Influence of light and leaf epicuticular wax layer on Phakopsora pachyrhizi infection in soybean

Influence of light and leaf epicuticular wax layer on Phakopsora pachyrhizi infection in soybean Asian rust, caused by the fungus Phakopsora pachyrhizi, is one of the most serious phytosanitary problems of soybean in Brazil, especially because no cultivars with satisfactory resistance levels as yet exist. The objective of this study was to evaluate the influence of luminosity and of leaf epicuticular wax on the infection of soybean by P. pachyrhizi. The adaxial and abaxial leaflet surfaces of the first trifoliate leaf from cultivar BRS 154, phenological stage V2, were inoculated with a suspension of 105 uredospores/mL. The plants were kept for 24 hours in a humid chamber at temperature of 23 degrees C, in light or dark conditions, using a factorial design. Subsequently, the plants were maintained for 14 days under a 12-hour photoperiod. The disease severity and density were evaluated. For in vitro experiments, in light or dark conditions, the evaluation was done in terms of uredospore germination and appressorium formation. The wax content of adaxial and abaxial leaflets was analyzed quantitatively using chloroform extraction and ultrastructurally using scanning electron microscope. Higher density and severity were observed when the adaxial surface was inoculated, with later incubation of the plants in the dark, with no significant interaction between these factors. Spore germination in the dark (40.7%) was statistically different from spore germination in the light (28.5%). The same effect was observed with appressorium formation, in the dark (24.7%) and in the light (12.8%). The quantity and the ultrastructural aspects of epicuticular wax content did not show differences between the adaxial and abaxial surfaces; nor did they show any effect on infection by Phakopsora pachyrhizi in the soybean cultivar studied.

Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion

Twenty endophytic bacteria were isolated from the meristematic tissues of three varieties of strawberry cultivated in vitro, and further identified, by FAME profile, into the genera Bacillus and Sphingopyxis. The strains were also characterized according to indole acetic acid production, phosphate solubilization and potential for plant growth promotion. Results showed that 15 strains produced high levels of IAA and all 20 showed potential for solubilizing inorganic phosphate. Plant growth promotion evaluated under greenhouse conditions revealed the ability of the strains to enhance the root number, length and dry weight and also the leaf number, petiole length and dry weight of the aerial portion. Seven Bacillus spp. strains promoted root development and one strain of Sphingopyxis sp. promoted the development of plant shoots. The plant growth promotion showed to be correlated to IAA production and phosphate solubilization. The data also suggested that bacterial effects could potentially be harnessed to promote plant growth during seedling acclimatization in strawberry.

Gas Chromatographic Analysis of Dimethyltryptamine and beta-Carboline Alkaloids in Ayahuasca, an Amazonian Psychoactive Plant Beverage

Introduction - Ayahuasca is obtained by infusing the pounded stems of Banisteriopsis caapi in combination with the leaves of Psychotria viridis. P. viridis is rich in the psychedelic indole N,N-dimethyltryptamine, whereas B. caapi contains substantial amounts of beta-carboline alkaloids, mainly harmine, harmaline and tetrahydroharmine, which are monoamine-oxidase inhibitors. Because of differences in composition in ayahuasca preparations, a method to measure their main active constituents is needed. Objective - To develop a gas chromatographic method for the simultaneous determination of dimethyltryptamine and the main beta-carbolines found in ayahuasca preparations. Methodology - The alkaloids were extracted by means of solid phase extraction (C(18)) and detected by gas chromatography with nitrogen/phosphorous detector. Results - The lower limit of quantification (LLOQ) was 0.02 mg/mL for all analytes. The calibration curves were linear over a concentration range of 0.02-4.0 mg/mL (r(2) > 0.99). The method was also precise (RSD < 10%). Conclusion - A simple gas chromatographic method to determine the main alkaloids found in ayahuasca was developed and validated. The method can be useful to estimate administered doses in animals and humans for further pharmacological and toxicological investigations of ayahuasca. Copyright (C) 2009 John Wiley & Sons, Ltd.

