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1.
Fitoterapia ; 155: 105056, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34626738

RESUMO

Three new meroterpenoid derivatives, furanocochlioquinol (1) and furanocochlioquinone (2), as well as nectrianolin D (3), together with two known biogenetically related compounds 4 and 5 were isolated from a mixed culture of two mangrove-derived fungi, Clonostachys rosea B5-2 and Nectria pseudotrichia B69-1. The structures of 1-3 were deduced based on the interpretation of HRMS and NMR data. Compounds 1-5 exhibited cytotoxicity against human promyelocytic leukemia (HL60) cells with IC50 values ranging from 0.47 to 10.16 µM.


Assuntos
Antineoplásicos/farmacologia , Hypocreales/química , Nectria/química , Rhizophoraceae/microbiologia , Terpenos/farmacologia , Antineoplásicos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Técnicas de Cocultura , Endófitos/química , Células HL-60 , Humanos , Indonésia , Estrutura Molecular , Terpenos/isolamento & purificação
2.
Sci Rep ; 10(1): 5597, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221468

RESUMO

During recent years, a new disease of Siberian fir (A. sibirica) emerged in Central Siberia, exhibiting symptoms of stem/branch deformation, cambium necrosis, and dieback of branches and twigs, the causal agent remaining unknown. The aim was to identify agent of the disease and to investigate its pathogenicity to A. sibirica and Norway spruce (Picea abies). Symptomatic tissues of fir were subjected to pure culture isolation of anticipated pathogen(s). Obtained isolates were subjected to molecular identification, phylogenetic analyses, and pathogenicity tests with A. sibirica saplings, and seeds and seedlings of A. sibirica and P. abies. The study demonstrated that, (i) most commonly isolated fungus from canker wounds of A. sibirica exhibited Acremonium-like anamorphs; (ii) phylogeny demonstrated that investigated fungi belong to genus Corinectria, but are genetically well separated from other worldwide known Corinectria spp.; (iii) one species of isolated fungi has the capacity to cause the disease and kill A. sibirica saplings and seedlings, but also seedlings of P. abies. Guidelines for future research were defined in order to generate needed information on species description, its origin and ecology, and estimation of potential risks upon the eventual invasion of the pathogen to new geographic areas, in particular of Europe.


Assuntos
Abies/microbiologia , Nectria/patogenicidade , Doenças das Plantas/microbiologia , Microscopia Eletrônica de Varredura , Nectria/genética , Nectria/ultraestrutura , Filogenia , Sibéria
3.
Phytopathology ; 110(4): 723-725, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31913742

RESUMO

Thelonectria rubi is the causal agent of Nectria canker of Rubus spp. Here, we report a high-quality draft genome sequence for this pathogen, which also represents the first genome sequence for a Thelonectria species. The genome assembly was 44.6 Mb in size, assembled into 669 scaffolds and consisting of 12,973 predicted protein-coding genes. The availability of genome data for T. rubi provides a critical additional resource for an important plant pathogen and will be useful for fungal biology, comparative genomic, taxonomic and population studies of this and related species.


Assuntos
Hypocreales , Nectria , Rubus , Genômica , Doenças das Plantas
4.
Nat Commun ; 10(1): 4972, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672969

RESUMO

Both lipid and ion translocation by Ca2+-regulated TMEM16 transmembrane proteins utilizes a membrane-exposed hydrophilic groove. Several conformations of the groove are observed in TMEM16 protein structures, but how these conformations form, and what functions they support, remains unknown. From analyses of atomistic molecular dynamics simulations of Ca2+-bound nhTMEM16 we find that the mechanism of a conformational transition of the groove from membrane-exposed to occluded from the membrane involves the repositioning of transmembrane helix 4 (TM4) following its disengagement from a TM3/TM4 interaction interface. Residue L302 is a key element in the hydrophobic TM3/TM4 interaction patch that braces the open-groove conformation, which should be changed by an L302A mutation. The structure of the L302A mutant determined by cryogenic electron microscopy (cryo-EM) reveals a partially closed groove that could translocate ions, but not lipids. This is corroborated with functional assays showing severely impaired lipid scrambling, but robust channel activity by L302A.


