Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite.

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-microl environment can be.

Inhibition of tumor cells interacting with stromal cells by xanthones isolated from a Costa Rican Penicillium sp.

CR1642D, an endophytic isolate of Penicillium sp. collected from a Costa Rican rainforest, was identified through a high-throughput approach to identify natural products with enhanced antitumor activity in the context of tumor-stromal interactions. Bioassay-guided separation led to the identification of five xanthones (1-5) from CR1642D. The structures of the xanthone dimer penexanthone A (1) and monomer penexanthone B (2) were elucidated on the basis of spectroscopic analyses, including 2D NMR experiments. All of the compounds were tested against a panel of tumor cell lines in the presence and absence of bone marrow stromal cells. Compound 3 was the most active, with IC(50) values of 1-17 µM, and its activity was enhanced 2-fold against tumor cell line RPMI8226 in the presence of stromal cells (IC(50) 1.2 µM, but 2.4 µM without stromal cells).

Subcutaneous antifungal screening of Latin American plant extracts against Sporothrix schenckii and Fonsecaea pedrosoi.

CONTEXT: Subcutaneous mycoses are chronic infections caused by slow growing environmental fungi. Latin American plants are used in folk medicine to treat these afflictions. Moreover, the potential of the rich Latin American biodiversity for this purpose has not been fully explored. OBJECTIVES: The aim of the study was to screen Latin American plant extracts against two species of subcutaneous fungi: Sporothrix schenckii and Fonsecaea pedrosoi. MATERIALS AND METHODS: One hundred ninety-five organic extracts from 151 Latin American plants were screened against two subcutaneous fungi by the agar dilution method at a concentration of 100 µg/mL, and minimum inhibitory concentrations (MICs) of active extracts were determined. Positive (amphothericin B) and negative (50% ethanol) controls were used. RESULTS AND DISCUSSION: Twenty eight extracts showed activity at ≤100 µg/mL. Of these, four extracts from Gnaphalium gaudichaudianum DC (Asteraceae), Plumeria rubra L (Apocynaceae), Tecoma stans (L.) Juss. ex Kunth. (Bignoniaceae), and Trichostigma octandum (L.), H. Walter showed activity against F. pedrosoi at MIC 12.5 µg/mL; and, four extracts from Bourreria huanita (Lex.) Hemsl. (Boraginaceae), Phytolacca bogotensis Kunth (Phytolaccaceae), Monnina xalapensis Kunth (Polygalaceae) and Crataegus pubescens (C. Presl) C. Presl (Rosaceae) against S. schenckii. This is the first report on antifungal activity of the Latin American plants against these two subcutaneous fungi. CONCLUSION: S. schenkii and F. pedrosoi were inhibited by B. huanita (MIC: 12.5 and 25 µg/mL), G. gaudichaudianum (MIC: 50 and 12.5 µg/mL) and T. triflora (MIC: 25 µg/mL).

Screening of Latin American plants for antiparasitic activities against malaria, Chagas disease, and leishmaniasis.

In order to explore rationally the medical potential of the plant biodiversity of the Central and South American region as a source of novel antiparasitic molecules, a multinational Organization of American States (OAS) project, which included the participation of multidisciplinary research centers from Argentina, Bolivia, Colombia, Costa Rica, Guatemala, Nicaragua and Panama, was carried out during the period 2001-2004. This project aimed at screening organic plant extracts for antitrypanosomal, antileishmanial and antimalarial activities and subsequently isolating and characterizing bioactive molecules. Plants for antiparasitic screening were selected from a database of ethnomedical uses of Latin American plants (PlanMedia) based on the amount of biological and chemical information available in the literature. We report here the evaluation of 452 extracts from 311 plant species in vitro screens against Plasmodium falciparum, Leishmania mexicana, and Trypanosoma cruzi. Out of 311 species tested, 17 plants (5.4%) showed antiparasitic activities at IC(50) values < or = 10 microg/mL. The most active plants were Acnistus arborescens (L.) Schltdl. (Solanaceae) (leaf, EtOH, IC(50): 4 microg/mL) Monochaetum myrtoideum Naudin (Melastomataceae) (leaf, MeOH, IC(50): 5 microg/mL) and Bourreria huanita (Lex.) Hemsl. (Boraginaceae) (branch, EtOH, IC(50): 6 microg/mL). These were selectively active against P. falciparum, L. mexicana and T. cruzi, respectively.

Molecular data indicate that Rhytidhysteron rufulum (ascomycetes, Patellariales) in Costa Rica consists of four distinct lineages corroborated by morphological and chemical characters.

Rhytidhysteron rufulum is a poorly known, common, pantropical species, capable of utilizing different substrata and occupying diverse habitats, and is the only species of its genus in Costa Rica. We have employed molecular, morphological, and chemical data to assess the variability and differentiation of R. rufulum in Costa Rica, including sites from the Pacific and Atlantic coast. Phylogenetic analyses of nuclear ITS rDNA sequences revealed the presence of four distinct lineages in the R. rufulum complex. Re-examination of the morphology and anatomy showed differences between these lineages in ascomatal, ascal, and ascospore size that have previously been regarded as intraspecific variations. In addition, there was a correlation between molecular phylogenies and chemical components as determined by hplc and nuclear magnetic resonance (NMR). Two lineages (clades I and II) produced the palmarumycins MK-3018, CJ-12372, and CR(1), whereas clade III produced dehydrocurvularin, and clade IV unidentified compounds. Our results based on a polyphasic approach contradict previous taxonomic interpretations of one morphologically variable species.