Transcriptional responses of host peripheral blood cells to tuberculosis infection

Host responses following exposure to Mycobacterium tuberculosis (TB) are complex and can significantly affect clinical outcome. These responses, which are largely mediated by complex immune mechanisms involving peripheral blood cells (PBCs) such as T-lymphocytes, NK cells and monocyte-derived macrophages, have not been fully characterized. We hypothesize that different clinical outcome following TB exposure will be uniquely reflected in host gene expression profiles, and expression profiling of PBCs can be used to discriminate between different TB infectious outcomes. In this study, microarray analysis was performed on PBCs from three TB groups (BCG-vaccinated, latent TB infection, and active TB infection) and a control healthy group. Supervised learning algorithms were used to identify signature genomic responses that differentiate among group samples. Gene Set Enrichment Analysis was used to determine sets of genes that were co-regulated. Multivariate permutation analysis (p < 0.01) gave 645 genes differentially expressed among the four groups, with both distinct and common patterns of gene expression observed for each group. A 127-probeset, representing 77 known genes, capable of accurately classifying samples into their respective groups was identified. In addition, 13 insulin-sensitive genes were found to be differentially regulated in all three TB infected groups, underscoring the functional association between insulin signaling pathway and TB infection. Published by Elsevier Ltd.

qNMR: An applicable method for the determination of dimethyltryptamine in ayahuasca, a psychoactive plant preparation

Ayahuasca is an Amazonian plant beverage obtained by infusing the pounded stems of Banisteriopsis caapi in combination with the leaves of Psychotria viridis. P. viridis contains the psychedelic indole N,N-dimethyltryptamine (DMT). This association has a wide range of use in religious rituals around the world. In the present work, an easy, fast and non-destructive method by Nuclear Magnetic Resonance of proton ((1)H NMR) for quantification of DMT in ayahuasca samples was developed and validated. 2,5-Dimethoxybenzaldehyde (DMBO) was used as internal standard (IS). For this purpose, the area ratios produced by protons of DMT (N(CH(3))(2)) at 2.70 ppm, singlet, (6H) and for DMBO (Ar(OCH(3))(2)) at 3.80 and 3.89 ppm, doublet, (6H) were used for quantification. The lower limit of quantification (LLOQ) was 12.5 mu g/mL and a good intra-assay precision was also obtained (relative standard deviation < 5.1%). The present (1)H NMR method is not time consuming and can be readily applied to monitor this tryptamine in plant preparations. We believe that qNMR can be used for identification and quantification of many plant-based products and metabolites with important advantages, while comparing with other analytical techniques. (C) 2010 Phytochemical Society of Europe. Published by Elsevier B. V. All rights reserved.

Host-guest complexation of a nitroheterocyclic compound with cyclodextrins: a spectrofluorimetric and molecular modeling study

Nitroheterocyclic compounds (NC) were candidate drugs proposed for Chagas disease chemotherapy. In this study, we investigated the complexation of hydroxymethylnitrofurazone (NFOH), a potential antichagasic compound, with alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), Hydroxypropyl-beta-cyclodextrin (HP-beta-CD), Dimethyl-beta-cyclodextrin (DM-beta-CD) and gamma-cyclodextrin (gamma-CD) by fluorescence spectroscopy and molecular modeling studies. Hildebrand-Benesi equation was used to calculate the formation constants of NFOH with cyclodextrins based on the fluorescence differences in the CDs solution. The complexing capacity of NFOH with different CDs was compared through the results of association constant according to the following order: DM-beta-CD > beta-CD > alpha-CD > HP-beta-CD > gamma-CD. Molecular modeling studies give support for the experimental assignments, in favor of the formation of an inclusion complex between cyclodextrins with NFOH. This is an important study to investigate the effects of different kinds of cyclodextrins on the inclusion complex formation with NFOH and to better characterize a potential formulations to be used as therapeutic options for the oral treatment of Chagas disease.

A survey on the sanitary condition of commercial foods of plant origin sold in Brazil

The objective of the study was to evaluate the hygienic conditions and practices of commercial foods of plant origin in establishments and street marketed by street vendors in cities in the State of Rio de Janeiro, Brazil. Forty different sales points were evaluated (establishments that prepared and sold fruit juices and street vendors that commercialized fresh coconut water, sugarcane juice and orange juice) using a questionnaire with 12 items, divided into three blocks (salesmen/handlers, operations, installations). The results indicated that the activities related to the commerce of fruit beverages in the cities of Sao Goncalo and Rio de Janeiro required the elaboration of a set of actions by the Sanitary Vigilance Agency in order to improve the hygienic and sanitary level and minimize health risk to consumers. Important requirements in the legislation relevant to this type of food are still not followed; adequate packaging and storage of the raw material, obtaining the raw material from registered suppliers, hygiene of the handlers and adequate management of wastes produced during the activities in question are amongst the main items deserving attention. (C) 2009 Elsevier Ltd. All rights reserved.

