RESUMO
Polyphenols are natural compounds with promising prophylactic and therapeutic applications. However, their methods of extraction, using organic solvents, may prove to be unsuitable for daily consumption or for certain medical indications. Here, we describe the neuroprotective effects of grape polyphenols extracted in an eco-sustainable manner in a rat model of neonatal hypoxia-ischemia (NHI). Polyphenols (resveratrol, pterostilben and viniferin) were obtained using a subcritical water extraction technology to avoid organic solvents and heavy metals associated with chemical synthesis processes. A resveratrol or a polyphenol cocktail were administered to pregnant females at a nutritional dose and different time windows, prior to induction of NHI in pups. Reduced brain edema and lesion volumes were observed in rat pups whose mothers were supplemented with polyphenols. Moreover, the preservation of motor and cognitive functions (including learning and memory) was evidenced in the same animals. Our results pave the way to the use of polyphenols to prevent brain lesions and their associated deficits that follow NHI, which is a major cause of neonatal death and disabilities.
Assuntos
Hipóxia-Isquemia Encefálica , Fármacos Neuroprotetores , Vitis , Animais , Animais Recém-Nascidos , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/prevenção & controle , Isquemia/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Polifenóis/farmacologia , Polifenóis/uso terapêutico , Gravidez , Ratos , Vitis/químicaRESUMO
The present work describes the isolation and anticancer activity of Tapinanthus sp. which is a hemi parasitic plant harvested on Combretum glutinosum, the host plant. Phytochemical study afforded a new flavonoid glycoside, tapinantoside (1) isolated for the first time from natural source, alongside six known compounds (2-7). Structure of compounds were elucidated by extensive spectroscopic analyses including 1 D and 2 D NMR, mass spectrometry and by comparison with literature data. The anticancer activity of extract and some isolated compounds were evaluated on glioblastoma (U87MG, C6) and prostate (PC-3) cancer cells. The methanol leaves extract showed good anticancer activity against U87 (IC50 = 21.40 µg/mL) and PC-3 cells (IC50 = 10.26 µg/mL). Compound 3 powerfully inhibits the proliferation of C6 (IC50 = 38.84 µM) and PC-3 cells (IC50 = 21.33 µM), while its effect was moderated on U87MG cells. Compound 1 and 7 were not active on all tested cancer cell lines.
Assuntos
Glicosídeos Cardíacos , Loranthaceae , Flavonoides/química , Flavonoides/farmacologia , Glicosídeos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de PlantaRESUMO
The polyphenol trans-ε-viniferin (viniferin) is a dimer of resveratrol, reported to hold antioxidant and anti-inflammatory properties. The aims of our study were to evaluate the neuroprotective potential of viniferin in the nerve growth factor (NGF)-differentiated PC12 cells, a dopaminergic cellular model of Parkinson's disease (PD) and assess its anti-inflammatory properties in a N9 microglia-neuronal PC12 cell co-culture system. The neuronal cells were pre-treated with viniferin, resveratrol or their mixture before the administration of 6-hydroxydopamine (6-OHDA), recognized to induce parkinsonism in rats. Furthermore, N9 microglia cells, in a co-culture system with neuronal PC12, were pre-treated with viniferin, resveratrol or their mixture to investigate whether these polyphenols could reduce lipopolysaccharide (LPS)-induced inflammation. Our results show that viniferin as well as a mixture of viniferin and resveratrol protects neuronal dopaminergic cells from 6-OHDA-induced cytotoxicity and apoptosis. Furthermore, when viniferin, resveratrol or their mixture was used to pre-treat microglia cells in our co-culture system, they reduced neuronal cytotoxicity induced by glial activation. Altogether, our data highlight a novel role for viniferin as a neuroprotective and anti-inflammatory molecule in a dopaminergic cellular model, paving the way for nutraceutical therapeutic avenues in the complementary treatments of PD.
