synthesis, enhancing the rate of translation by facilitating the addition of amino acids to the growing peptide chain. Using purified Staphylococcus aureus EF-P and a reconstituted Escherichia coli ribosomal system, an assay monitoring the addition of radiolabeled N-formyl methionine to biotinylated puromycin was developed. Reaction products were captured with streptavidin-coated scintillation proximity assay (SPA) beads and quantified by scintillation counting. Data from the assay were used to create a kinetic model of the reaction scheme. In this model, EF-P binding to the ribosome essentially doubled the rate of the ribosomal peptidyl transferase reaction. As described here, EF-P bound to the ribosomes with an apparent K(a) of 0.75 microM, and the substrates N-fMet-tRNA and biotinylated puromycin had apparent K(m)s of 19 microM and 0.5 microM, respectively. The assay was shown to be sensitive to a number of antibiotics known to target ribosomal peptide bond synthesis, such as chloramphenicol and puromycin, but not inhibitors that target other stages of protein synthesis, such as fusidic acid or thiostrepton.

Irreversible thermal denaturation of elongation factor Ts from Thermus thermophilus effect of the residual structure and intermonomer disulfide bond.

The homodimeric wild-type elongation factor Ts, EF-Ts(wt), and its C190A mutant, EF-Ts(C190A), from Thermus thermophilus goes through thermal denaturation in a way consistent with a two state irreversible model with a relatively high activation energy, approximately 530 kJ/mol (Supplemental materials provides a list of 98 activation energies from 54 proteins in various solvent conditions). Removing the intermonomeric disulfide bond by substituting alanine for cysteine 190 affects the rate constant of the irreversible thermal transition. At physiological temperatures, the half-life of the native conformations was estimated to be approximately 21 days for wt and 1.3 days for C190A. Thermally denatured EF-Ts refolds into a molten-globule-like state as indicated by its native-like circular dichroism spectrum in the far UV region and the enhanced fluorescence of the hydrophobic probe, 1-anilinonaphtalene-8-sulphonate. The residual secondary structure observed in the thermally denatured state of EF-Ts at high temperatures affects its apparent temperature of thermal transition, T(trs), independent of the presence or absence of the intermonomeric disulfide bond. The effect of the GdmHCl concentration on the activation energy, E(a), and the temperature, T*, i.e., the temperature at which the rate of the irreversible step is 1 min(-1), indicates that the intermonomeric disulfide bond contributes to the irreversibility of thermal transition of EF-Ts.

[Effects of the Radix Ranuncoli Ternati extracts on Mycobacterium tuberculosis proteome profiling revealed by 2D electrophoresis].

Radix Ranuncoli Ternati is clinically effective traditional Chinese medicine for multidrug resistant tuberculosis. Its active components and mechanism of action remain unsolved. Two dimensional gel electrophoresis (2-DE) was employed to address this problem. Globlal proteome of Mycobacterium tuberculosis untreated and treated with Radix Ranuncoli Ternati were compared, and 22 protein spots were found to be expressed differentially. 3 protein spots which remarkably decreased in Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati were subjected to matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. The data obtained from peptide mass finger printing were used for database search. The 3 protein spots in gel were identified as cysA2 (thiosulfate sulfurtransferase), tsf (elongation factor EF-Ts) and hspX (heat shock protein X). These data provide insights into the changed global protein patterns of Mycobacterium tuberculosis treated with Radix Ranuncoli Ternati and may prove useful for further study in the mechanism in how Radix Ranuncoli Ternati influence the life of Mycobacterium tuberculosis. The differentially expressed proteins may be potential novel antituberculosis drug targets.

Inactivation of the selB gene in Methanococcus maripaludis: effect on synthesis of selenoproteins and their sulfur-containing homologs.