Evaluation of Antigenotoxic Effects of Plant Flavonoids Quercetin and Rutin on HepG2 Cells

The flavonoid quercetin and its derivative rutin were investigated for genotoxicity/antigenotoxicity activity in human hepatoma HepG2 cells using the comet assay. The extract cytotoxicity was evaluated using the trypan blue exclusion dye method with quercetin and rutin concentrations ranging from 0.1 to 200.0 mu g/mL of culture medium. Three minor non-cytotoxic concentrations were chosen to evaluate the genotoxicity and antigenotoxicity of the flavonoids (0.1, 1.0 and 5.0 mu g/mL) through comet assay. The cultures were treated with three different concentrations of rutin or quercetin (genotoxicity) or their association with Aflatoxin B1 (AFB1), methyl methanesulfonate (MMS) or doxorubicin (DXR) (antigenotoxicity test) in three protocols: pretreatment, simultaneous treatment and post-treatment. The cell cultures were also treated with 1% DMSO (control group), AFB1, MMS and DXR (positive-control). Statistical analyses were performed using ANOVA and Dunnett`s test (p <= 0.05). Quercetin at concentrations higher than 10.0 mu g/mL or rutin higher than 50.0 mu g/mL exhibited a cytotoxic effect on the cells, showing that quercetin is more cytotoxic than rutin. Furthermore, neither compound was able to induce genotoxicity in the concentrations evaluated. On the other hand, both flavonoids reduced DNA damage induced by AFB1, MMS and DXR in all treatment protocols. Copyright (C) 2011 John Wiley & Sons, Ltd.

Screening of plant extracts from the Brazilian Cerrado for their in vitro trypanocidal activity

In this study we report the screening of the in vitro trypanocidal activity of 20 extracts obtained from 10 different plant species growing in the Brazilian Cerrado: Aspidosperma macrocarpum Mart. (Apocynaceae), Aegiphila sellowiano Cham. (Verbenaceae), Byrsonima intermedia Juss. (Malpighiaceae), Cyperus rotundus L. (Cyperaceae), Leandra lacunosa Cogn. (Melastomataceae), Miconia ligustroides (DC.) Naudin. (Melastomataceae), Miconia sellowiana Naudin.(Melastomataceae),Myrcia variabilis Mart.ex DC. (Myrtaceae), Solanum lycocarpum St. Hil. (Solanaceae), and Tibouchina stenocarpa Cogn. (Melastomataceae). The most active extracts were submitted to phytochemical analyses. High-resolution gas chromatography analysis of the n-hexane extract of T. stenocarpa (IC(50) = 23.6 mu g/mL), the most active extract amongst all the tested samples, allowed the identification of beta-amyrin, alpha-amyrin, lupeol, friedelin, beta-friedelanol, campesterol, stigmasterol, and beta-sitosterol. Oleanolic and ursolic acids were isolated from the methylene chloride extract of T stenocarpa (IC(50) = 51.5 mu g/mL), while ursolic acid was isolated from the methylene chloride extract of M. variabilis (IC(50)=38.4 mu g/mL). Solasonine and solamargine were identified as major compounds by mass spectrometry analysis in the hydroalcoholic extract of the fruits of S. lycocarpum (IC(50)=57.1 mu g/mL).The results showed that the trypanocidal activity may be related to the major compounds identified in the crude active extracts.

Bioactive Chemical Constituents and Comparative Antimicrobial Activity of Callus Culture and Adult Plant Extracts from Alternanthera tenella

Crude extracts of a callus culture (two culture media) and adult plants (two collections) from Alternanthera tenella Colla (Amaranthaceae) were evaluated for their antibacterial and antifungal activity, in order to investigate the maintenance of antimicrobial activity of the extracts obtained from plants in vivo and in vitro. The antibacterial and antifungal activity was determined against thirty strains of microorganisms including Gram-positive and Gram-negative bacteria, yeasts and dermatophytes. Ethanolic and hexanic extracts of adult plants collected during the same period of the years 1997 and 2002 [Ribeirao Preto (SP), collections 1 and 2] and obtained from plant cell callus culture in two different hormonal media (AtT43 and AtT11) inhibited the growth of bacteria, yeasts and dermatophytes with inhibition halos between 6 and 20 mm. For the crude extracts of adult plants bioassay-guided fractionation, purification, and isolation were performed by chromatographic methods, and the structures of the isolated compounds were established by analysis of chemical and spectral evidences (UV, IR, NMR and ES-MS). Steroids, saponins and flavonoids (aglycones and C-glycosides) were isolated. The minimum inhibitory concentration (MIC) of the isolated compounds varied from 50 to 500 mu g/mL.