Assuntos
Anoctaminas/metabolismo , Cálcio/metabolismo , Cloretos/metabolismo , Proteínas Fúngicas/metabolismo , Fosfolipídeos/metabolismo , Anoctaminas/ultraestrutura , Transporte Biológico , Microscopia Crioeletrônica , Proteínas Fúngicas/ultraestrutura , Interações Hidrofóbicas e Hidrofílicas , Transporte de Íons , Simulação de Acoplamento Molecular , Nectria , Conformação Proteica
5.
Int J Mol Sci ; 20(21)2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31661772

RESUMO

Our previous research has shown that a fungal immunomodulatory protein from Nectria haematococca (FIP-nha) possesses a wide spectrum of anti-tumor activities, and FIP-nha induced A549 apoptosis by negatively regulating the PI3K/Akt signaling pathway based on comparative quantitative proteomics. This study further confirmed that the anti-lung cancer activity of FIP-nha was significantly stronger than that of the reported LZ-8 and FIP-fve. Subsequently, 1H NMR-based metabolomics was applied to comprehensively investigate the underlying mechanism, and a clear separation of FIP-nha-treated and untreated groups was achieved using pattern recognition analysis. Four potential pathways associated with the anti-tumor effect of FIP-nha on A549 cells were identified, and these were mainly involved in glycolysis, taurine and hypotaurine metabolism, fructose and mannose metabolism, and glycerolipid metabolism. Metabolic pathway analysis demonstrated that FIP-nha could induce A549 cell apoptosis partly by regulating the p53 inhibition pathway, which then disrupted the Warburg effect, as well as through other metabolic pathways. Using RT-PCR analysis, FIP-nha-induced apoptosis was confirmed to occur through upregulation of p53 expression. This work highlights the possible use of FIP-nha as a therapeutic adjuvant for lung cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Fúngicas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Nectria , Proteína Supressora de Tumor p53/metabolismo , Células A549 , Antineoplásicos/uso terapêutico , Vias Biossintéticas , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/uso terapêutico , Células HEK293 , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
6.
J Phys Chem Lett ; 10(20): 6351-6354, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31566982

RESUMO

The cell membrane and many organellar membranes are asymmetric and highly curved. In experiments, it is challenging to reconstitute and characterize membranes that differ in the lipid composition of their leaflets. Here we use molecular dynamics simulations to study the large-scale membrane shape changes associated with lipid shuttling between asymmetric leaflets. We exploit leaflet asymmetry to create a stable, near-spherical vesicle bud connected to a flat bilayer under periodic boundary conditions. Then we demonstrate how the lipid scramblase nhTMEM16 relaxes the lipid-number asymmetry. By mediating the flipping of lipids, this transmembrane protein dissipates the mechanochemical gradient between the leaflets and drives a large-scale membrane reorganization, converting the vesicle bud into a flat membrane. Our procedure to exploit bilayer asymmetry for simulations of highly curved membranes can be used to study the function of other lipid transporters and membrane-shaping proteins.


Assuntos
Proteínas Fúngicas/química , Bicamadas Lipídicas/química , Proteínas de Transferência de Fosfolipídeos/química , Simulação de Dinâmica Molecular , Nectria/química , Fosfatidilcolinas/química
7.
Biochim Biophys Acta Gen Subj ; 1863(6): 1070-1078, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936018