RESUMO
Ischemia reperfusion injury is a complex process consisting of a seemingly chaotic but actually organized and compartmentalized shutdown of cell function, of which oxidative stress is a key component. Studying oxidative stress, which results in an imbalance between reactive oxygen species (ROS) production and antioxidant defense activity, is a multi-faceted issue, particularly considering the double function of ROS, assuming roles as physiological intracellular signals and as mediators of cellular component damage. Herein, we propose a comprehensive overview of the tools available to explore oxidative stress, particularly in the study of ischemia reperfusion. Applying chemistry as well as biology, we present the different models currently developed to study oxidative stress, spanning the vitro and the silico, discussing the advantages and the drawbacks of each set-up, including the issues relating to the use of in vitro hypoxia as a surrogate for ischemia. Having identified the limitations of historical models, we shall study new paradigms, including the use of stem cell-derived organoids, as a bridge between the in vitro and the in vivo comprising 3D intercellular interactions in vivo and versatile pathway investigations in vitro. We shall conclude this review by distancing ourselves from "wet" biology and reviewing the in silico, computer-based, mathematical modeling, and numerical simulation options: (a) molecular modeling with quantum chemistry and molecular dynamic algorithms, which facilitates the study of molecule-to-molecule interactions, and the integration of a compound in a dynamic environment (the plasma membrane...); (b) integrative systemic models, which can include many facets of complex mechanisms such as oxidative stress or ischemia reperfusion and help to formulate integrated predictions and to enhance understanding of dynamic interaction between pathways.
Assuntos
Modelos Animais de Doenças , Estresse Oxidativo , Traumatismo por Reperfusão/metabolismo , Animais , Linhagem Celular , Humanos , Modelos Moleculares , Espécies Reativas de OxigênioRESUMO
Capitalizing on a recently reported iminosugar-based aza-crown (ISAC) accessed by a double Staudinger azaWittig coupling reaction, we have expanded the structural diversity of this new family of sweet cyclam analogs. Replacement of the two secondary amines linking the iminosugar units by two amide bonds obtained a cyclodimerization by with BOP and DIPEA led to a macrocycle that did not demonstrate efficient Zn2+ chelation unlike the parent ISAC. Introduction of two pyrene moieties on the secondary amines of the parent ISAC yielded a new fluoroionophore that selectively binds Hg2+ in methanol.
Assuntos
Quelantes/síntese química , Coronantes/química , Corantes Fluorescentes/síntese química , Glicopeptídeos/síntese química , Imino Açúcares/química , Quelantes/química , Corantes Fluorescentes/química , Glicopeptídeos/química , Conformação MolecularRESUMO
In a previous study, we showed that viniferin decreased amyloid deposits and reduced neuroinflammation in APPswePS1dE9 transgenic mice between 3 and 6 months of age. In the present study, wild type and APPswePS1dE9 transgenic mice were treated from 7 to 11 or from 3 to 12 months by a weekly intraperitoneal injection of either 20 mg/kg viniferin or resveratrol or their vehicle, the polyethylene glycol 200 (PEG 200). The cognitive status of the mice was evaluated by the Morris water maze test. Then, amyloid burden and neuroinflammation were quantified by western-blot, Enzyme-Linked ImmunoSorbent Assay (ELISA), immunofluorescence, and in vivo micro-Positon Emission Tomography (PET) imaging. Viniferin decreased hippocampal amyloid load and deposits with greater efficiency than resveratrol, and both treatments partially prevented the cognitive decline. Furthermore, a significant decrease in brain uptake of the TSPO PET tracer [18F]DPA-714 was observed with viniferin compared to resveratrol. Expression of GFAP, IBA1, and IL-1ß were decreased by viniferin but PEG 200, which was very recently shown to be a neuroinflammatory inducer, masked the neuroprotective power of viniferin.
RESUMO
Through superacid activation, N-(arenesulfonyl)-aminoalcohols derived from readily available ephedrines or amino acids undergo an intramolecular Friedel-Crafts reaction to afford enantiopure benzosultams bearing two adjacent stereocenters in high yields with fully controlled diastereoselectivity. Low-temperature NMR spectroscopy demonstrated the crucial role played by the conformationally restricted chiral dicationic intermediates.
