Synthesis of New Thiosemicarbazones and Semicarbazones Containing the 1,2,3-1H-triazole-isatin Scaffold: Trypanocidal, Cytotoxicity, Electrochemical Assays, and Molecular Docking.

BACKGROUND: Chagas disease, also known as American trypanosomiasis, is classified as one of the 17 most important neglected diseases by the World Health Organization. The only drugs with proven efficacy against Chagas disease are benznidazole and nifurtimox, however both show adverse effects, poor clinical efficacy, and development of resistance. For these reasons, the search for new effective chemical entities is a challenge to research groups and the pharmaceutical industry. OBJECTIVE: Synthesis and evaluation of antitrypanosomal activities of a series of thiosemicarbazones and semicarbazones containing 1,2,3-1H triazole isatin scaffold. METHOD: 5'-(4-alkyl/aryl)-1H-1,2,3-triazole-isatins were prepared by Huisgen 1,3-dipolar cycloaddition and the thiosemicarbazones and semicarbazones were obtained by the 1:1 reactions of the carbonylated derivatives with thiosemicarbazide and semicarbazide hydrochloride, respectively, in methanol, using conventional reflux or microwave heating. The compounds were assayed for in vitro trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Beyond the thio/semicarbazone derivatives, isatin and triazole synthetic intermediates were also evaluated for comparison. RESULTS: A series of compounds were prepared in good yields. Among the 37 compounds evaluated, 18 were found to be active, in particular thiosemicarbazones containing a non-polar saturated alkyl chain (IC50 = 24.1, 38.6, and 83.2 µM; SI = 11.6, 11.8, and 14.0, respectively). To further elucidate the mechanism of action of these new compounds, the redox behaviour of some active and inactive derivatives was studied by cyclic voltammetry. Molecular docking studies were also performed in two validated protein targets of Trypanosoma cruzi, i.e., cruzipain (CRZ) and phosphodiesterase C (TcrPDEC). CONCLUSION: A class of thio/semicarbazones structurally simple and easily accessible was synthesized. Compounds containing thiosemicarbazone moieties showed the best results in the series, being more active than the corresponding semicarbazones. Our results indicated that the activity of these compounds does not originate from an oxidation-reduction pathway but probably from the interactions with trypanosomal enzymes.

Molecular characterization of lipoamide dehydrogenase gene in Trypanosoma cruzi populations susceptible and resistant to benznidazole.

Lipoamide dehydrogenase (LipDH) is a flavin-containing disulfide oxidoreductase from the same group of thioredoxin reductase, glutathione reductase and trypanothione reductase. This enzyme is found in the mitochondria of all aerobic organisms where it takes part in at least three important multienzyme complexes from the citric acid cycle. In this study, we performed a phylogenetic analysis comparing the amino acid sequence of the LipDH from Trypanosoma cruzi (TcLipDH) with the LipDH from other organisms. Subsequently, the copy number of the TcLipDH gene, the mRNA and protein levels, and the enzymatic activity of the LipDH were determined in populations and strains of T. cruzi that were either resistant or susceptible to benznidazole (BZ). In silico analysis showed the presence of two TcLipDH alleles in the T. cruzi genome. It also showed that TcLipDH protein has less than 55% of identity in comparison to the human LipDH, but the active site is conserved in both of them. Southern blot results suggest that the TcLipDH is a single copy gene in the genome of the T. cruzi samples analyzed. Northern blot assays showed one transcript of 2.4 kb in all T. cruzi populations. Northern blot and Real Time RT-PCR data revealed that the TcLipDH mRNA levels were 2-fold more expressed in the BZ-resistant T. cruzi population (17LER) than in its susceptible pair (17WTS). Western blot results revealed that the TcLipDH protein level is 2-fold higher in 17LER sample in comparison to 17WTS sample. In addition, LipDH activity was higher in the 17LER population than in the 17WTS. Sequencing analysis revealed that the amino acid sequences of the TcLipDH from 17WTS and 17LER populations are identical. Our findings show that one of the mechanisms associated with in vitro-induced BZ resistance to T. cruzi correlates with upregulation of LipDH enzyme.

Biological activities of ophiobolin K and 6-epi-ophiobolin K produced by the endophytic fungus Aspergillus calidoustus.