RESUMO

BACKGOUND: Xylan is the second most abundant plant cell wall polysaccharide after cellulose with α-L-arabinofuranose (L-Araf) as one of the major side substituents. Capacity to degrade xylan is characteristic of many plant pathogens; and corresponding enzymes that debranch arabinoxylan provide tools to tailor xylan functionality or permit its full hydrolysis. METHOD: Three GH62_2 family α-arabinofuranosidases (Abfs) from plant pathogenic fungi, NhaAbf62A from Nectria haematococca, SreAbf62A from Sporisorium reilianum and GzeAbf62A from Gibberella zeae, were recombinantly produced in Escherichia coli. Their biochemical properties and substrate specificities were characterized in detail. Particularly with 1H NMR, the regioselectivity and debranching preference of the three Abfs were directly compared. RESULTS: The activities of selected Abfs towards arabinoxylan were all optimal at pH 6.5. Their preferred substrates were wheat arabinoxylan, followed by soluble oat spelt xylan. The Abfs displayed selectivity towards either α-(1 → 2) or α-(1 → 3)-L- Araf mono-substituents in arabinoxylan. Specifically, SreAbf62A and GzeAbf62A removed m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substituents with a similar rates, whereas NhaAbf62A released m-α-(1 → 3)-L-Araf 1.9 times faster than m-α-(1 → 2)-L-Araf. MAJOR CONCLUSIONS: Building upon the known selectivity of GH62 family α-arabinofuranosidases towards L-Araf mono-substituents in xylans, the current study uncovers enzyme-dependent preferences towards m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substitutions. Comparative sequence-structure analyses of Abfs identified an arginine residue in the xylose binding +2R subsite that was correlated to the observed enzyme-dependent L-Araf debranching preferences. GENERAL SIGNIFICANCE: This study expands the limited pool of characterized GH62 Abfs particularly those from plant pathogenic fungi, and provides biochemical details and methodology to evaluate regioselectivity within this glycoside hydrolase family.


Assuntos
Proteínas Fúngicas/química , Gibberella/enzimologia , Glicosídeo Hidrolases/química , Nectria/enzimologia , Xilanos/química , Cinética , Doenças das Plantas/microbiologia , Especificidade por Substrato
8.
J Asian Nat Prod Res ; 21(10): 961-969, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29911892

RESUMO

Two new cyclohexanone derivatives, nectriatones A-B (1-2), and one new natural product, nectriatone C (3), together with three known phenolic sesquiterpene derivatives (4-6), were isolated from the culture of Nectria sp. B-13 obtained from high-latitude soil of the Arctic. The structures of all compounds were unambiguously elucidated by extensive spectroscopic analysis, as well as by comparison with the literature. These compounds were evaluated in cytotoxic and antibacterial activities. Compounds 1-6 showed cytotoxicities against SW1990, HCT-116, MCF-7, and K562 cells, with IC50 values in the range of 0.43 to 42.64 µM. Only compound 4 exhibited antibacterial activity against Escherichisa coli, Bacillus subtilis, and Staphylococcus aureus (MIC 4.0, 2.0, and 4.0 µg/ml, respectively).


Assuntos
Nectria/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antibióticos Antineoplásicos/isolamento & purificação , Antibióticos Antineoplásicos/farmacologia , Regiões Árticas , Bactérias/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Sesquiterpenos/química , Microbiologia do Solo
9.
Int J Mol Sci ; 19(11)2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388826

RESUMO

Lung cancer is a common disease that is associated with poor prognosis. Fungal immunomodulatory protein from Nectria haematococca (FIP-nha) has potential as a lung cancer therapeutic; as such, illuminating its anti-tumor mechanism is expected to facilitate novel treatment options. Here, we showed that FIP-nha affects lung adenocarcinoma growth ex vivo and in vivo. Comparative quantitative proteomics showed that FIP-nha negatively regulates PI3K/Akt signaling and induces cell cycle arrest, autophagy, and apoptosis. We further demonstrated that FIP-nha suppresses Akt phosphorylation, leading to upregulation of p21 and p27 and downregulation of cyclin B1, cyclin D1, CDK2, and CDK4 expression, ultimately resulting in G1/S and G2/M cell cycle arrest. Meanwhile, FIP-nha-induced PI3K/Akt downregulation promotes A549 apoptosis by increasing the expression ratio of Bax/Bcl-2 and c-PARP and autophagy by decreasing the phosphorylation of mTOR. Thus, we comprehensively revealed the anti-tumor mechanism of FIP-nha, which inhibits tumor growth by modulating PI3K/Akt-regulated cell cycle arrest, autophagy, and apoptosis, and provided the basis for further application of fungal immunomodulatory proteins, especially FIP-nha.


