Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Composition and antifungal activity against Candida albicans, Candida parapsilosis, Candida krusei and Cryptococcus neoformans of essential oils from leaves of Piper and Peperomia species

This study was addressed to investigate the composition and antifungal activity of essential oils from leaves of Piperaceae species (Piper aduncum, Piper amalago, Piper cernuum, Piper diospyrifolium, Piper crassinervium, Piper gaudichaudianum, Piper solmsianum, Piper regnellii, Piper tuberculatum, Piper umbelata and Peperomia obtusifolia) against Candida albicans, C. parapsilosis, C. krusei and Cryptococcus neoformans. The essential oils from P. aduncum, P. gaudichaudianum and P. solmsianum showed the highest antifungal activity against Cryptococcus neoformans with the MIC of 62.5, 62.5 and 3.9 mg.mL-1, respectively. The oil from P. gaudichaudianum showed activity against C. krusei with MIC of 31.25 mg.mL-1.

Antifungal activity of tri- and tetra-thioureido amino derivatives against different Candida species

The in vitro antifungal activity of six thioureido substituted amines (P1-P6) was evaluated against Candida species, including Candida albicans, C. glabrata, C. krusei and C. parapsilosis. These tri- and tetra-thioureido amino derivatives with different methylation levels were synthesised through easy synthetic routes to evaluate their antifungal properties against Candida species. Among all studied derivatives, the tri-(2-thioureido-ethyl)-amine (P1) was the most active compound inhibiting C. albicans and C. glabrata at a concentration of 0.49 mu g ml(-1); P3, the N,N `,N ``,N ```-hexamethyl-derivative, also showed inhibitory activity against C. albicans and C. glabrata, but in higher concentrations (250 mu g ml(-1)). The N,N `,N ``,N ```-tetramethylated amine (P5) only inhibited the growth of C. glabrata, but its corresponding N,N `,N ``,N ```-octamethyl derivative (P6) was also active against C. glabrata (125 mu g ml(-1)) and it was the only compound active against C. parapsilosis. P2 and P4 showed no significant antifungal activity. The structure-activity relationship of the thioureido-substituted derivatives indicates that the molecular branching and the alkylation levels can influence the antifungal activity. This study demonstrated that thioureido derivatives exhibited significant antifungal activity against Candida species and that they can be considered as a very promising bioactive lead compound to develop novel antifungal agents.

Osmotin from Calotropis procera latex: New insights into structure and antifungal properties

This study aimed at investigating the structural properties and mechanisms of the antifungal action of CpOsm, a purified osmotin from Calotropis procera latex. Fluorescence and CD assays revealed that the CpOsm structure is highly stable, regardless of pH levels. Accordingly, CpOsm inhibited the spore germination of Fusarium solani in all pH ranges tested. The content of the secondary structure of CpOsm was estimated as follows: alpha-helix (20%), beta-sheet (33%), turned (19%) and unordered (28%). RMSD 1%. CpOsm was stable at up to 75 degrees C, and thermal denaturation (T(m)) was calculated to be 77.8 degrees C. This osmotin interacted with the negatively charged large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol (POPG), inducing vesicle permeabilization by the leakage of calcein. CpOsm induced the membrane permeabilization of spores and hyphae from Fusarium solani, allowing for propidium iodide uptake. These results show that CpOsm is a stable protein, and its antifungal activity involves membrane permeabilization, as property reported earlier for other osmotins and thaumatin-like proteins. (C) 2011 Elsevier B.V. All rights reserved.

Experimental paracoccidioidomycosis: alternative therapy with ajoene, compound from Allium sativum, associated with sulfamethoxazole/trimethoprim

Ajoene has been described as an antithrombotic, anti-tumour, antifungal, antiparasitic and antibacterial agent. This study deals with the efficacy of ajoene to treat mice intratracheally infected with Paracoccidioides brasiliensis. The results indicate that ajoene therapy is effective in association with antifungal drugs (sulfametoxazol/trimethoprim), showing a positive additive effect. Ajoene-treated mice developed Th1-type cytokine responses producing higher levels of IFN-gamma and IL-12 when compared to the infected but untreated members of the control group. Antifungal activity of ajoene involves a direct effect on fungi and a protective pro-inflammatory immune response. Reduction of fungal load is additive to chemotherapy and therefore the combined treatment is mostly effective against experimental paracoccidioidomycosis.