RESUMO
A new family of chiral C2 symmetric tetraazamacrocycles, coined ISAC for IminoSugar Aza-Crown, incorporating two iminosugars adopting a 4C1 conformation is disclosed. Multinuclear NMR experiments on the corresponding Cd2+ complex show that the ISAC is a strong chelator in water and its tetramine cavity adopts a conformation similar to that of the parent Cd-cyclam complex. Similar behavior is observed with Cu2+ in solution, with enhanced stability compared to the Cu-cyclam complex.
RESUMO
BACKGROUND AND PURPOSE: Ischemia-reperfusion injury is encountered in numerous processes such as cardiovascular diseases or kidney transplantation; however, the latter involves cold ischemia, different from the warm ischemia found in vascular surgery by arterial clamping. The nature and the intensity of the processes induced by ischemia types are different, hence the therapeutic strategy should be adapted. Herein, we investigated the protective role of tannic acid, a natural polyphenol in a rat model reproducing both renal warm ischemia and kidney allotransplantation. The follow-up was done after 1 week. EXPERIMENTAL APPROACH: To characterize the effect of tannic acid, an in vitro model of endothelial cells subjected to hypoxia-reoxygenation was used. KEY RESULTS: Tannic acid statistically improved recovery after warm ischemia but not after cold ischemia. In kidneys biopsies, 3h after warm ischemia-reperfusion, oxidative stress development was limited by tannic acid and the production of reactive oxygen species was inhibited, potentially through Nuclear Factor erythroid-2-Related factor 2 (NRF2) activation. In vitro, tannic acid and its derivatives limited cytotoxicity and the generation of reactive oxygen species. Molecular dynamics simulations showed that tannic acid efficiently interacts with biological membranes, allowing efficient lipid oxidation inhibition. Tannic acid also promoted endothelial cell migration and proliferation during hypoxia. CONCLUSIONS: Tannic acid was able to improve renal recovery after renal warm ischemia with an antioxidant effect putatively extended by the production of its derivatives in the body and promoted cell regeneration during hypoxia. This suggests that the mechanisms induced by warm and cold ischemia are different and require specific therapeutic strategies.
Assuntos
Rim , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismo por Reperfusão , Taninos/farmacologia , Animais , Modelos Animais de Doenças , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Testes de Função Renal , Ratos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologiaRESUMO
Candida albicans can form biofilm on tissues and medical devices, becoming, in that case, less susceptible to antifungal agents. Treatment of candidiasis associated with the formation of C. albicans biofilms is restricted to echinocandins and lipid forms of amphotericin B. This study investigated the activity of micafungin and resveratrol modified molecule (EB487) against C. albicans biofilms. The anti-biofilm growth (Bgrowth) and anti-preformed biofilm (Bpreformed) activities of micafungin (0 to 3.94 µM) and EB487 (0 to 20.32 mM) were comparatively studied separately and combined, using XTT, flow cytometry and cell counts approaches. Concentrations causing 50% inhibition of the studied steps (IC50) were evaluated. When tested separately, IC50 Bgrowth was obtained for 4.8 mM and 0.13 µM of EB487 and micafungin respectively, and IC50 Bpreformed for 3.6 mM and 0.06 µM of EB487 and micafungin respectively. Micafungin used alone was not able to totally eradicate fungi. Micafungin combined with EB487 displayed synergistic activity (both anti-growth- and anti-preformed biofilm-activities). Optimal combination concentrations were EB487 (≤9.12 mM -strain ATCC 28367™ or ≤8.12 mM -strain CAI4-p), micafungin (≤0.05 µM for both) and caused a total eradication of fungi. Dose reduction indexes obtained using these concentrations were at least 9 (micafungin) and 3.2 (EB487) for both anti-biofilm growth- and anti-preformed biofilm-activities. Combinations indexes were consistently below one, demonstrating a synergistic relationship between micafungin and EB487 in these conditions. This study demonstrated the strong anti-biofilm activity of EB487 and highlighted its synergistic potential when combined with micafungin. EB487 is a promising semi-synthetic molecule with prophylactic and curative interests in fighting C. albicans biofilms.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Resveratrol/farmacologia , Antifúngicos/síntese química , Biofilmes/crescimento & desenvolvimento , Candida albicans/classificação , Sinergismo Farmacológico , Concentração Inibidora 50 , Micafungina/farmacologia , Testes de Sensibilidade Microbiana , Estudo de Prova de Conceito , Resveratrol/síntese químicaRESUMO