Endophytic fungi represent ubiquitous microbial organisms able to live in the tissues of different plants around the world and represent a prolific source of bioactive metabolites. In the present study, the endophytic fungus Aspergillus calidoustus was isolated from the medicinal plant Acanthospermum australe (Asteraceae), and identified using molecular, physiological and morphological methods. A methylene chloride crude extract of A. calidoustus has been produced and subjected to antifungal bioassay-directed fractionation which resulted in the isolation of the two bioactive compounds: ophiobolin K and 6-epi-ophiobolin K. These pure compounds displayed antifungal activity against fungal plant pathogens, protozoal activity against Trypanosoma cruzi, and cytotoxic activity against human tumoral cell lines. The results show that A. calidoustus was able to produce the antifungal and cytotoxic metabolites ophiobolin K and 6-epi-ophiobolin K, which may help the fungus to colonise and occupy the substratum as well as survive in natural environments.

Fungi associated with rocks of the Atacama Desert: taxonomy, distribution, diversity, ecology and bioprospection for bioactive compounds.

This study assessed the diversity of cultivable rock-associated fungi from Atacama Desert. A total of 81 fungal isolates obtained were identified as 29 Ascomycota taxa by sequencing different regions of DNA. Cladosporium halotolerans, Penicillium chrysogenum and Penicillium cf. citrinum were the most frequent species, which occur at least in four different altitudes. The diversity and similarity indices ranged in the fungal communities across the latitudinal gradient. The Fisher-α index displayed the higher values for the fungal communities obtained from the siltstone and fine matrix of pyroclastic rocks with finer grain size, which are more degraded. A total of 23 fungal extracts displayed activity against the different targets screened. The extract of P. chrysogenum afforded the compounds α-linolenic acid and ergosterol endoperoxide, which were active against Cryptococcus neoformans and methicillin-resistance Staphylococcus aureus respectively. Our study represents the first report of a new habitat of fungi associated with rocks of the Atacama Desert and indicated the presence of interesting fungal community, including species related with saprobes, parasite/pathogen and mycotoxigenic taxa. The geological characteristics of the rocks, associated with the presence of rich resident/resilient fungal communities suggests that the rocks may provide a favourable microenvironment fungal colonization, survival and dispersal in extreme conditions.

Diversity and bioprospection of fungal community present in oligotrophic soil of continental Antarctica.

We surveyed the diversity and capability of producing bioactive compounds from a cultivable fungal community isolated from oligotrophic soil of continental Antarctica. A total of 115 fungal isolates were obtained and identified in 11 taxa of Aspergillus, Debaryomyces, Cladosporium, Pseudogymnoascus, Penicillium and Hypocreales. The fungal community showed low diversity and richness, and high dominance indices. The extracts of Aspergillus sydowii, Penicillium allii-sativi, Penicillium brevicompactum, Penicillium chrysogenum and Penicillium rubens possess antiviral, antibacterial, antifungal, antitumoral, herbicidal and antiprotozoal activities. Bioactive extracts were examined using (1)H NMR spectroscopy and detected the presence of secondary metabolites with chemical shifts. Our results show that the fungi present in cold-oligotrophic soil from Antarctica included few dominant species, which may have important implications for understanding eukaryotic survival in cold-arid oligotrophic soils. We hypothesize that detailed further investigations may provide a greater understanding of the evolution of Antarctic fungi and their relationships with other organisms described in that region. Additionally, different wild pristine bioactive fungal isolates found in continental Antarctic soil may represent a unique source to discover prototype molecules for use in drug and biopesticide discovery studies.

Diversity patterns, ecology and biological activities of fungal communities associated with the endemic macroalgae across the Antarctic peninsula.

We surveyed diversity patterns and engaged in bioprospecting for bioactive compounds of fungi associated with the endemic macroalgae, Monostroma hariotii and Pyropia endiviifolia, in Antarctica. A total of 239 fungal isolates were obtained, which were identified to represent 48 taxa and 18 genera using molecular methods. The fungal communities consisted of endemic, indigenous and cold-adapted cosmopolitan taxa, which displayed high diversity and richness, but low dominance indices. The extracts of endemic and cold-adapted fungi displayed biological activities and may represent sources of promising prototype molecules to develop drugs. Our results suggest that macroalgae along the marine Antarctic Peninsula provide additional niches where fungal taxa can survive and coexist with their host in the extreme conditions. We hypothesise that the dynamics of richness and dominance among endemic, indigenous and cold-adapted cosmopolitan fungal taxa might be used to understand and model the influence of climate change on the maritime Antarctic mycota.