Molecular Characterization of Propolis-Induced Cell Death in Saccharomyces cerevisiae

Propolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in folk medicine for centuries. Here, we apply the power of Saccharomyces cerevisiae as a model organism for studies of genetics, cell biology, and genomics to determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochrome c but not endonuclease G (Nuc1p) is involved in propolis-mediated cell death in S. cerevisiae. We also observed that the metacaspase YCA1 gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required for propolis sensitivity in eukaryotes, the full collection of about 4,800 haploid S. cerevisiae deletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular response to starvation. Validation studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis.

Structural characterization of the interaction of the polyene antibiotic Amphotericin B with DODAB bicelles and vesicles

Amphotericin B (AmB) is widely used in the treatment of systemic fungal infections, despite its toxic effects. Nephrotoxicity, ascribed as the most serious toxic effect, has been related to the state of aggregation of the antibiotic. In search of the increase in AmB antifungal activity associated with low toxicity, several AmB-amphiphile formulations have been proposed. This work focuses on the structural characterization of a specific AmB formulation: AmB associated with sonicated dioctadecyl dimethylammonium bromide (DODAB) aggregates. Here, it was confirmed that sonicated DODAB dispersion is constituted by DODAB bicelles, and that monomeric AmB is much more soluble in bicelles than in DODAB vesicles. A new optical parameter is proposed for the estimation of the relative amount of amphiphile-bound monomeric AmB. With theoretical simulations of the spectra of spin labels incorporated in DODAB bicelles it was possible to prove that monomeric AmB binds preferentially to lipids located at the edges of DODAB bicelles, rigidifying them, and decreasing the polarity of the region. That special binding of monomeric AmB along the borders of bicelles, where the lipids are highly disorganized, could be used in the formulation of other carriers for the antibiotic, including mixtures of natural lipids which are known to form bicelles. (C) 2011 Elsevier B.V. All rights reserved.

Copper(II) and zinc(II) complexes with 2-benzoylpyridine-methyl hydrazone

2-Benzoylpyridine-methyl hydrazone (HBzMe) has been obtained as well as its copper(II) [Cu(HBzMe)Cl(2)] (1) and zinc(II) [Zn(HBzMe)Cl(2)] (2) complexes. Upon re-crystallization in 1 - 9 DMSO:acetone conversion of I into dimeric [Cu(BzMe)Cl](2) (1a) occurred. The crystal structures of HBzMe, 1, 1a, and 2 were determined. HBzMe adopts the ZE conformation in the solid. In all complexes the hydrazone adopts the E configuration to attach to the metal through the N(py)-N2-O chelating system. In 1 and 2 a neutral hydrazone coordinates to the metal center while in 1a deprotonation occurs with coordination of an anionic ligand. la presents a dimeric structure. having two copper(II) ions per asymmetric unit. Two chlorides are also present in the copper coordination sphere, which act as bridging ligands and connect the copper centers to each other. (C) 2008 Elsevier B.V. All rights reserved.

Ruthenium(II) complexes containing N(4)-tolyl-2-acetylpyridine thiosemicarbazones and phosphine ligands: NMR and electrochemical studies of cis-trans isomerization

[Ru(HL)(PPh3)(2)Cl]Cl complexes have been obtained in which HL = N(4)-ortho (complex 1), N(4)-meta (complex 2) and N(4) pctratolyl 2-acetylpyridine thiosemicarbazone (complex 3). NMR and electrochemical studies indicate that both cis and trans isomers exist in solution, and that the cis isomers are converted into the trans isomers with time. Crystal structure determination of (1) reveals that the traps isomer is formed in the solid state. (c) 2007 Elsevier B.V. All rights reserved.

