Origin of the Giant Enhanced Raman Scattering by Sulfur Chains Encapsulated inside Single-Wall Carbon Nanotubes.

In this work, we explain the origin and the mechanism responsible for the strong enhancement of the Raman signal of sulfur chains encapsulated by single-wall carbon nanotubes by running resonance Raman measurements in a wide range of excitation energies for two nanotube samples with different diameter distributions. The Raman signal associated with the vibrational modes of the sulfur chain is observed when it is confined by small-diameter metallic nanotubes. Moreover, a strong enhancement of the Raman signal is observed for excitation energies corresponding to the formation of excited nanotube-chain-hybrid electronic states. Our hypothesis was further tested by high pressure Raman measurements and confirmed by density functional theory calculations of the electronic density of states of hybrid systems formed by sulfur chains encapsulated by different types of single-wall carbon nanotubes.

PvD1 defensin, a plant antimicrobial peptide with inhibitory activity against Leishmania amazonensis.

Plant defensins are small cysteine-rich peptides and exhibit antimicrobial activity against a variety of both plant and human pathogens. Despite the broad inhibitory activity that plant defensins exhibit against different micro-organisms, little is known about their activity against protozoa. In a previous study, we isolated a plant defensin named PvD1 from Phaseolus vulgaris (cv. Pérola) seeds, which was seen to be deleterious against different yeast cells and filamentous fungi. It exerted its effects by causing an increase in the endogenous production of ROS (reactive oxygen species) and NO (nitric oxide), plasma membrane permeabilization and the inhibition of medium acidification. In the present study, we investigated whether PvD1 could act against the protozoan Leishmania amazonensis. Our results show that, besides inhibiting the proliferation of L. amazonensis promastigotes, the PvD1 defensin was able to cause cytoplasmic fragmentation, formation of multiple cytoplasmic vacuoles and membrane permeabilization in the cells of this organism. Furthermore, we show, for the first time, that PvD1 defensin was located within the L. amazonensis cells, suggesting the existence of a possible intracellular target.

Characterization of Peptides from Capsicum annuum Hybrid Seeds with Inhibitory Activity Against α-Amylase, Serine Proteinases and Fungi.

Over the last several years, the activity of antimicrobial peptides (AMPs), isolated from plant species, against different microorganisms has been demonstrated. More recently, some of these AMPs have been described as potent inhibitors of α-amylases and serine proteinases from insects and mammals. The aim of this work was to obtain AMPs from protein extracts of a hybrid Capsicum (Ikeda × UENF 1381) seeds and to evaluate their microbial and enzyme inhibitory activities. Initially, proteins were extracted from the Capsicum hybrid seeds in buffer (sodium phosphate pH 5.4,) and precipitated with ammonium sulfate (90% saturated). Extract of hybrid seeds was subjected to size exclusion chromatography, and three fractions were obtained: S1, S2 and S3. The amino acid sequence, obtained by mass spectrometry, of the 6 kDa peptide from the S3 fraction, named HyPep, showed 100% identity with PSI-1.2, a serine protease inhibitor isolated from C. annuum seeds, however the bifunctionality of this inhibitor against two enzymes is being shown for the first time in this work. The S3 fraction showed the highest antifungal activity, inhibiting all the yeast strains tested, and it also exhibited inhibitory activity against human salivary and Callosobruchus maculatus α-amylases as well as serine proteinases.

The toxicity of a lipid transfer protein (Cc-LTP1) from Coffea canephora Seeds on the larval development of Callosobruchus maculatus (Coleoptera: Bruchidae).

In this work, we analyzed the effects of coffee seed proteins, especially Cc-LTP1 on the larval development of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), a bruchid pest of beans and the most important insect pest of Vigna unguiculata (L.) Walp. Artificial seed assay, which incorporated the F/0-90 fraction from Coffea canephora seeds, resulted in the reduction of oviposition and caused an inhibition of C. maculatus larval development in a dose-dependent manner. The F/0-90 fraction used at a 4 % concentration resulted in the survival of no larvae. The purified Cc-LTP1, at a concentration of 0.5 %, also demonstrated effective inhibition of larval development, reducing both females oviposition and the weight and number of larvae. Cc-LTP1 was also able to inhibit the C. maculatus gut α-amylase activity, and immunolabeling by an anti-LTP serum was observed in the midgut tissues of the C. maculatus larvae. Cc-LTP1 has shown binding affinity towards microvillar cells, endoplasmic reticulum and mitochondria, as demonstrated by micrographic images taken by a transmission electron microscope. The results from this study indicate that Cc-LTP1 has insecticidal actions toward C. maculatus and exerts anti-nutritional effects with direct actions on intestinal tissues.

Vicilin-like peptides from Capsicum baccatum L. seeds are α-amylase inhibitors and exhibit antifungal activity against important yeasts in medical mycology.

