Biosynthesis of silver nanoparticles using actinomycetes, phytotoxicity on rice seeds, and potential application in the biocontrol of phytopathogens.

To find effective silver nanoparticles (AgNPs) for control of phytopathogens, in this study, two strains of actinomycetes isolated from the soil of the Brazilian biome Caatinga (Caat5-35) and from mangrove sediment (Canv1-58) were utilized. The strains were identified by using the 16S rRNA gene sequencing as Streptomyces sp., related to Streptomyces mimosus species. The obtained AgNPs were coded as AgNPs 35 and AgNPs58 and characterized by size and morphology using dynamic light scattering, zeta potential, transmission electron microscopy, and Fourier transformed infrared (FTIR). The antifungal activity of the AgNPs35 and AgNPs58 was evaluated in vitro by the minimal inhibitory concentration (MIC) assay on the phytopathogens, Alternaria solani, Alternaria alternata, and Colletotrichum gloeosporioides. The phytotoxic effect was evaluated by the germination rate and seedling growth of rice (Oryza sativa). AgNPs35 and AgNPs58 showed surface plasmon resonance and average sizes of 30 and 60 nm, respectively. Both AgNPs presented spherical shape and the FTIR analysis confirmed the presence of functional groups such as free amines and hydroxyls of biomolecules bounded to the external layer of the nanoparticles. Both AgNPs inhibited the growth of the three phytopathogens tested, and A. alternate was the most sensible (MIC ≤ 4 µM). Moreover, the AgNPs35 and AgNPs58 did not induce phytotoxic effects on the germination and development of rice seedlings. In conclusion, these AgNPs are promising candidates to biocontrol of these phytopathogens without endangering rice plants.

Biogenic antimicrobial silver nanoparticles produced by fungi.

Aspergillus tubingensis and Bionectria ochroleuca showed excellent extracellular ability to synthesize silver nanoparticles (Ag NP), spherical in shape and 35 ± 10 nm in size. Ag NP were characterized by transmission electron microscopy, X-ray diffraction analysis, and photon correlation spectroscopy for particle size and zeta potential. Proteins present in the fungal filtrate and in Ag NP dispersion were analyzed by electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Ag NP showed pronounced antifungal activity against Candida sp, frequently occurring in hospital infections, with minimal inhibitory concentration in the range of 0.11-1.75 µg/mL. Regarding antibacterial activity, nanoparticles produced by A. tubingensis were more effective compared to the other fungus, inhibiting 98.0 % of Pseudomonas. aeruginosa growth at 0.28 µg/mL. A. tubingensis synthesized Ag NP with surprisingly high and positive surface potential, differing greatly from all known fungi. These data open the possibility of obtaining biogenic Ag NP with positive surface potential and new applications.

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine.

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the ß-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 µg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 µg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 µg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 µg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.

Antimycobacterial activity of 2-phenoxy-1-phenylethanone, a synthetic analogue of neolignan, entrapped in polymeric microparticles.

Conventional treatment of tuberculosis (TB) demands a long course therapy (6 months), known to originate multiple drug resistant strains (MDR-TB), which emphasizes the urgent need for new antituberculous drugs. The purpose of this study was to investigate a novel treatment for TB meant to improve patient compliance by reducing drug dosage frequency. Polymeric microparticles containing the synthetic analogue of neolignan, 1-phenyl-2-phenoxiethanone (LS-2), were obtained by a method of emulsification and solvent evaporation and chemically characterized. Only representative LS-2-loaded microparticles were considered for further studies involving experimental murine TB induced by Mycobacterium tuberculosis H37Rv ATCC 27294. The LS-2-loaded microparticles were spherical in shape, had a smooth wall and showed an encapsulation efficiency of 93% in addition to displaying sustained release. Chemotherapeutic potential of LS-2 entrapped in microparticles was comparable to control groups. These findings are encouraging and indicate that LS-2-loaded microparticles are a potential alternative to conventional chemotherapy of TB.

3-[4'-bromo-(1,1'-biphenyl)-4-yl]-N, N-dimethyl-3-(2-thienyl)-2-propen-1-amine: synthesis, cytotoxicity, and leishmanicidal, trypanocidal and antimycobacterial activities.

Current therapies for Chagas' disease, leishmaniasis and tuberculosis are unsatisfactory because of the failure rates, significant toxicity and/or drug resistance. In this study, the compound 3-[4'-bromo-(1,1'-biphenyl)-4-yl]-N,N-dimethyl-3-(2-thienyl)-2-propen-1-amine (IV) was synthesized and its trypanocidal, leishmanicidal and antimycobacterial activities were investigated. The cytotoxicity was determined on V79 cells with three endpoints: nucleic acid content, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide reduction and Neutral Red uptake. This compound was active against different species of mycobacteria and different life cycle stages of Trypanosoma cruzi. In experiments with trypomastigotes performed at 4 degrees C in the presence of blood, the activity was 8.8-fold more active than the standard drug, Crystal Violet. Higher activity was achieved against Leishmania amazonensis, with an ED(50)/24 h of 3.0 +/- 0.3 micro mol/L. The effect against trypanosomatids, which suggests high activity of compound IV against promastigotes of L. amazonensis and amastigotes of T. cruzi, stimulated further studies in vitro with amastigotes interiorized in macrophages and with in vivo models. Our results indicate that mammalian V79 cells are less susceptible to the action of compound IV than promastigotes of L. amazonensis (8.0-13.3-fold) and axenic amastigotes of T. cruzi (3.5-5.9-fold).

Vaccines of the future: from rational design to clinical development. 17-19 October 2001, Paris, France.

The Institut Pasteur (France) sponsors and organises a series of Euroconferences on important topics in biology, medicine and environmental sciences which facilitate a large exchange of ideas between basic and applied scientists from the Institut Pasteur, other academic institutions, and pharmaceutical companies. This Euroconference focused on the most recent advances currently affecting the way vaccine development is approached. The identification of new target antigens for vaccination purposes is presently facilitated by the availability of genome sequences from a growing number of important pathogens and tumours. The identification of T-cell epitopes and the subsequent optimisation of these target sequences allows the design of vaccines which are significantly improved with respect to their capacity to stimulate cellular immunity. In parallel, various antigen formulations, adjuvants and delivery systems are being developed and tested with the aim of eliciting broad immune responses in humans. These new orientations capitalise on recent major advances in the understanding of the use of therapeutic vaccines and expands on their use from being solely profilactic. Specific issues related to the development of therapeutic vaccines directed against diseases such as cancer or chronic infectious disease were presented.