As Alzheimer's disease (AD) induces several cellular and molecular damages, it could be interesting to use multi-target molecules for therapeutics. We previously published that trans ε-viniferin induced the disaggregation of Aß42 peptide and inhibited the inflammatory response in primary cellular model of AD. Here, effects of this stilbenoid were evaluated in transgenic APPswePS1dE9 mice. We report that trans ε-viniferin could go through the blood brain barrier, reduces size and density of amyloid deposits and decreases reactivity of astrocytes and microglia, after a weekly intraperitoneal injection at 10 mg/kg from 3 to 6 months of age.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Benzofuranos/uso terapêutico , Inflamação/tratamento farmacológico , Placa Amiloide/tratamento farmacológico , Estilbenos/uso terapêutico , Doença de Alzheimer/patologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Benzofuranos/farmacocinética , Modelos Animais de Doenças , Feminino , Inflamação/patologia , Masculino , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/patologia , Placa Amiloide/patologia , Estilbenos/farmacocinéticaRESUMO
Mounting evidence indicates free radicals as toxic species causing damage to human cells leading to the pathogenesis of many diseases such as neurodegenerative disease. Plant derived antioxidants are considered as promising strategy to prevent free radical toxicity. In this study, the crude extract (CE), 50%MeOH, Petroleum Ether (PE) and Ethyl acetate (EA) fractions of Lawsonia inermis leaves were investigated for their antioxidant activity and their ability to counteract amyloid-ß42 (Aß42) aggregation. Elution of the most bioactive fraction (EA) on silica gel column chromatography led to six sub-fractions. The most active sub-fraction (1) was further resolved on silica gel column chromatography. A new compound with powerful antioxidant and anti-Aß42 aggregation properties was purified and characterised by spectroscopic methods as 1,2,4-trihydroxynaphthalene-2-O-ß-D-glucopyranoside (THNG). This finding suggests that the antioxidant and anti-Aß42 aggregation activities of L. inermis leaves are strongly correlated to this compound.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Antioxidantes/farmacologia , Lawsonia (Planta)/química , Naftalenos/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Extratos Vegetais/farmacologia , Folhas de Planta/química , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/química , Antioxidantes/isolamento & purificação , Radicais Livres , Humanos , Naftalenos/química , Naftalenos/isolamento & purificação , Fragmentos de Peptídeos/metabolismoRESUMO
Mounting evidence indicates soluble Aß42 oligomers as the most toxic species causing neuronal death which leads to the onset and progression of Alzheimer disease (AD). Recently, it has been found that neurotoxic Aß42 oligomers grow from monomeric species or arise following secondary nucleation by preformed mature fibrils. Thus, the use of natural compounds such as polyphenols to hinder the growth or to remodel Aß42 fibrils is one of the most promising strategies for AD treatment. In our previous study, we showed that 1, 2, 4-trihydroxynaphthalene-2-O-ß-d-glucopyranoside (THNG) inhibits Aß42 aggregation during the early steps of the aggregation process, inhibits its conformational change to a ß-sheet-rich structure, decreases its polymerization, inhibits its fibrillogenisis and reduces oxidative stress and aggregate cytotoxicity. Here, we used different spectroscopic and cell culture methods to check the effect of THNG on fibrils disaggregation. We showed that THNG binds to mature Aß42 fibrils, rearrange their secondary structure, and remodels them into non-amyloid, less toxic, species by inhibiting their interaction with the plasma membrane. Our findings reveal that THNG is a good agent to remodel amyloid fibrils and could be used as a starting molecular scaffold to design new anti-AD drugs.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/efeitos dos fármacos , Lawsonia (Planta)/química , Fragmentos de Peptídeos/química , Amiloide/toxicidade , Peptídeos beta-Amiloides/toxicidade , Linhagem Celular Tumoral , Dicroísmo Circular , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Gangliosídeo G(M1)/análise , Humanos , Microdomínios da Membrana , Microscopia Eletrônica , Estrutura Molecular , Neuroblastoma/patologia , Fragmentos de Peptídeos/toxicidade , Folhas de Planta/químicaRESUMO