Assuntos
Adenocarcinoma de Pulmão/patologia , Proteínas Fúngicas/farmacologia , Fatores Imunológicos/farmacologia , Nectria/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células A549 , Adenocarcinoma de Pulmão/ultraestrutura , Animais , Apoptose/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Proteômica , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Biol Chem ; 293(47): 18318-18327, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30287690

RESUMO

Members of the G protein-coupled receptor and TMEM16 (transmembrane protein 16) protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner. On reconstitution into large unilamellar vesicles, these proteins scramble more than 10,000 lipids/protein/s as measured with co-reconstituted fluorescent nitrobenzoxadiazole (NBD)-labeled phospholipids. Although NBD-labeled phospholipids are ubiquitously used as reporters of scramblase activity, it remains unclear whether the NBD modification influences the quantitative outcomes of the scramblase assay. We now report a refined biochemical approach for measuring the activity of scramblase proteins with radiolabeled natural phosphatidylinositol ([3H]PI) and exploiting the hydrolytic activity of bacterial PI-specific phospholipase C (PI-PLC) to detect the transbilayer movement of PI. PI-PLC rapidly hydrolyzed 50% of [3H]PI in large symmetric, unilamellar liposomes, corresponding to the lipid pool in the outer leaflet. On reconstitution of a crude preparation of yeast endoplasmic reticulum scramblase, purified bovine opsin, or purified Nectria haematococca TMEM16, the extent of [3H]PI hydrolysis increased, indicating that [3H]PI from the inner leaflet had been scrambled to the outer leaflet. Using transphosphatidylation, we synthesized acyl-NBD-PI and used it to compare our PI-PLC-based assay with conventional fluorescence-based methods. Our results revealed quantitative differences between the two assays that we attribute to the specific features of the assays themselves rather than to the nature of the phospholipid. In summary, we have developed an assay that measures scrambling of a chemically unmodified phospholipid by a reconstituted scramblase.


Assuntos
Anoctaminas/metabolismo , Proteínas Fúngicas/metabolismo , Opsinas/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Anoctaminas/química , Anoctaminas/genética , Transporte Biológico , Bovinos , Fluorescência , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hidrólise , Cinética , Nectria/enzimologia , Opsinas/química , Opsinas/genética , Fosfatidilinositóis/química , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipases Tipo C/química , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
11.
J Biol Chem ; 293(31): 12248-12258, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-29903908

RESUMO

Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca2+-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse physiological processes. The nhTMEM16 scramblase (from the fungus Nectria hematococca) is a homodimer with a large cytoplasmic region and a hydrophilic, membrane-exposed groove in each monomer. The groove provides the transbilayer conduit for lipids, but the mechanism by which Ca2+ regulates it is not clear. Because fusion of large protein tags at either the N or C terminus abolishes nhTMEM16 activity, we hypothesized that its cytoplasmic portion containing both termini may regulate lipid translocation via a Ca2+-dependent conformational change. To test this hypothesis, here we used fluorescence methods to map key distances within the nhTMEM16 homodimer and between its termini and the membrane. To this end, we developed functional nhTMEM16 variants bearing an acyl carrier protein (ACP) tag at one or both of the termini. These constructs were fluorescently labeled by ACP synthase-mediated insertion of CoA-conjugated fluorophores and reconstituted into vesicles containing fluorescent lipids to obtain the distance of closest approach between the labeled tag and the membrane via FRET. Fluorescence lifetime measurements with phasor analysis were used to determine the distance between the N and C termini of partnering monomers in the nhTMEM16 homodimer. We now report that the measured distances do not vary significantly between Ca2+-replete and EGTA-treated samples, indicating that whereas the cytoplasmic portion of the protein is important for function, it does not appear to regulate scramblase activity via a detectable conformational change.


Assuntos
Anoctaminas/química , Anoctaminas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Bicamadas Lipídicas/metabolismo , Nectria/enzimologia , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/genética , Transporte Biológico , Cálcio/metabolismo , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Dimerização , Fluorescência , Proteínas Fúngicas/genética , Bicamadas Lipídicas/química , Nectria/química , Nectria/genética , Proteínas de Transferência de Fosfolipídeos/genética
12.
Fitoterapia ; 127: 356-361, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29621598

RESUMO

Four new compounds, namely, nectriapyrones A (2) and B (3), nectriaquinone B (5), and zythiostromic acid C (8), were isolated from the brown rice culture of Nectria pseudotrichia 120-1NP together with four known compounds (1, 4, 6, and 7). To the best of our knowledge, this is the first report of 4 from a natural source. Their structures were determined on the basis of 1D/2D-NMR spectroscopy and HRESITOFMS data. In addition, the absolute configuration of secondary alcohols in 8 were determined using modified Mosher's ester method. All isolated compounds were evaluated for their antimicrobials activity, phytotoxicity, and cytotoxicity.