Synthesis and evaluation of the antiparasitic activity of bis-(arylmethylidene) cycloalkanones.

A series of bis-(arylmethylidene)-cycloalkanones was synthesized by cross-aldol condensation. The activity of the compounds was evaluated against amastigotes forms of Trypanosoma cruzi and promastigotes forms of Leishmania amazonensis. The cytotoxicity of the active compounds on uninfected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their antiparasitic effects. Six compounds displayed trypanocidal activity against amastigotes intracellular forms of T. cruzi with IC50 values ranging from 7.0 to 249 µM. Besides these six compounds, eight other molecules exhibited significant leishmanicidal activity (IC50 values ranging from 0.6 to 110.4 µM). Two compounds can be considered as promising antiparasitic lead molecules because they showed IC50 values in the low-micromolar range (≤1.2 µM) with an adequate SI (≥19.9). To understand the mechanism of action of these compounds, two possible molecular targets were investigated: trypanothione reductase (TR) and cruzain.

In vitro cytotoxic, antifungal, trypanocidal and leishmanicidal activities of acetogenins isolated from Annona cornifolia A. St. -Hil. (Annonaceae).

Annona cornifolia A. St. -Hil. is a small annual perennial tree found in the Brazilian savannah; their green fruit is popularly used in the treatment of ulcers. The acetogenins isolated from the seeds of Annona cornifolia previously showed to possess antioxidant activity. In continuation of our investigations on the biological activities of acetogenins, four binary mixtures and ten pure adjacent bis-tetrahydrofuran annonaceous acetogenins were evaluated: the cytotoxic (against three human tumor cell lines), antifungal (against Paracoccidioides brasiliensis), trypanocidal (against Trypanosoma cruzi) and leishmanicidal (against Leishmania amazonensis) activities. Acetogenins presented cytotoxic activity confirming their potential use in anti-cancer therapy. Regarding leishmanicidal and trypanocidal activities, an inhibition of 87% of L. amazonensis amastigotes and 100% of T. cruzi amastigotes and trypomastigotes was observed, when tested at the concentration of 20 µg mL-1. Moreover, six acetogenins showed more activity against all the three tested isolates of P. brasiliensis than trimethoprim-sulfamethoxazole, a drug used for treating paracoccidioidomycosis. Thus, acetogenins may be an alternative in treating a number of diseases that have a huge impact on millions of people worldwide. This paper reports for the first time the antifungal, leishmanicidal and trypanocidal activities for these acetogenins.

Diversity and bioprospecting of fungal communities associated with endemic and cold-adapted macroalgae in Antarctica.

We surveyed the distribution and diversity of fungi associated with eight macroalgae from Antarctica and their capability to produce bioactive compounds. The collections yielded 148 fungal isolates, which were identified using molecular methods as belonging to 21 genera and 50 taxa. The most frequent taxa were Geomyces species (sp.), Penicillium sp. and Metschnikowia australis. Seven fungal isolates associated with the endemic Antarctic macroalgae Monostroma hariotii (Chlorophyte) displayed high internal transcribed spacer sequences similarities with the psychrophilic pathogenic fungus Geomyces destructans. Thirty-three fungal singletons (66%) were identified, representing rare components of the fungal communities. The fungal communities displayed high diversity, richness and dominance indices; however, rarefaction curves indicated that not all of the fungal diversity present was recovered. Penicillium sp. UFMGCB 6034 and Penicillium sp. UFMGCB 6120, recovered from the endemic species Palmaria decipiens (Rhodophyte) and M. hariotii, respectively, yielded extracts with high and selective antifungal and/or trypanocidal activities, in which a preliminary spectral analysis using proton nuclear magnetic resonance spectroscopy indicated the presence of highly functionalised aromatic compounds. These results suggest that the endemic and cold-adapted macroalgae of Antarctica shelter a rich, diversity and complex fungal communities consisting of a few dominant indigenous or mesophilic cold-adapted species, and a large number of rare and/or endemic taxa, which may provide an interesting model of algal-fungal interactions under extreme conditions as well as a potential source of bioactive compounds.