Counterregulation of Th2 immunity by interleukin 12 reduces host defenses against Strongyloides venezuelensis infection

The aim of this study was to investigate the role of interleukin 12 (IL-12) during Strongyloides venezuelensis infection. IL-12(-/-) and wildtype C57BL/6 mice were subcutaneously infected with 1500 larvae of S. venezuelensis. On days 7, 14, and 21 post-infection, we determined eosinophil and mononuclear cell numbers in the blood and broncoalveolar lavage fluid (BALF), Th2 cytokine secretion in the lung parenchyma, and serum antibody levels. The numbers of eggs in the feces and worm parasites in the duodena were also quantified. The eosinophil and mononuclear cell counts and the concentrations of IL-3, IL-5, IL-10, IL-13, and IgG1 and IgE antibodies increased significantly in infected IL-12(-/-) and wild-type mice as compared with uninfected controls. However, the number of eosinophils and mononuclear cells in the blood and BALF and the Th2 cytokine levels in the lungs of infected IL-12-/- mice were greater than in infected wild-type C57BL/6 mice. In addition, serum IgE and IgG1 levels were also significantly enhanced in the infected mice lacking IL-12. Meanwhile, parasite burden and fecal egg counts were significantly decreased in infected IL-12-/- mice. Together, our results showed that the absence of IL-12 upregulates the Th2 immune response, which is important for control of S. venezuelensis infection. (C) 2009 Elsevier Masson SAS. All rights reserved.

Cytotoxic and mutagenic evaluation of extracts from plant species of the Miconia genus and their influence on doxorubicin-induced mutagenicity: An in vitro analysis

The Miconia genus, a plant widely used for medicine, occurs in tropical America and its extracts and isolated compounds have demonstrated antibiotic, antitumoral, analgesic and antimalarial activities. However, no study concerning its genotoxicity has been conducted and it is necessary to determine its potential mutagenic effects to develop products and chemicals from these extracts. This study assessed the cytotoxicity, mutagenicity and the protective effects of methanolic extracts from Miconia species on Chinese hamster lung fibroblast cell cultures (V79). The cytotoxicity was evaluated using a clonogenic assay. Cultures exposed to the extract of Miconia albicans up to a concentration of 30 mu g/mL, M. cabucu up to 40 mu g/mL, M. albicans up to 40 mu g/mL and M. stenostachya up to 60 mu g/mL exhibited a cytotoxic effect on the cells. The clonogenic assay used three non-cytotoxic concentrations (5, 10 and 20 mu g/mL) to evaluate mutagenicity and antimutagenicity of the extracts. Cultures were treated with these three extract concentrations (mutagenicity test) or the extract associated with doxorubicin (DXR) (antimutagenicity test) in three protocols (pre-, simultaneous and post-treatments). Distilled water and DXR were used as negative and positive controls, respectively. In the micronucleus (MN) test, a significant reduction was observed in MN frequency in cultures treated with DXR and extracts compared to those receiving only DXR; a significant reduction was also observed for the presence of mutagenicity in all treatments. This study confirmed the safe use of Miconia extracts at the concentrations tested and reinforced the therapeutic properties previously described for Miconia species by showing their protective effects on doxorubicin-induced mutagenicity. (C) 2010 Elsevier GmbH. All rights reserved.

Wolbachia replication and host cell division in Aedes albopictus

Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species. The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing. Like other vertically transmitted intracellular symbionts, Wolbachiarsquos replication rate must not outpace that of its host cells if it is to remain benign. The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis. During diapause in mosquito eggs, no host cell division is believed to occur. Further development is triggered only by subsequent exposure of the egg to water. This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate. We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.

Wolbachia infections of tephritid fruit flies: molecular evidence for five distinct strains in a single host species

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and can induce cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing or feminization in their hosts. Here, we report phylogenetic relationships of Wolbachia in tephritid fruit flies based on wsp gene sequences. We also report, for the first time, five distinct strains of Wolbachia in Bactrocera ascita sp. B. Four of the five Wolbachia strains found in this species were in the same groups as those found in other tephritid fruit flies, suggesting possible horizontal transmission of Wolbachia from other fruit flies into B. ascita sp. B. The unreliability of wsp-specific group primers demonstrated in this study suggests that these primers might be useful only for preliminary identification of Wolbachia. Final determination of group affiliation needs to be verified with wsp sequence data.

Host age effect and expression of cytoplasmic incompatibility in field populations of Wolbachia-superinfected Aedes albopictus

The Asian tiger mosquito, Aedes albopictus (Skuse), is a known vector of dengue in South America and Southeast Asia. It is naturally superinfected with two strains of Wolbachia endosymbiont that are able to induce cytoplasmic incompatibility (CI). In this paper, we report the strength of CI expression in crosses involving field-caught males. CI expression was found to be very strong in all crosses between field males and laboratory-reared uninfected or wAlbA infected young females. In addition, crossing experiments with laboratory colonies showed that aged super- infected males could express strong CI when mated with young uninfected or wAlbA infected females. These results provide additional evidence that the CI properties of Wolbachia infecting Aedes albopictus are well suited for applied strategies that seek to utilise Wolbachia for host population modification.