Assuntos
Diterpenos/isolamento & purificação , Isocumarinas/isolamento & purificação , Naftoquinonas/isolamento & purificação , Nectria/química , Células HL-60 , Humanos , Alface/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular
13.
PLoS One ; 13(2): e0192653, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29420618

RESUMO

Kievitone hydratase catalyzes the addition of water to the double bond of the prenyl moiety of plant isoflavonoid kievitone and, thereby, forms the tertiary alcohol hydroxy-kievitone. In nature, this conversion is associated with a defense mechanism of fungal pathogens against phytoalexins generated by host plants after infection. As of today, a gene sequence coding for kievitone hydratase activity has only been identified and characterized in Fusarium solani f. sp. phaseoli. Here, we report on the identification of a putative kievitone hydratase sequence in Nectria haematococca (NhKHS), the teleomorph state of F. solani, based on in silico sequence analyses. After heterologous expression of the enzyme in the methylotrophic yeast Pichia pastoris, we have confirmed its kievitone hydration activity and have assessed its biochemical properties and substrate specificity. Purified recombinant NhKHS is obviously a homodimeric glycoprotein. Due to its good activity for the readily available chalcone derivative xanthohumol (XN), this compound was selected as a model substrate for biochemical studies. The optimal pH and temperature for hydratase activity were 6.0 and 35°C, respectively, and apparent Vmax and Km values for hydration of XN were 7.16 µmol min-1 mg-1 and 0.98 ± 0.13 mM, respectively. Due to its catalytic properties and apparent substrate promiscuity, NhKHS is a promising enzyme for the biocatalytic production of tertiary alcohols.


Assuntos
Hidroliases/metabolismo , Nectria/enzimologia , Sequência de Aminoácidos , Reatores Biológicos , Glicosilação , Hidroliases/química , Hidroliases/genética , Hidroliases/isolamento & purificação , Cinética , Ressonância Magnética Nuclear Biomolecular , Pichia/genética , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta
14.
Mem Inst Oswaldo Cruz ; 113(2): 102-110, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29236928

RESUMO

BACKGROUND In a screen of extracts from plants and fungi to detect antileishmanial activity, we found that the ethyl acetate extract of the fungus Nectria pseudotrichia, isolated from the tree Caesalpinia echinata (Brazilwood), is a promising source of bioactive compounds. OBJECTIVES The aims of this study were to isolate and determine the chemical structures of the compounds responsible for the antileishmanial activity of the organic extract from N. pseudotrichia. METHODS Compounds were isolated by chromatographic fractionation using semi-preparative high-performance liquid chromatography, and their chemical structures were determined by analytical and spectral data and by comparison with published data. The antileishmanial activity of the isolated compounds was evaluated in intracellular amastigote forms of Leishmania (Viannia) braziliensis expressing firefly luciferase as reporter gene, and cytotoxicity was determined in Vero and THP-1 mammalian cell lines by MTT assay. FINDINGS Fractionation of the extract yielded seven compounds: 10-acetyl trichoderonic acid A (1), 6'-acetoxy-piliformic acid (2), 5',6'-dehydropiliformic acid (3), piliformic acid (4), hydroheptelidic acid (5), xylaric acid D (6), and cytochalasin D (7). Compounds 1, 2 and 3 are reported here for the first time. Compounds 1, 2, and 5 were more active, with IC50 values of 21.4, 28.3, and 24.8 µM, respectively, and showed low toxicity to Vero and THP-1 cells. MAIN CONCLUSIONS N. pseudotrichia produces secondary metabolites that are more toxic to intracellular amastigote forms of L. (V.) braziliensis than to mammalian cells.


