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Avenacina is a hydrolytic enzyme that can degrade the oat saponin avenacine, found and was first recognized as an essential pathogenicity factor in the take all fungus Gaeumannomyces graminis var. avenae. Saponins, gly cosides Inhibitors,Modulators,Libraries with soap like properties that disrupt mem branes, are a class of phytoanticipins. Inhibitors,Modulators,Libraries The role of saponin detoxification remains controversial in other plant pathogen interactions. However, the saponin degrading tomatinase from F. oxysporum f. sp. lycopersici has recently been confirmed as a virulence factor in Inhibitors,Modulators,Libraries tomato, by targeted disruption and over expression of the corresponding gene. In melon, we found that the avenacinase transcript is not only expressed specifically in planta, but is also differen tially expressed between the two 1,2 strains, with higher levels produced by ISPaVe1018.
Inhibitors,Modulators,Libraries To our knowl edge, this is the first evidence to support a role for saponin detoxifying enzymes in FOM infection. The siderophore iron transporter mirB gene may also represent a virulence factor because siderophores are crucial for fungal pathogenicity in both animals and plants, and also maintain plant fungal symbioses. The final group of FOM genes expressed only in planta includes several involved in transport and intracellular trafficking, and three related to signal transduction, with similarity to a calnexin involved in calcium regulated protein folding, a phosphoserine phosphatase and a MADS box protein. Although expressed both in planta and in vitro, a per oxisomal biogenesis factor PEX11 and an arginase coding gene are also worth mentioning.
Peroxisomes are single membrane bound organelles which, in fila mentous fungi, are involved in the b oxidation of fatty acids, peroxide detoxification and the occlusion of septal pores. Peroxisomal function Inhibitors,Modulators,Libraries and fatty acid metabo lism are required for fungal virulence. In F. oxysporum, four different Pex genes were identified as potential pathogenicity genes in a recent insertional mutagenesis screen, and the requirement for full pathogenicity was verified for two of them by complementation with the intact genes. Arginase regulates the production of nitric oxide, which is induced in a jasmonate dependent manner in response to wounding and is strongly implicated in the activation of disease resistance genes. In microorganisms, arginase activity has been correlated with pathogenicity and was shown to act as a bacterial survival mechanism by downregulat ing host nitric oxide production.
Other transcripts expressed by FOM in planta, specifically or otherwise, are involved in ubiquitinylation and protein degradation, Pazopanib chemical structure both of which are necessary for pathogenicity in F. oxy sporum f. sp. lycopersici, and in different aspects of fungal metabolism. Differentially expressed genes among F. oxysporum f. sp. melonis strains in vitro One major problem in FOM diagnosis is the identifica tion of isolates at the race level.
Saponin was used as positive control. Efficacy to protect against AAPH induced ROS generation Seliciclib The ability of crude extracts and polyphenolic rich fractions to attenuate Inhibitors,Modulators,Libraries AAPH induced ROS generation was mea sured using the 2, 7 dichlorodihydrofluorescein diacetate method as described by Jakubowski and Bartosz. Into pre seeded U937 white plates was pipetted a 20 ul medium, crude extract, polyphenolic rich fraction or 1 mM Trolox and 5 uM DCFHDA, which was incubated for 1 h at 37?C and 5% CO2. Plates were washed with PBS and treated with 1. 5 mM AAPH. Fluorescence was mea sured over a period of 3 h at ex 485 nm and em 520 nm. Percentage inhibition was determined using the following equation where, AUC average area under curve of AAPH exposed cells. AUC average area under curve of sample treated, AAPH exposed cells.
Efficacy to protect against AAPH induced apoptosis The ability of crude extracts and polyphenolic rich frac tions to attenuate AAPH induced apoptosis was mea sured using the caspase 3 activity assay as described by Banjerdpongchai et al. Staurosporine was employed Inhibitors,Modulators,Libraries as positive control. Pre seeded U937 AAPH exposed plates were centrifuged, medium replaced with 25 ul cold Inhibitors,Modulators,Libraries lysis buffer and incubated for 15 min on ice. Thereafter, a 100 ul caspase 3 substrate buffer containing Ac DEVD AMC was added and plates were incubated for 3 h at 37?C. Fluorescence was mea sured at ex 355 nm and em 460 nm. Efficacy to protect against AAPH induced lipid peroxidation The ability of crude extracts and polyphenolic rich frac tions to attenuate AAPH induced lipid peroxidation was measured using the thiobarbituric acid assay as described by Stern et al.
Hydrogen peroxide was used as positive control. From pre seeded U937 AAPH exposed plates were Inhibitors,Modulators,Libraries taken aliquots of supernatant, which were mixed with 200 ul trichloroacetic acid and 400 ul TBA and in cubated at 95?C for 20 min. 3 Methyl butan 1 ol was added to the mixture, vortex mixed and the organic layer was left to separate from the aqueous layer. Into a white 96 well plate was transferred 100 ul of the organic layer and the fluorescence measured at ex 544 nm and em 590 nm. Efficacy to protect against AAPH induced GSH depletion The ability of crude extracts and polyphenolic rich frac tions to attenuate AAPH induced GSH depletion was measured using the monochlorobimane assay as de scribed by Fernandez Checa and Kaplowitz.