Caldensinic acid, a prenylated benzoic acid from Piper caldense

The CH(2)Cl(2) and MeOH extracts from leaves of Piper caldense were subjected to chromatographic separation procedures to afford the new prenylated benzoic acid, caldensinic acid (3-[(2`E,6`E,10`E)-11`-carboxy-3`,7`,15`-trimethylhexadeca-2`,6`,10`,14`-tetraenyl]-4,5-dihydroxybenzoic acid) whose structure was determined by spectral analysis, mainly NMR ((1)H, (13)C, HSQC, HMBC) and ESI-MS. The natural compound and derivatives displayed antifungal activity against the phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum by direct bioautography. (C) 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Differential Acetyl Cholinesterase Inhibition by Volatile Oils from two Specimens of Marlierea racemosa (Myrtaceae) Collected from Different Areas of the Atlantic Rain Forest

The volatile oil composition and anti-acetyl cholinesterase activity were analyzed in two specimens of Marlierea racemosa growing in different areas of the Atlantic Rain Forest (Cananeia and Caraguatatuba, SP, Brazil). Component identifications were performed by GUMS and their acetyl cholinesterase inhibitory activity was measured through colorimetric analysis. The major constituent in both specimens was spathulenol (25.1 % in Cananeia and 31.9% in Caraguatatuba). However, the first one also presented monoterpenes (41.2%), while in the Carguatatuba plants, this class was not detected. The oils from the plants collected in Cananeia were able to inhibit the acetyl cholinesterase activity by LIP to 75%, but for oils from the other locality the maximal inhibition achieved was 35%. These results suggested that the monoterpenes are more effective in the inhibition of acetyl cholinesterase activity than sesquiterpenes as these compounds are present in higher amounts in the M. racemosa plants collected in Cananeia.

Isoswertisin flavones and other constituents from Peperomia obtusifolia

A phytochemical investigation of the leaves and stems of Peperomia obtusifolia (Piperaceae) yielded a new flavone C-diglycoside isoswertisin-4`-methyl-ether-2 ``alpha-L-rhamnoside (1), along with four known compounds: isoswertisin-2 ``alpha-L-rhamnoside (2), (+)-diayangambin (3), 2-episesalatin (4) and corchoionoside C (5). The structures of the two flavone C-diglycosides (1, 2) were elucidated on the basis of 1D and 2D NMR spectroscopy and MS spectrometric data. These flavones were evaluated by bioautographic assay against Cladosporium cladosporioides and C. sphaerospermum and showed weak antifungal activity.

Sesquiterpenes from Xylaria sp., an endophytic fungus associated with Piper aduncum (Piperaceae)

Two new presilphiperfolane sesquiterpenes, 1 and 2, were isolated from the ethyl acetate extract of Xylaria sp., obtained from the leaves of Piper aduncum, along with two known eremophilane sesquiterpenes, phaseolinone (3) and phomenone (4). Chemical structures of 1 and 2 were established by analysis of spectroscopic data. The four compounds were tested in vitro for antifungal and cytotoxicity activities using CHO (Chinese hamster ovary). Compounds 1 and 2 did not show any antifungal and cytotoxic activity. Compounds 3 and 4 displayed moderate cytotoxic activities, as well as 4 antifungal activity. (C) 2010 Phytochemical Society of Europe. Published by Elsevier B. V. All rights reserved.

In Vitro Activity of the Antifungal Plant Defensin RsAFP2 against Candida Isolates and Its In Vivo Efficacy in Prophylactic Murine Models of Candidiasis

We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.

Correlation of the in vitro antifungal drug susceptibility with the in vivo activity of fluconazole in a murine model of cerebral infection caused by Cryptococcus gattii

Forty Cryptococcus gattii strains were submitted to antifungal susceptibility testing with fluconazole, itraconazole, amphotericin B and terbinafine. The minimum inhibitory concentration (MIC) ranges were 0.5-64.0 for fluconazole, < 0.015-0.25 for itraconazole, 0.015-0.5 for amphotericin B and 0.062-2.0 for terbinafine. A bioassay for the quantitation of fluconazole in murine brain tissue was developed. Swiss mice received daily injections of the antifungal, and their brains were withdrawn at different times over the 14-day study period. The drug concentrations varied from 12.98 to 44.60 mu g/mL. This assay was used to evaluate the therapy with fluconazole in a model of infection caused by C. gattii. Swiss mice were infected intracranially and treated with fluconazole for 7, 10 or 14 days. The treatment reduced the fungal burden, but an increase in fungal growth was observed on day 14. The MIC for fluconazole against sequential isolates was 16 mu g/mL, except for the isolates obtained from animals treated for 14 days (MIC = 64 mu g/mL). The quantitation of cytokines revealed a predominance of IFN-gamma and IL-12 in the non-treated group and elevation of IL-4 and IL-10 in the treated group. Our data revealed the possibility of acquired resistance during the antifungal drug therapy.