Assuntos
Caesalpinia/microbiologia , Leishmania braziliensis/efeitos dos fármacos , Nectria/química , Tripanossomicidas/farmacologia , Animais , Sobrevivência Celular , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Concentração Inibidora 50 , Testes de Sensibilidade Parasitária , Testes de Toxicidade , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/toxicidade , Células Vero
15.
Mar Drugs ; 14(5)2016 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-27136568

RESUMO

Four new polyketides: nectriacids A-C (1-3) and 12-epicitreoisocoumarinol (4), together with three known compounds: citreoisocoumarinol (5), citreoisocoumarin (6), and macrocarpon C (7) were isolated from the culture of the endophytic fungus Nectria sp. HN001, which was isolated from a fresh branch of the mangrove plant Sonneratia ovata collected from the South China Sea. Their structures were determined by the detailed analysis of NMR and mass spectroscopic data. The absolute configuration of the stereogenic carbons for compound 4 was further assigned by Mosher's ester method. All of the isolated compounds were tested for their α-glucosidase inhibitory activity by UV absorbance at 405 nm, and new compounds 2 and 3 exhibited potent inhibitory activity with IC50 values of 23.5 and 42.3 µM, respectively, which were more potent than positive control (acarbose, IC50, 815.3 µM).


Assuntos
Endófitos/química , Fungos/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Nectria/química , Policetídeos/farmacologia , Rhizophoraceae/microbiologia , alfa-Glucosidases/metabolismo , China , Inibidores de Glicosídeo Hidrolases/química , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Policetídeos/química
16.
Mitochondrial DNA A DNA Mapp Seq Anal ; 27(6): 4670-4671, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27159696

RESUMO

The complete nucleotide sequence of the mitochondrial genome of the important phytopathogic fungus Nectria cinnabarina was determined using the next-generation sequencing technology. The circular molecule is 69 895 bp long with a GC content of 28.71%. Gene prediction revealed 42 genes encoding 15 conserved proteins, 25 tRNAs, the large and small ribosomal RNAs. All genes are located on the same strand. Compared with previously sequenced mitochondrial genomes of the other members of Nectriaceae, the composition and order of the protein and rRNA genes are highly conserved; however, the quantity and order of tRNA genes are different. The phylogenetic analysis confirmed N. cinnabarina as a basal lineage in Nectriaceae. The mitochondrial genome of N. cinnabarina will contribute to the understanding of phylogeny and evolution of Nectriaceae and Hypocreales.


Assuntos
Genes Fúngicos , Genes Mitocondriais , Genoma Mitocondrial , Nectria/genética , Composição de Bases , Evolução Molecular , Nectria/classificação , Filogenia , Análise de Sequência de DNA
17.
Fungal Biol ; 119(10): 929-939, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26399187

RESUMO

PDA1-conditionally dispensable chromosome (CDC) of Nectria haematococca MP VI has long served as a model of supernumerary chromosomes in plant pathogenic fungi because of pathogenicity-related genes located on it. In our previous study, we showed the dosage effects of PDA1-CDC on pathogenicity and homoserine utilization by exploiting tagged PDA1-CDC with a marker gene. CDC content of mating partners and progenies analyzed by PCR, PFGE combined with Southern analysis and chromosome painting via FISH. In this study, we analyzed mode of meiotic inheritance of PDA1-CDC in several mating patterns with regard to CDC content and found a correlation between CDC content of parental strains with fertility of crosses. The results showed non-Mendelian inheritance of this chromosome followed by duplication or loss of the CDC in haploid genome through meiosis that probably were due to premature centromere division, not by nondisjunction as reported for the supernumerary chromosomes in other species. Correlation of CDC with fertility is the first time to be examined in fungi in this study.


Assuntos
Cromossomos Fúngicos , Meiose , Nectria/genética , Nectria/fisiologia , Poliploidia , Testamentos , Cruzamentos Genéticos
18.
J Asian Nat Prod Res ; 17(6): 633-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26001271

RESUMO

Two new monoterpenoid α-pyrones, named nectriapyrones C and D (1 and 2), along with a known α-pyrone (nectriapyrone, 3) were isolated from a marine-derived fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa collected from the South China Sea. Their structures were determined on the basis of 1D NMR, 2D NMR, HR-ESI-MS methods.