Molecular characterization of the hexose transporter gene in benznidazole resistant and susceptible populations of Trypanosoma cruzi.

BACKGROUND: Hexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell. Previous studies employing the Differential Display technique have shown that the transcription level of the HT gene from T. cruzi (TcrHT) is higher in an in vitro-induced benznidazole (BZ)-resistant population of the parasite (17 LER) than in its susceptible counterpart (17 WTS). METHODS: In the present study, TcrHT has been characterized in populations and strains of T. cruzi that are resistant or susceptible to BZ. We investigated the copy number and chromosomal location of the gene, the levels of TcrHT mRNA and of TcrHT activity, and the phylogenetic relationship between TcrHT and HTs from other organisms. RESULTS: In silico analyses revealed that 15 sequences of the TcrHT gene are present in the T. cruzi genome, considering both CL Brener haplotypes. Southern blot analyses confirmed that the gene is present as a multicopy tandem array and indicated a nucleotide sequence polymorphism associated to T. cruzi group I or II. Karyotype analyses revealed that TcrHT is located in two chromosomal bands varying in size from 1.85 to 2.6 Mb depending on the strain of T. cruzi. The sequence of amino acids in the HT from T. cruzi is closely related to the HT sequences of Leishmania species according to phylogenetic analysis. Northern blot and quantitative real-time reverse transcriptase polymerase chain reaction analyses revealed that TcrHT transcripts are 2.6-fold higher in the resistant 17 LER population than in the susceptible 17 WTS. Interestingly, the hexose transporter activity was 40% lower in the 17 LER population than in all other T. cruzi samples analyzed. This phenotype was detected only in the in vitro-induced BZ resistant population, but not in the in vivo-selected or naturally BZ resistant T. cruzi samples. Sequencing analysis revealed that the amino acid sequences of the TcrHT from 17WTS and 17LER populations are identical. This result suggests that the difference in glucose transport between 17WTS and 17LER populations is not due to point mutations, but probably due to lower protein expression level. CONCLUSION: The BZ resistant population 17 LER presents a decrease in glucose uptake in response to drug pressure.

Intraspecies variation in Trypanosoma cruzi GPI-mucins: biological activities and differential expression of α-galactosyl residues.

The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T. cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. The significance of these variations was assessed after analysis of the terminal α-galactosyl residues. Enzymatic treatment with α-galactosidase indicated a differential expression of O-linked α-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T. cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T. cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I).

NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection.

(•)NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91(phox) (-/-) or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with (•)NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.

Leishmanicidal and antitumoral activities of endophytic fungi associated with the Antarctic angiosperms Deschampsia antarctica Desv. and Colobanthus quitensis (Kunth) Bartl.

A total of 564 isolates of endophytic fungi were recovered from the plants Deschampsia antarctica and Colobanthus quitensis collected from Antarctica. The isolates were screened against parasites Leishmania amazonensis and Trypanosoma cruzi and against the human tumour cell lines. Of the 313 fungal isolates obtained from D. antarctica and 251 from C. quitensis, 25 displayed biological activity. Nineteen extracts displayed leishmanicidal activity, and six inhibited the growth of at least one tumour cell line. These fungi belong to 19 taxa of the genera Alternaria, Antarctomyces, Cadophora, Davidiella, Helgardia, Herpotrichia, Microdochium, Oculimacula, Phaeosphaeria and one unidentified fungus. Extracts of 12 fungal isolates inhibited the proliferation of L. amazonesis at a low IC(50) of between 0.2 and 12.5 µg ml(-1). The fungus Phaeosphaeria herpotrichoides displayed only leishmanicidal activity with an IC(50) of 0.2 µg ml(-1), which is equivalent to the inhibitory value of amphotericin B. The extract of Microdochium phragmitis displayed specific cytotoxic activity against the UACC-62 cell line with an IC(50) value of 12.5 µg ml(-1). Our results indicate that the unique angiosperms living in Antarctica shelter an interesting bioactive fungal community that is able to produce antiprotozoal and antitumoral molecules. These molecules may be used to develop new leishmanicidal and anticancer drugs.