[Arthropod collection for biodiversity prospection in the Guanacaste Conservation Area, Costa Rica]. / Recolecta de artrópodos para prospección de la biodiversidad en el area de Conservación Guanacaste, Costa Rica.

This study describes the results and collection practices for obtaining arthropod samples to be studied as potential sources of new medicines in a bioprospecting effort. From 1994 to 1998, 1800 arthropod samples of 6-10 g were collected in 21 sites of the Area de Conservaci6n Guancaste (A.C.G) in Northwestern Costa Rica. The samples corresponded to 642 species distributed in 21 orders and 95 families. Most of the collections were obtained in the rainy season and in the tropical rainforest and dry forest of the ACG. Samples were obtained from a diversity of arthropod orders: 49.72% of the samples collected corresponded to Lepidoptera, 15.75% to Coleoptera, 13.33% to Hymenoptera, 11.43% to Orthoptera, 6.75% to Hemiptera, 3.20% to Homoptera and 7.89% to other groups. Different life stages per arthropod species were obtained in most samples, 54.26% of them were adults, 19.90% corresponded to larvae, 6.46% to pupae, 6.12% to pre-pupae, 2.07% to nymphs and 3.74% to other stages. Other materials associated to insects like frass represented 11.20% of the samples collected. Several collecting methods were explored, based on the possibility of accessing the necessary amount of material causing the less impact. Most of the samples were obtained by manual collection (44.38%),. followed by insects breeding (25.73%), light traps (18.80%), different types of nets (10.52%) and other methods (0.16%). In general, collecting methods and practices excluded the use of solvents, mixing different species or life stages in the same bag, which might have introduced undesirable effects in the screening systems for new compounds. Based on the possibility of finding new chemicals in similar samples associated to one arthropod species, the collecting strategy included the generation of several samples from same species, separated according to differences in life stages, collecting sites, ecosystems. seasons, feeding materials or behavioral aspects. This strategy allowed the generation a larger number of samples submitted to bioassays in different areas of pharmaceutical research.

A high-throughput screen identifies a new natural product with broad-spectrum antibacterial activity.

Due to the inexorable invasion of our hospitals and communities by drug-resistant bacteria, there is a pressing need for novel antibacterial agents. Here we report the development of a sensitive and robust but low-tech and inexpensive high-throughput metabolic screen for novel antibiotics. This screen is based on a colorimetric assay of pH that identifies inhibitors of bacterial sugar fermentation. After validation of the method, we screened over 39,000 crude extracts derived from organisms that grow in the diverse ecosystems of Costa Rica and identified 49 with reproducible antibacterial effects. An extract from an endophytic fungus was further characterized, and this led to the discovery of three novel natural products. One of these, which we named mirandamycin, has broad-spectrum antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, methicillin-resistant Staphylococcus aureus, and Mycobacterium tuberculosis. This demonstrates the power of simple high throughput screens for rapid identification of new antibacterial agents from environmental samples.

Asterogynins: secondary metabolites from a Costa Rican endophytic fungus.

An endophytic fungus isolated from the small palm Asterogyne martiana produced two unusual steroid-like metabolites, asterogynin A (1) and asterogynin B (2), along with the known compounds viridiol (3) and viridin (4). Asterogynins A and B were characterized by NMR and MS spectroscopic analysis.

Value of the ethnomedical information for the discovery of plants with antifungal properties. A survey among seven Latin American countries.

ETHNOPHARMACOLOGICAL RELEVANCE: This study reports the antifungal evaluation of 327 plant species (92 families and 251 genera) from seven Latin American countries which were selected on the basis of their reported ethnomedical uses and compared them with plants selected at random. AIM OF THE STUDY: (a) The main aim of this study was to investigate whether the probability of detecting antifungal plants is higher when plants have reports of ethnopharmacological uses related to fungal infections (PAU group) than when they are selected at random (PNAU group). (b) The second objective was to determine, within the PAU group, whether the probability of obtaining a positive result will be higher when the plants are tested against dermatophytes, than against yeasts or Aspergillus spp. (c) The third goal was to investigate, within all MICs0.05). (c) Within the detected antifungal plants from both groups, plants of the PAU group displayed higher activities (lower MICs) than those of PNAU group against dermatophytes (p<0.05) but not against yeasts or Aspergillus spp. CONCLUSIONS: Considering that dermatophytes are the cause of superficial fungal infections, which can be easily detected and followed by traditional healers, our findings suggest that the ethnopharmacological approach is useful in guiding the detection of antifungal plants in Latin America mainly for infections in which the pathological expression is obvious and, therefore, the cure can be clearly observed.

Natural product based inhibitors of the thioredoxin-thioredoxin reductase system.

Spiroketal naphthodecalins are readily assembled by Barton's base mediated Ullmann binaphthyl ether coupling, Dakin reactions and hypervalent iodine spirocyclization. The core structures can be further diversified by enone addition and Stille coupling reactions. Nanomolar inhibitors for the Trx/TrxR redox control system were prepared by this approach and compared to series of natural product isolates. Cytotoxicity in MCF-7 cell assays ranged from an IC50 of 1.6 to >100 microM.

Unusual microbial xylanases from insect guts.

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted beta-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.