Assuntos
Monoterpenos/isolamento & purificação , Nectria/química , Poríferos/microbiologia , Pironas/isolamento & purificação , Animais , Biologia Marinha , Estrutura Molecular , Monoterpenos/química , Ressonância Magnética Nuclear Biomolecular , Pironas/química
19.
Mol Biotechnol ; 57(5): 466-74, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25652193

RESUMO

The application of arylacetonitrilases from filamentous fungi to the hydrolysis of high concentrations of (R,S)-mandelonitrile (100-500 mM) was demonstrated for the first time. Escherichia coli strains expressing the corresponding genes were used as whole-cell catalysts. Nitrilases from Aspergillus niger, Neurospora crassa, Nectria haematococca, and Arthroderma benhamiae (enzymes NitAn, NitNc, NitNh, and NitAb, respectively) exhibited different degrees of enantio- and chemoselectivity (amide formation). Their enantio- and chemoselectivity was increased by increasing pH (from 8 to 9-10) and adding 4-10% (v/v) toluene as the cosolvent. NitAn and NitNc were able to convert an up to 500 mM substrate in batch mode. NitAn formed a very low amount of the by-product, amide (<1% of the total product). This enzyme produced up to >70 g/L of (R)-mandelic acid (e.e. 94.5-95.6%) in batch or fed-batch mode. Its volumetric productivities were the highest in batch mode [571 ± 32 g/(L d)] and its catalyst productivities in fed-batch mode (39.9 ± 2.5 g/g of dcw). NitAb hydrolyzed both enantiomers of 100 mM (R,S)-mandelonitrile at pH 5.0 and is therefore promising for the enantioretentive transformation of (S)-mandelonitrile. Sequence analysis suggested that fungal arylacetonitrilases with similar properties (enantioselectivity, chemoselectivity) were clustered together.


Assuntos
Aminoidrolases/química , Aminoidrolases/metabolismo , Ácidos Mandélicos/metabolismo , Aminoidrolases/genética , Arthrodermataceae/enzimologia , Aspergillus niger/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Nectria/enzimologia , Neurospora crassa/enzimologia , Filogenia , Especificidade da Espécie
20.
Appl Microbiol Biotechnol ; 99(6): 2613-21, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25267157

RESUMO

Compared to (S)-selective amine transaminase ((S)-AT), the (R)-selective counterpart ((R)-AT) has been less studied. As such, a simplified "Motif Sequence Blast" search (Höhne et al. Nat Chem Biol 6:807-813, 2010) was carried out to identify new (R)-ATs from the protein databases. The combined conserved sequence motifs of (R)-ATs based on the previous in silico method of predicting (R)-selective amine transaminase were used as the template sequence for BLASTP search at default settings in NCBI, and six candidate sequences were identified. These putative (R)-AT genes were synthesized and overexpressed in Escherichia coli. Among them, five new (R)-ATs were expressed as soluble protein and showed unusual substrate specificity and high stereoselectivity. Furthermore, several unnatural amino acids, such as D-alanine, D-2-aminobutyric acid, and D-norvaline, were synthesized via the (R)-AT-catalyzed amino transfer reaction to the corresponding keto acids. Optically pure (S)-amines were also obtained by kinetic resolution of racemic amines catalyzed with these new (R)-ATs. Therefore, the Motif Sequence Blast search offers a quick and effective method for in silico identification of new (R)-ATs, and the newly identified (R)-ATs are attractive additions to the toolbox of (R)-ATs for further study and industrial application.


Assuntos
Transaminases/genética , Transaminases/metabolismo , Alanina/metabolismo , Aminobutiratos/metabolismo , Cordyceps/genética , Cordyceps/metabolismo , Bases de Dados de Proteínas , Fusarium/genética , Fusarium/metabolismo , Cetoácidos/metabolismo , Nectria/genética , Nectria/metabolismo , Estrutura Terciária de Proteína , Especificidade por Substrato , Trichoderma/genética , Trichoderma/metabolismo , Valina/análogos & derivados , Valina/metabolismo
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