Synthesis and evaluation of the anti parasitic activity of aromatic nitro compounds.

A series of nitroaromatic compounds was synthesized and evaluated as potential antileishmanial and trypanocidal agents. Five compounds exerted significant anti-leishmanial activity in vitro against promastigotes forms of Leishmania (L.) amazonensis, with IC(50) in the range of 23-59 µmol L(-1), but none were active against amastigotes intracellular forms of Trypanosoma cruzi. In vitro cytotoxicity on the proliferation of human peripheral blood mononuclear cells (PBMC) stimulated with phytohemaglutinin (PHA) was also evaluated. Two compounds, 6 and 7, were found to present a promising anti-leishmanial activity with IC(50) values of 59.5 and 50.6 µM, respectively, without affecting the lymphocyte proliferation in PBMCs (selectivity index of 16.1 and 21.7, respectively), indicating low toxicity to human cells.

Evaluation of the bactericidal and trypanocidal activities of triterpenes isolated from the leaves, stems, and flowers of Lychnophora pinaster

The phytochemical investigation on the aereal parts of Lychnophora pinaster Mart., Asteraceae, was carried to isolation of triterpenes. 3-O-Acetyl-lupeol (1), 3-O-acetyl-pseudotaraxasterol (2), and 3-O-acetyl-α-amyrin (3) were isolated from hexanic extract and 4,4-dimethyl-cholesta-22,24-dien-5-ol (4), α-amyrin (5), and lupeol (6) were isolated from hexanic/dichlorometanic extract of the leaves. Compounds Δ7-bauerenyl acetate (7), friedelin (8), stigmasterol (9), and sitosterol (10) were isolated from the hexanic/dichlorometanic extract of the stems. The steroids 9 and 10 were also isolated from the hexanic/dichlorometanic extract of the flowers. Triterpenes 1, 3, 4, and 7 are described for the first time in the genus Lychnophora. The apolar fractions of the leaf and stem extracts and some isolated triterpenes showed low trypanocidal activity. Moreover, apolar fractions of the leaf and stem extracts and 5 showed antibacterial action against Staphylococcus aureus.

Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil

One hundred and twenty-one isolates of endophytic fungi were recovered from leaves of the bioactive Brazilian plant species Ageratum myriadenia, Palicourea tetraphylla, Piptadenia adiantoides, and Trixis vauthieri. All fungal isolates were cultivated in liquid media and crude extracts were obtained with ethyl acetate. The crude extracts were tested in bioassay panels using Leishmania amazonensis, Trypanosoma cruzi, the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and three human cancer cell lines. Thirty-three extracts (27.2 percent) exhibited at least one biological activity. Seventeen extracts (14 percent) were cytotoxic against one or more human cancer cell line with the IC50 values ranged of >0.2 to 25 µg/mL. Twenty-four extracts (19.8 percent) inhibited the activity of TryR, and three showed ability to inhibit the growth of T. cruzi above 60 percent and their IC50 values ranged among 1 to 10 µg/mL. Eleven extracts (9 percent) were able to inhibit the growth of L. amazonensis and showed with IC50 values ranged among 4.6 to 24.4 µg/mL. The endophytic fungi were identified as belonging to the genera Alternaria, Arthrinium, Cochliobolus, Colletotrichum, Penicillium, Fusarium, and Gibberella. An interesting result was obtained for the bioactive isolates UFMGCB 508, 537, 899 and 903, which were related to fungi associated with medicinal plants native to Asia, Australia, Africa, and Polynesia. These results indicate that bioactive plants living in Brazilian ecosystems are a potential host of endophytic fungi able to produce bioactive prototype molecules for drug development against neglected tropical diseases.

Role of CCL3/MIP-1alpha and CCL5/RANTES during acute Trypanosoma cruzi infection in rats.