Presently, misfolding and aggregation of amyloid-ß42 (Aß42 ) are considered early events in Alzheimer's disease (AD) pathogenesis. The use of natural products to inhibit the aggregation process and to protect cells from cytotoxicity of early aggregate grown at the onset of the aggregation path is one of the promising strategies against AD. Recently, we have purified a new powerful antioxidant and inhibitor of Aß42 aggregation from the leaves of Lawsonia inermis. The new compound was identified as a new Lawsoniaside; 1,2,4-trihydroxynaphthalene-2-O-ß-D-glucopyranoside (THNG). Herein, we show that THNG interferes with Aß42 aggregation, inhibits its conformational change to a ß-sheet-rich structure, decreases its polymerization into large fibrillar species, reduces oxidative stress, and aggregate cytotoxicity. These results indicate that THNG has great potential as a neuroprotective and therapeutic agent against AD. © 2018 BioFactors, 44(3):272-280, 2018.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Glucosídeos/farmacologia , Lawsonia (Planta)/química , Naftalenos/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/farmacologia , Benzotiazóis , Cálcio/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Glucosídeos/química , Glucosídeos/isolamento & purificação , Humanos , Naftalenos/química , Naftalenos/isolamento & purificação , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Extratos Vegetais/química , Agregados Proteicos/efeitos dos fármacos , Conformação Proteica em Folha beta , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Fluorescência , Tiazóis/químicaRESUMO
Alzheimer's disease (AD) is marked by several cellular and molecular damage. Therefore, the therapeutic interest of multi-target molecules is increasingly justified. Polyphenols presenting multiple pharmacological effects would be more efficient. In this study, beneficial effects of trans ε-viniferin, a natural polyphenol were thus evaluated. This study reported that this stilbenoid (1) induced the disaggregation of amyloid ß (Aß) peptide and (2) rescued inflammation in murine primary neuronal cultures. These both effects are higher than those of resveratrol, and so, trans ε-viniferin could be a good therapeutic multi-target candidate.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Anti-Inflamatórios/uso terapêutico , Benzofuranos/uso terapêutico , Neurônios/efeitos dos fármacos , Estilbenos/uso terapêutico , Animais , Células Cultivadas , Modelos Animais de Doenças , Camundongos , Neurônios/metabolismoRESUMO
This work describes the synthesis and biological evaluation of a new heterocyclic hybrid derived from the ellipticine and the marine alkaloid makaluvamine A. Pyridoquinoxalinedione 12 was obtained in seven steps with 6.5% overall yield. 12 and its intermediates 1-11 were evaluated for their in vitro cytotoxic activity against different cancer cell lines and tested for their inhibitory activity against the human DNA topoisomerase II. The analysis by electrophoresis shows that the pentacycle 12 inhibits the topoisomerase II like doxorubicine at 100 µM. Compound 9 was found to have an interesting profile, having a cytotoxicity of 15, 15, 15 and 10 µM against Caco-2, HCT-116, Pc-3 and NCI cell lines respectively, without any noticeable toxicity against human fibroblast.
Assuntos
Antineoplásicos/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Células CACO-2/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Desenho de Fármacos , Células HCT116/efeitos dos fármacos , Humanos , Estrutura Molecular , Pirróis/química , Quinolonas/química , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologiaRESUMO
trans-Resveratrol (1a) is a phytoalexin produced by plants in response to infections by pathogens. Its potential activity against clinically relevant opportunistic fungal pathogens has previously been poorly investigated. Evaluated herein are the candidacidal activities of oligomers (2a, 3-5) of 1a purified from Vitis vinifera grape canes and several analogues (1b-1j) of 1a obtained through semisynthesis using methylation and acetylation. Moreover, trans-ε-viniferin (2a), a dimer of 1a, was also subjected to methylation (2b) and acetylation (2c) under nonselective conditions. Neither the natural oligomers of 1a (2a, 3-5) nor the derivatives of 2a were active against Candida albicans SC5314. However, the dimethoxy resveratrol derivatives 1d and 1e exhibited antifungal activity against C. albicans with minimum inhibitory concentration (MIC) values of 29-37 µg/mL and against 11 other Candida species. Compound 1e inhibited the yeast-to-hyphae morphogenetic transition of C. albicans at 14 µg/mL.