The objective of this study was to isolate antimicrobial peptides from Capsicum baccatum seeds and evaluate their antimicrobial activity and inhibitory effects against α-amylase. Initially, proteins from the flour of C. baccatum seeds were extracted in sodium phosphate buffer, pH 5.4, and precipitated with ammonium sulfate at 90% saturation. The D1 and D2 fractions were subjected to antifungal tests against the yeasts Saccharomyces cerevisiae, Candida albicans, Candida tropicalis, and Kluyveromyces marxiannus, and tested against α-amylases from Callosobruchus maculates and human saliva. The D2 fraction presented higher antimicrobial activity and was subjected to further purification and seven new different fractions (H1-H7) were obtained. Peptides in the H4 fraction were sequenced and the N-terminal sequences revealed homology with previously reported storage vicilins from seeds. The H4 fraction exhibited strong antifungal activity and also promoted morphological changes in yeast, including pseudohyphae formation. All fractions, including H4, inhibited mammalian α-amylase activity but only the H4 fraction was able to inhibit C. maculatus α-amylase activity. These results suggest that the fractions isolated from the seeds of C. baccatum can act directly in plant defenses against pathogens and insects.

Functional expression and activity of the recombinant antifungal defensin PvD1r from Phaseolus vulgaris L. (common bean) seeds.

BACKGROUND: Defensins are basic, cysteine-rich antimicrobial peptides that are important components of plant defense against pathogens. Previously, we isolated a defensin, PvD1, from Phaseolus vulgaris L. (common bean) seeds. RESULTS: The aim of this study was to overexpress PvD1 in a prokaryotic system, verify the biologic function of recombinant PvD1 (PvD1r) by comparing the antimicrobial activity of PvD1r to that of the natural defensin, PvD1, and use a mutant Candida albicans strain that lacks the gene for sphingolipid biosynthesis to unravel the target site of the PvD1r in C. albicans cells. The cDNA encoding PvD1, which was previously obtained, was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform bacterial cells (Rosetta Gami 2 (DE3) pLysS) leading to recombinant protein expression. After expression had been induced, PvD1r was purified, cleaved with enterokinase and repurified by chromatographic steps. N-terminal amino acid sequencing showed that the overall process of the recombinant production of PvD1r, including cleavage with the enterokinase, was successful. Additionally, modeling revealed that PvD1r had a structure that was similar to the defensin isolated from plants. Purified PvD1 and PvD1r possessed inhibitory activity against the growth of the wild-type pathogenic yeast strain C. albicans. Both defensins, however, did not present inhibitory activity against the mutant strain of C. albicans. Antifungal assays with the wild-type C. albicans strains showed morphological changes upon observation by light microscopy following growth assays. PvD1r was coupled to FITC, and the subsequent treatment of wild type C. albicans with DAPI revealed that the labeled peptide was intracellularly localized. In the mutant strain, no intracellular labeling was detected. CONCLUSION: Our results indicate that PvD1r retains full biological activity after recombinant production, enterokinase cleavage and purification. Additionally, our results from the antimicrobial assay, the microscopic analysis and the PvD1r-FITC labeling assays corroborate each other and lead us to suggest that the target of PvD1 in C. albicans cells is the sphingolipid glucosylceramide.

Antimicrobial peptides from Adenanthera pavonina L. seeds: characterization and antifungal activity.

In this study, the antifungal activity of peptides extracted from Adenanthera pavonina seeds was assessed. Peptides were extracted and fractionated by DEAE-Sepharose chromatography. The non-retained D1 fraction efficiently inhibited the growth of the pathogenic fungi. This fraction was later further fractionated by reversed-phase chromatography, resulting in 23 sub-fractions. All separation processes were monitored by tricine-SDS-PAGE. Fractions H11 and H22 strongly inhibited the growth of Saccharomyces cerevisiae and Candida albicans. Fraction H11 caused 100% death in S. cerevisiae in an antimicrobial assay. The complete amino acid sequence of the peptide in fraction P2 was determined, revealing homology to plant defensins, which was named ApDef1. Peptides from fraction H22 were also sequenced.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel α-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 α-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 α-amylase activity in vitro.

Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect alpha-amylase inhibitory activity.

Plant defensins make up a family of cationic antimicrobial peptides with a characteristic three-dimensional folding pattern stabilized by four disulfide bridges. The aim of this work was the purification and functional expression of a defensin from cowpea seeds and the assessment of its alpha-amylase inhibitory activity. The cDNA encoding the cowpea defensin was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform Escherichia coli cells. The recombinant peptide was purified via affinity chromatography on a Ni Sepharose column and by reverse-phase chromatography on a C2/C18 column using HPLC. N-terminal amino acid sequencing revealed that the recombinant peptide had a similar sequence to that of the defensin isolated from seeds. The natural and recombinant defensins were submitted to the alpha-amylase inhibition assay. The cowpea seed defensin was found to inhibit alpha-amylases from the weevils Callosobruchus maculatus and Zabrotes subfasciatus. alpha-Amylase inhibition assays also showed that the recombinant defensin inhibited alpha-amylase from the weevil C. maculatus. The cowpea seed defensin and its recombinant form were unable to inhibit mammalian alpha-amylases. The three-dimensional structure of the recombinant defensin was modeled, and the resulting structure was found to be similar to those of other plant defensins.