The genome of Methanococcus maripaludis harbors genes for at least six selenocysteine-containing proteins and also for homologs that contain a cysteine codon in the position of the UGA selenocysteine codon. To investigate the synthesis and function of both the Se and the S forms, a mutant with an inactivated selB gene was constructed and analyzed. The mutant was unable to synthesize any of the selenoproteins, thus proving that the gene product is the archaeal translation factor (aSelB) specialized for selenocysteine insertion. The wild-type form of M. maripaludis repressed the synthesis of the S forms of selenoproteins, i.e., the selenium-independent alternative system, in selenium-enriched medium, but the mutant did not. We concluded that free selenium is not involved in regulation but rather a successional compound such as selenocysteyl-tRNA or some selenoprotein. Apart from the S forms, several enzymes from the general methanogenic route were affected by selenium supplementation of the wild type or by the selB mutation. Although the growth of M. maripaludis on H(2)/CO(2) is only marginally affected by the selB lesion, the gene is indispensable for growth on formate because M. maripaludis possesses only a selenocysteine-containing formate dehydrogenase.

Translation elongation-3-like factors: are they rational antifungal targets?

The occurrence of fungal infection has escalated significantly in recent years and is expected to continue to increase for the foreseeable future. Unfortunately, only a limited number of antifungal drugs are currently available partially due to a lack of suitable targets. The most commonly used antifungals target the same molecule in the cell membrane and, while efficacious, are either extremely toxic or susceptible to resistance. This article examines elongation factor-3, which is unique to fungi and essential for fungal cell survival and, thus, an attractive antifungal target. A search for inhibitors of this 'perfect target' led to identification of compounds (sordarins) which inhibited elongation factor-2, a protein with a mammalian homologue. Molecular analysis demonstrated why sordarins can specifically act against fungal elongation factor-2. This data questions the validity of pursuing genes as targets only if they are unique to fungi. Proteins that are homologous to elongation factor-3 are also discussed. The advances in molecular techniques and bioinformatics will allow the re-evaluation of targets previously thought to be unattractive. In addition, molecular genetics provides new and novel information on cellular processes that can potentially introduce new targets.

Distinctive features in the SelB family of elongation factors for selenoprotein synthesis. A glimpse of an evolutionary complexified translation apparatus.

The last ten years have seen a dramatic increase in our understanding of the molecular mechanism allowing specific incorporation of selenocysteine into selenoproteins. Whether in prokaryotes or eukaryotes, this incorporation requires several gene products, among which the specialized elongation factor SelB and the tRNA(Sec) play a pivotal role. While the molecular actors have been discovered and their role elucidated in the eubacterial machinery, recent data from our and other laboratories pointed to a higher degree of complexity in archaea and eukaryotes. These findings also revealed that more needs to be discovered in this area. This review will focus on phylogenetic aspects of the SelB proteins. In particular, we will discuss the concerted evolution that occurred within the SelB/tRNA(Sec) couples, and also the distinctive roles carried out by the SelB C-terminal domains in eubacteria on the one side, and archaea and eukaryotes, on the other.

Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes.

Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts.

Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha.

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

mRNA stability and selenocysteine insertion sequence efficiency rank gastrointestinal glutathione peroxidase high in the hierarchy of selenoproteins.

The recently described gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes glutathione peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical glutathione peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.

Effect of the components extracted from the needles of Taxus baccata on protein biosynthesis in a cell-free rat liver system.

Various species of Taxus contain taxanes that promote polymerization and stabilization of microtubules. They have been reported as antineoplastic compounds with highly effective chemiotherapeutic application. A decrease in incorporation of the radiolabelled precursors into DNA, RNA and proteins in vivo has been reported too. The preliminary results have shown that also the other compounds present in the aqueous extract from Taxus baccata needles, participate in the inhibition of the protein biosynthesis. The binding site of eEF-2 on the ribosome seems to be the target of this inhibition process.

Emerging targets for the development of novel antifungal therapeutics.

Invasive mycoses have become important causes of morbidity and mortality in immunocompromised patients. New approaches for antifungal therapy are required to meet the challenges imposed by these life-threatening infections. Such approaches are being developed through identification of novel biochemical and molecular targets of pathogenic fungi.