H2O2 was used as positive control. Into pre seeded U937 AAPH exposed plates was pipetted Inhibitors,Modulators,Libraries 50 uM monochlorobimane and plates were incubated for 1 h. Plates were regardless washed twice with PBS after which the fluorescence was measured at ex 355 nm and em 460 nm. Statistical analyses All experiments were performed in triplicate on three separate days and results expressed as mean SEM using GraphPad Prism 4.
Inhibitors,Modulators,Libraries The group of putative virulence fac tors identified in our analysis included plant cell wall degrading enzymes, represented by five tran scripts encoding pectate lyases, endo 1,4 beta xylanases and endo 1,4 beta glucanases, possibly activated by interaction with the host. Among these transcripts, an endo 1,4 beta xylanase 2 precursor is the only sequence peculiar to race 1, induced in the incompatible interac tion, while the other four TDFs are specific Inhibitors,Modulators,Libraries to the race 1,2 strains. Like most fungi, F. oxysporum secretes CWDEs during either penetration or colonization. Although the inactivation of individual CWDE or pro tease encoding genes might not have a detectable impact on virulence, possibly because of functional redundancy, their activity is crucial in the process of fungal colonization.
Active fungal growth is also documented by the specific in planta expression of several genes related to carbohydrate and lipid metabo lism, among them a squalene synthase involved in sterol biosynthesis. Sterols facilitate normal membrane func tion Inhibitors,Modulators,Libraries by controlling their fluidity, Inhibitors,Modulators,Libraries but they have also been implicated as ligands for nuclear receptors directly affecting transcription and signal transduction pathways. Other examples include genes for cytoskeleton components and a chitin synthase gene. Class V chitin synthase is a pathogenicity determinant in F. oxysporum and a mediator of protection against plant defense compounds. Three other in planta specific TDFs seem particularly important in terms of virulence. These represent genes encoding homologs of an avenacinase, a fumonisin 16p, and a siderophore iron transporter.
There is increasing evidence that mycotoxin production may enhance pathogen virulence, especially fumonisins and some trichothecenes. Fumonisin enhances the abil ity of F. graminearum to cause wheat head blight, one of the most important wheat kinase inhibitor Gefitinib diseases in the world. It has been reported that mycotoxin production can be induced in fungi following the perception of the oxida tive burst produced by the plant in response to infec tion, and could enhance pathogenicity by reducing the oxidative status of the fungal cell.
The U937 human pro monocytic cell line was obtained from European Collection of Cell Cultures. Non adherent U937 cells were cultured www.selleckchem.com/products/PD-0332991.html in 10% FCS supplemented Inhibitors,Modulators,Libraries Roswell Park Memorial Institution 1640 with penicillin and strepto mycin at 37?C and 5% CO2. Cells were washed, counted using the trypan blue ex clusion assay and diluted to 1 106 cells ml in 10% FCS supplemented RPMI 1640. Cells were differentiated for 48 h with 32 nM phorbol 12 myristate 13 acetate at 37?C and 5% CO2. Cells were harvested and recounted using the trypan blue exclusion assay after differentiation and diluted to 1 106 cells ml in 2% FCS supplemented RPMI 1640. Into a 96 well plate was pipetted a 100 ul cell suspen sion and 80 ul 2% FCS supplemented medium. Plates were incubated at 37?C and 5% CO2 for 1 h or 24 h for non adherent or adherent cell lines, respectively.
Cytotoxicity of crude extracts and polyphenolic Inhibitors,Modulators,Libraries rich fractions Cytotoxicity was determined using the neutral red up take assay as described by Borenfreund et al. The final concentration of ethanol used in the cellular assays Inhibitors,Modulators,Libraries for the methanolic extract and polyphenolic rich fraction did not exceed 0. 1%. The cytotoxicity of crude extracts and polyphenolic rich samples was determined in pre seeded plates by addition of 20 ul medium, crude extracts or polyphenolic rich fractions and incubation for 72 h at 37?C and 5% CO2. Medium was replaced with 100 ul neutral red medium and incubated for 3 h after which plates were washed with PBS. Plates were left to dry, the dye dissolved using 100 ul neutral red eluent and the absorbance measured at 540 nm.
Attenuation of oxidative stress induced parameters in U937 cells The oxidant 2,2 azobis dihydro chloride is able to generate free radicals such as hydroxyls during thermolysis reactions. During generation of ROS, cells undergo cytotoxicity that can be detected as GSH depletion, Inhibitors,Modulators,Libraries apoptosis and lipid peroxida tion. These parameters can be measured using fluoromet ric and spectrophotometric assays. Induction of AAPH induced oxidative stress Into pre seeded U937 plates was pipetted 20 ul medium, Inhibitors,Modulators,Libraries positive control, crude extract, polyphenolic rich fraction or 10 mM Trolox and incubated for 1 h at 37 C and 5% CO2. In all experiments, except the ROS generation assays de scribed in Section Efficacy to protect against AAPH induced ROS generation, plates were washed with RPMI 1640, treated with 1. 5 mM AAPH and incubated for 48 h at 37?C and 5% CO2. All values were adjusted by subtraction of the blank. The results for percentage viability, apoptosis, lipid per oxidation and GSH depletion were expressed relative to the negative control using the following equation where, A intensity Imatinib Mesylate purchase of triplicate negative con trol. A triplicate intensity of sample at a given concentration.