Polygodial, a sesquiterpene isolated from Drimys brasiliensis (Winteraceae), triggers glucocorticoid-like effects on pancreatic ß-cells.

Despite its common use, the synthetic glucocorticoid dexamethasone can cause several adverse effects, such as diabetes and insulin-related metabolic impairment. Thus, research on molecules that could provide the same anti-inflammatory response with milder side effects is constant. In this work the anti-inflammatory activity of the natural sesquiterpene polygodial, extracted from the endemic Brazilian plant Drimys brasiliensis Miers (Winteraceae), was investigated. Employing a pancreatic ß-cell model (INS 1E), the effect of polygodial on signaling pathways is similar to that caused by dexamethasone - both increased MKP1 and decreased ERK1/2 expression in a dose-response and time-dependent manner. Relating to such finding, nuclear translocation of the glucocorticoid receptor was also discovered to be induced by the sesquiterpene. Molecular modeling results indicated that polygodial was capable of docking to the glucocorticoid receptor, but presented preference for the Arg611 binding site rather than Thr739 when set to bind freely inside the pocket. At last, fragmentation of DNA was verified as consequence of sesquiterpene-induced cell death. Altogether, our results suggest that, like dexamethasone, polygodial interacts the glucocorticoid receptor ligand binding domain but create fewer ligand-protein interactions at the site, yielding a weaker effector response. Such property provides an advantage when regarding the adverse effects resulting from stronger affinity ligands of the glucocorticoid receptor, such as in the case of the current standard dexamethasone-based treatment. This aspect, also, turns polygodial an interesting hit compound to the development of new drugs based on its backbone structure providing less harmful anti-inflammatory treatments.

Antiprotozoal activity of extracts and isolated triterpenoids of 'carnauba' (Copernicia prunifera) wax from Brazil.

CONTEXT: 'Carnauba' wax is a natural product obtained from the processing of the powder exuded from Copernicia prunifera (Miller) H. E. Moore (Arecaceae). This material is widely used in the Brazilian folk medicine, including the treatment of rheumatism and syphilis. OBJECTIVE: To investigate the antiprotozoal activity of hexane and EtOH extracts from the 'carnauba' wax as well as from the isolated compounds from the bioactive extracts. MATERIAL AND METHODS: Two different samples of 'carnauba' (C. prunifera) waxes - types 1 and 4 - were individually extracted using hexane (EH) and EtOH (EE). Aliquots of hexane (type 1 - EH-1 and EH-4) and EtOH (type 4 - EE-1 and EE-4) extracts were tested against promastigote (2-200 µg/mL in DMSO during 48 h at 24 °C) and amastigote (3-150 µg/mL in DMSO during 120 h at 37 °C) forms of Leishmania infantum as well as against trypomastigote (3-150 µg/mL in DMSO during 24 h at 37 °C) forms of Trypanosoma cruzi. Bioactive extracts EH-1 and EE-4 were subjected to a bioactivity-guided fractionation to afford three dammarane-type triterpenoids (1-3). The in vitro antiprotozoal activities of the obtained compounds were evaluated as described above. Additionally, the cytotoxicity activity of compounds 1-3 against mammalian conjunctive cells (NCTC - 2-200 µg/mL in DMSO during 48 h at 37 °C) was determined. RESULTS: From the bioactive hexane and EtOH extracts from the 'carnauba' (C. prunifera) wax, were isolated three dammarane-type triterpenoids: (24R*)-methyldammar-25-ene-3ß,20-diol (carnaubadiol, 1), (24R*)-methyldammara-20,25-dien-3-one (2) and (24R*)-methyldammara-20,25-dien-3α-ol (3). These compounds were identified based on the analysis of NMR and MS spectroscopic data. Compounds 1-3 were effective against the intracellular amastigotes of L. infantum, with IC50 values ranging from 8 to 52 µM, while compounds 1 and 3 displayed activity against trypomastigote forms of T. cruzi with IC50 values of 15 and 35 µM, respectively. The mammalian cytotoxicity assay demonstrated no damage to NCTC conjunctive cells up to 200 µM, except for compound 1, which demonstrated a CC50 value of 34 µM. CONCLUSION: Based on the results, it was possible to conclude that the detected antiprotozoal bioactivity of 'carnauba' (C. prunifera) wax extracts could be related to the presence of the natural dammarane triterpenoid derivatives. The results suggested that these compounds could be used as promising scaffolds for drug design studies for leishmaniasis and Chagas disease.

Immobilization of primary cultures of insulin-releasing human pancreatic cells.

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type1 Diabetes, however, it is severely limited by the shortage of organ donors. Ex-vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 Diabetes. It has recently been shown that, even in the absence of fibrotic overgrowth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells.  Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing.  Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

Oxygen- and glucose-dependent expression of Trhxt1, a putative glucose transporter gene of Trichoderma reesei.

The filamentous fungus Trichoderma reesei is adapted to nutrient-poor environments, in which it uses extracellular cellulases to obtain glucose from the available cellulose biomass. We have isolated and characterized Trhxt1, a putative glucose transporter gene, as judged by the glucose accumulation phenotype of a DeltaTrhxt1 mutant. This gene is repressed at high glucose concentrations and expressed at micromolar levels and in the absence of glucose. The gene is also induced during the growth of T. reesei on cellulose when the glucose concentration generated from the hydrolysis of cellulose present in the culture medium is in the micromolar range. We also show that oxygen availability controls the expression of the Trxht1 gene. In this regard, the gene is down-regulated by hypoxia and also by the inhibition of the flow of electrons through the respiratory chain using antimycin A. Intriguingly, anoxia but not hypoxia strongly induces the expression of the gene in the presence of an otherwise repressive concentration of glucose. These results indicate that although the absence of repressing concentrations of glucose and an active respiratory chain are required for Trhxt1 expression under normoxic conditions these physiological processes have no effect on the expression of this gene under an anoxic state. Thus, our results highlight the presence of a novel coordinated interaction between oxygen and the regulatory circuit for glucose repression under anoxic conditions.

Transcriptional response of the obligatory aerobe Trichoderma reesei to hypoxia and transient anoxia: implications for energy production and survival in the absence of oxygen.

Oxygen is essential for the survival of obligatorily aerobic eukaryotic microorganisms, such as the multicellular fungus Trichoderma reesei. However, the molecular basis for the inability of such cells to survive for extended periods under anoxic conditions is not fully understood. Using cDNA microarray analysis, we show that changes in oxygen availability have a drastic effect on gene expression in T. reesei. The expression levels of 392 (19.6%) out of 2000 genes examined changed significantly in response to hypoxia, transient anoxia, and reoxygenation. In addition to modulating many genes with no previously assigned function, cells respond to hypoxia by readjusting the balance of expression between genes required for energy production and consumption, and altering the expression of genes involved in protective mechanisms and signaling pathways. Moreover, we show that transient anoxia strongly represses genes for enzymes that are critical for glycolysis, and are essential for energy production under anaerobic conditions. Our study thus reveals crucial differences between the facultative anaerobe Saccharomyces cerevisiae and T. reesei with regard to the oxygen-dependent transcriptional control of the glycolytic pathway, which can account for the differential survival of the two species in the absence of oxygen.

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays.

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.