Chagas' disease is caused by Trypanosoma cruzi infection and is characterized by chronic fibrogenic inflammation and heart dysfunction. Chemokines are produced during infection and drive tissue inflammation. In rats, acute infection is characterized by intense myocarditis and regression of inflammation after control of parasitism. We investigated the role of CCL3 and CCL5 during infection by using DNA vaccination encoding for each chemokine separately or simultaneously. MetRANTES treatment was used to evaluate the role of CCR1 and CCR5, the receptors for CCL3 and CCL5. Vaccination with CCL3 or CCL5 increased heart parasitism and decreased local IFN-gamma production, but did not influence intensity of inflammation. Simultaneous treatment with both plasmids or treatment with MetRANTES enhanced cardiac inflammation, fibrosis and parasitism. In conclusion, chemokines CCL3 and CCL5 are relevant, but not essential, for control of T. cruzi infection in rats. On the other hand, combined blockade of these chemokines or their receptors enhanced tissue inflammation and fibrosis, clearly contrasting with available data in murine models of T. cruzi infection. These data reinforce the important role of chemokines during T. cruzi infection but suggest that caution must be taken when expanding the therapeutic modulation of the chemokine system in mice to the human infection.

Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil.

One hundred and twenty-one isolates of endophytic fungi were recovered from leaves of the bioactive Brazilian plant species Ageratum myriadenia , Palicourea tetraphylla , Piptadenia adiantoides, and Trixis vauthieri. All fungal isolates were cultivated in liquid media and crude extracts were obtained with ethyl acetate. The crude extracts were tested in bioassay panels using Leishmania amazonensis , Trypanosoma cruzi, the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and three human cancer cell lines. Thirty-three extracts (27.2%) exhibited at least one biological activity. Seventeen extracts (14%) were cytotoxic against one or more human cancer cell line with the IC50 values ranged of >0.2 to 25 µg/mL. Twenty-four extracts (19.8%) inhibited the activity of TryR, and three showed ability to inhibit the growth of T. cruzi above 60% and their IC50 values ranged among 1 to 10 µg/mL. Eleven extracts (9%) were able to inhibit the growth of L. amazonensis and showed with IC50 values ranged among 4.6 to 24.4 µg/mL. The endophytic fungi were identified as belonging to the genera Alternaria , Arthrinium , Cochliobolus , Colletotrichum , Penicillium , Fusarium, and Gibberella. An interesting result was obtained for the bioactive isolates UFMGCB 508, 537, 899 and 903, which were related to fungi associated with medicinal plants native to Asia, Australia, Africa, and Polynesia. These results indicate that bioactive plants living in Brazilian ecosystems are a potential host of endophytic fungi able to produce bioactive prototype molecules for drug development against neglected tropical diseases.

Characterization of a gene encoding alcohol dehydrogenase in benznidazole-susceptible and -resistant populations of Trypanosoma cruzi.

Alcohol dehydrogenases (ADH) are a class of oxidoreductases that catalyse the reversible oxidation of ethanol to acetaldehyde. In the human parasite Trypanosoma cruzi the TcADH gene was identified through microarray analysis as having reduced transcription in an in vitro induced benznidazole (BZ)-resistant population. In the present study, we have extended these results by characterizing the TcADH gene from 11 strains of T. cruzi that were either susceptible or naturally resistant to benznidazole and nifurtimox or had in vivo selected or in vitro induced resistance to BZ. Sequence comparisons showed that TcADH was more similar to prokaryotic ADHs than to orthologs identified Leishmania spp. Immunolocalisation using confocal microscopy revealed that TcADH is present in the kinetoplast region and along the parasite body, consistent with the mitochondrial localization predicted by sequence analysis. Northern blots showed a 1.9kb transcript with similar signal intensity in all T. cruzi samples analysed, except for the in vitro selected resistant population, where transcript levels were 2-fold lower. These findings were confirmed by quantitative real-time PCR. In Western blot analysis, anti-TcADH polyclonal antisera recognised a 42kDa protein in all T. cruzi strains tested. The level of expression of this polypeptide was approximately 2-fold lower in the in vitro induced benznidazole-resistant strain, than in the susceptible parental strain. The chromosomal location of the TcADH gene was variable, but was not associated with the zymodeme or with the drug resistance phenotype. The data presented here show that the TcADH enzyme has a decreased level of expression in the in vitro induced BZ-resistant T. cruzi population, a situation that has not been observed in the in vivo selected BZ-resistant and naturally resistant strains.

Trypanosoma cruzi: multiplex PCR to detect and classify strains according to groups I and II.

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.