Fusarium graminearum as a producer of xylanases with low cellulases when grown on wheat bran.

The xylanolytic potential of endophytic fungi isolated from leaves of Theobroma cacao was explored for the first time. Four fungal strains showed significant amounts of xylanase activity and low cellulase levels when grown on wheat bran as the sole carbon source. Strain Ec220 of Fusarium graminearum had the highest xylanase production (1.79 U/ml), whereas its cellulase activity was minimal (0.24 U/ml). Optimal conditions for xylanase production were: 154 h of incubation time, pH 5.79 and 29.8 °C. Furthermore, two protein spots detected by two-dimensional gel electrophoresis showed molecular weights (26.05 and 27.70 kDa) and isoelectric points (6.18 and 9.20) corresponding to previously reported F. graminearum xylanases, Xyl A and Xyl B, respectively. Therefore, endophytic fungi of T. cacao can be an important source of xylanolytic activities when cultured on wheat bran, and xylanases with low cellulases found in strain Ec220 require further characterization as they show promise for possible industrial applications.

Envenomation by the scorpion Tityus breweri in the Guayana Shield, Venezuela: report of a case, efficacy and reactivity of antivenom, and proposal for a toxinological partitioning of the Venezuelan scorpion fauna.

OBJECTIVES: Scorpion envenomation is a common public health problem in Venezuela. We report an envenoming case by Tityus breweri, endemic to the Guayana Shield, southeast Venezuela, and the outcome of its treatment with antivenom anti-Tityus discrepans. Toxin composition and antigenic reactivity of T breweri venom were also explored. T breweri distribution range was re-evaluated. METHODS: Clinical signs and symptoms in an adult male were recorded after envenoming and treatment with antivenom. Toxin composition and antigenicity of T breweri venom were investigated by polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometry. T breweri distribution range was reassessed by mapping new records of the species. RESULTS: The moderately severe case (a 21-year-old man) presented autonomic manifestations, including cardiopulmonary and gastrointestinal effects. Full recovery was achieved after anti-T discrepans antivenom administration. T breweri venom contains toxins in the 6-8 kd range that affect voltage-sensitive sodium channels. Based on new records, T breweri distribution area reaches 12 155 km.(2) Inclusion of southeast Venezuela as an endemic area of scorpionism prompted the examination of clinical, immunological, and phylogenetic evidence for suggesting a partitioning of the Venezuelan Tityus fauna into toxinological provinces. CONCLUSIONS: The severity of the case reinforces categorization of the Guayana Shield region as a macroendemic area of scorpionism in Venezuela and allows classification of T breweri as a species of medical importance, with toxins immunologically related to central-eastern Venezuelan Tityus. Partitioning of the territory incorporating multiple criteria may help health authorities establish and implement preventive and therapeutic measures for scorpion envenoming in this region.

Localization and developmental regulation of a dispersed gene family 1 protein in Trypanosoma cruzi.

The dispersed gene family 1 (DGF-1) is the fifth largest gene family in the Trypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N-glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.

Snake venomics and antivenomics of Bothrops colombiensis, a medically important pitviper of the Bothrops atrox-asper complex endemic to Venezuela: Contributing to its taxonomy and snakebite management.

The taxonomic status of the medically important pitviper of the Bothrops atrox-asper complex endemic to Venezuela, which has been classified as Bothrops colombiensis, remains incertae cedis. To help resolving this question, the venom proteome of B. colombiensis was characterized by reverse-phase HPLC fractionation followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom contained proteins belonging to 8 types of families. PI Zn(2+)-metalloproteinases and K49 PLA(2) molecules comprise over 65% of the venom proteins. Other venom protein families comprised PIII Zn(2+)-metalloproteinases (11.3%), D49 PLA(2)s (10.2%), l-amino acid oxidase (5.7%), the medium-sized disintegrin colombistatin (5.6%), serine proteinases (1%), bradykinin-potentiating peptides (0.8%), a DC-fragment (0.5%), and a CRISP protein (0.1%). A comparison of the venom proteomes of B. colombiensis and B. atrox did not support the suggested synonymy between these two species. The closest homologues to B. colombiensis venom proteins appeared to be toxins from B. asper. A rough estimation of the similarity between the venoms of B. colombiensis and B. asper indicated that these species share approximately 65-70% of their venom proteomes. The close kinship of B. colombiensis and B. asper points at the ancestor of B. colombiensis as the founding Central American B. asper ancestor. This finding may be relevant for reconstructing the natural history and cladogenesis of Bothrops. Further, the virtually indistinguishable immunological crossreactivity of a Venezuelan ABC antiserum (raised against a mixture of B. colombiensis and Crotalus durissus cumanensis venoms) and the Costa Rican ICP polyvalent antivenom (generated against a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) towards the venoms of B. colombiensis and B. asper, supports this view and suggests the possibility of indistinctly using these antivenoms for the management of snakebites by any of these Bothrops species. However, our analyses also evidenced the limited recognition capability or avidity of these antivenoms towards a number of B. colombiensis and B. asper venom components, most notably medium-size disintegrins, bradykinin-potentiating peptides, PLA(2) proteins, and PI Zn(2+)-metalloproteinases.

Cytotoxic and apoptosis-inducing effect of ent-15-oxo-kaur-16-en-19-oic acid, a derivative of grandiflorolic acid from Espeletia schultzii.

ent-Kaurenic acid and many natural derivatives of this diterpene are known to have interesting biological properties. ent-15-Oxo-kaur-16-en-19-oic acid can be easily obtained from grandiflorolic acid which was first isolated from Espeletia grandiflora. The present work describes the proapoptotic effect of ent-15-oxo-kaur-16-en-19-oic acid on the human prostate carcinoma epithelial cell line PC-3 as evidenced by the changes in the expression level of proteins associated with the execution and regulation of apoptosis. Cell viability was affected upon exposure to the compound, the IC(50) were determined as 3.7 microg/ml, which is 4 times lower than that corresponding to a primary cell culture of fibroblasts (14.8 microg/mL). Through Western blot analysis, active forms of caspace-3 associated with the specific proteolysis of Poly(ADP-ribose) polymerase (PARP) were detected. Reduced levels of the antiapoptotic protein Bcl-2, as well as the appearance of internucleosomal DNA fragmentation, were also demonstrated. Thus, ent-15-oxo-kaur-16-en-19-oic acid may be a promising lead compound for new chemopreventive strategies, alone or in combination with traditional chemotherapy agents to overcome drug resistance in tumoral cells.