Assuntos
Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Estilbenos/farmacologia , Vitis/química , Antifúngicos/química , Benzofuranos/química , Benzofuranos/farmacologia , Candida albicans/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Resveratrol , Estereoisomerismo , Estilbenos/químicaRESUMO
The mirror image of natural product (+)-adenophorine along with its 1-epi-, 1-homo-analogs and other derivatives have been synthesized and evaluated as glycosidase inhibitors. The synthetic strategy is based on the skeletal rearrangement of tetrahydroxylated C-alkyl azepanes obtained via a Staudinger/azaWittig/alkylation sequence starting from a sugar-derived azidolactol. Several organometallic species have been investigated for the alkylation step including organomagnesium, organolithium, organozinc, organoaluminum and organocerium reagents. While diallylzinc proved to be the most efficient to introduce an allyl substituent, disappointing results were obtained with the other organometallic species leading either to lower yields or no reaction. Enzymatic assays indicate that (-)-adenophorine is a moderate α-l-fucosidase inhibitor.
Assuntos
Compostos Aza/síntese química , Inibidores Enzimáticos/síntese química , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Açúcares/química , Alquilação , Animais , Aspergillus niger/enzimologia , Compostos Aza/química , Compostos Aza/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glicosídeo Hidrolases/metabolismo , Imino Açúcares/síntese química , Imino Açúcares/metabolismo , Oryza/enzimologia , Ligação Proteica , Ratos , Saccharomyces cerevisiae/enzimologia , Estereoisomerismo , alfa-L-Fucosidase/antagonistas & inibidores , alfa-L-Fucosidase/metabolismoRESUMO
Candida guilliermondii is an interesting biotechnological model for the industrial production of value-added metabolites and also remains an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both an ATCC 6260-derived recipient strain and a recyclable selection marker. We first disrupted the TRP5 gene in the wild-type strain and demonstrated that trp5 mutants were tryptophan auxotroph as well as being resistant to the antimetabolite 5-fluoroanthranilic acid (FAA). Following an FAA selection of spontaneous mutants derived from the ATCC 6260 strain and complementation analysis, we demonstrated that trp5 genotypes could be directly recovered on FAA-containing medium. The TRP5 wild-type allele, flanked by two short repeated sequences of its 3'UTR, was then used to disrupt the FCY1 gene in C. guilliermondii trp5 recipient strains. The resulting fcy1 mutants displayed strong flucytosine resistance and a counter-selection on FAA allowed us to pop-out the TRP5 allele from the FCY1 locus. To illustrate the capacity of this blaster system to achieve a second round of gene disruption, we knocked out both the LEU2 and the HOG1 genes in the trp5, fcy1 background. Although all previously described yeast "TRP blaster" disruption systems used TRP1 as counter-selectable marker, this study demonstrated the potential of the TRP5 gene in such strategies. This newly created "TRP5 blaster" disruption system thus represents a powerful genetic tool to study the function of a large pallet of genes in C. guilliermondii.
Assuntos
Candida/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Família Multigênica , Canais de Cátion TRPC/genética , ortoaminobenzoatos/metabolismo , Candida/metabolismo , Proteínas Fúngicas/metabolismo , Marcadores Genéticos , Mutação , Canais de Cátion TRPC/metabolismoRESUMO
A flexible synthetic access to six-membered L- and D-iminosugar C-glycosides is reported starting from the easily available 6-azido-6-deoxy-2,3,4-tri-O-benzyl-D-glucopyranose precursor. This methodology involves a highly diastereoselective tandem ring enlargement/alkylation and a stereocontrolled ring contraction. It allows an efficient synthesis of iminosugar C-glycosides displaying structural diversity at both C-1 and C-6.