Metabolites from endophytes of the medicinal plant Erythrina crista-galli

Erythrina crista-galli (Fabaceae) is used in Argentinean ethnopharmacology as anti-inflammatory medication, narcotic, desinfectant, and for the treatment of wounds. The common name of the tree is "ceibo" or coral tree. The dominating endophytes in E. crista-galli all belong to the genus Phomopsis as identified by microscopic features and the analysis of their ITS sequences. To investigate a possible contribution of Phomopsis spp. to the metabolites found in the plant, twelve different isolates were cultivated in different media. Besides several new metabolites a number of known compounds were detected: mellein, nectriapyrone, 4-hydroxymellein, scytalone, tyrosol, clavatol, mevinic acid, and mevalonolactone.     

Electrophoresis karyotype and chromosome-length polymorphism of Histoplasma capsulatum clinical isolates from Latin America

Intact chromosomes of 19 clinical isolates of Histoplasma capsulatum recently obtained in Argentina, Mexico and Guatemala and the laboratory reference strain G186B from Panama were analyzed using pulsed-field gel electrophoresis. Chromosomal banding patterns of the human isolates revealed 5-7 bands, ranging from 1.3 to 10 Mbp in size. Strain G186B showed five bands of approximately 1.1, 2.8, 3.3, 5.4 and 9.7 Mbp. Thirteen different electrokaryotypes were identified, indicating that the genome of H. capsulatum varies widely in nature, as observed previously in laboratory strains. No definite association was found between electrokaryotype and geographical or clinical source.     

Mechanism and structure-activity relationships of norspermidine-based peptidic inhibitors of trypanothione reductase

A library of polyamine-peptide conjugates based around some previously identified inhibitors of trypanothione reductase was synthesised by parallel solid-phase chemistry and screened. Kinetic analysis of library members established that subtle structural changes altered their mechanism of action, switching between competitive and non-competitive inhibition. The mode of action of the non-competitive inhibitors was investigated in detail by a variety of techniques including enzyme kinetic analysis (looking at both NADPH and trypanothione disulfide substrates), gel filtration chromatography and analytical ultracentrifugation, leading to the identification of an allosteric mode of inhibition.     

Heat shock triggers MAPK activation and MKP-1 induction in Leydig testicular cells

Testicular function is highly dependent on temperature control. In Leydig testicular cells, the signaling pathway activated by heat stress is poorly defined. Here we describe the molecular events triggered by heat shock (HS, 10 min, 45 degrees C) in MA-10 cells. HS produced a rapid and transient activation of ERK1/2 and JNK kinases, and also increased MAP kinase phosphatase-1 (MKP-1) protein and mRNA levels. The effect of HS on MKP-1 messenger reached significance at 15 min, peaked (3.5-fold) at 60 min, and was partially dependent on ERK1/2 activity. The temporal profiles of MKP-1 protein levels and MAPKs phospho-dephosphorylation suggest that MKP-1 induction could contribute to ERK1/2 and JNK inactivation after HS. In summary, this study indicates that the response to heat stress in Leydig cells includes the activation of MAPKs related to cell survival (ERK1/2) and death (JNK), and the induction of a MAPK activity inhibitory loop.     

Genotype of inflammatory cytokines in limbal stem cell graft in Italian patients

We tested the hypothesis that the genetic capability to mount an inflammatory response might contribute to the inter-individual variability of limbal stem cell graft (LSCG) outcome. Two functional polymorphisms in the IL-6 and TNF-alpha promoter regions were genotyped in 35 patients. A new score system (clinical assessment score, CAS) was set up in order to classify patients' clinical profile, and the main parameters relevant for LSCG as well as for the follow-up of the patients. Patients carrying at both loci a genotype associated with a lower production of both cytokines were classified as "low producers" (LP), while all the others were classified as "intermediate or high producers" (HP). LP patients did not show any difference in CAS before and after transplantation while a significant difference was present in HP patients. A similar trend was evident in the 35 months of follow-up. Polymorphisms of IL-6 and TNF-alpha can be used to identify subgroups of patients with higher risk of unsuccessful outcome.     

Characteristics of in vivo murine erythropoietic response to sodium orthovanadate

Current knowledge about the effects of vanadium compounds on erythropoiesis is still reduced and even contradictory. The aim of this work was to evaluate the in vivo effects of a single dose of sodium orthovanadate (OV, 33 mg/kg i.p.) on CF-1 mice in a time course study (0-8 days). Murine erythropoiesis was assessed through a combinatory of experimental approaches. Classical peripheral and bone marrow (BM) hematological parameters were determined. Erythroid maturation in blood stream and hemopoietic tissues (59Fe uptake assays), BM erythroid progenitor frequency (clonogenic assays) and erythroid crucial protein expressions for commitment and survival: GATA-1, erythropoietin receptor (Epo-R) and Bcl-xL (immunoblottings) were evaluated. Neither BM cellularities nor BM viabilities changed noticeably during the study. Peripheral reticulocytes showed a biphasic increment on days 2 and 8 post-OV. hematocrits enhanced transiently between days 2 and 4. 59Fe uptake percentages enhanced in peripheral blood nearly two-fold over control values between 4 and 8 days (p<0.01) without changes in BM and spleen. Additionally, mature erythroid BM compartments: polychromatophilic erythroblasts and orthochromatic normoblasts increased by the eighth day. BFU-E colonies remained near basal values during the whole experience, whilst CFU-E colonies raised 60% over control at 8 days post-OV (p<0.05). GATA-1 and Epo-R were significantly over-expressed from the third until the end of the experimental protocol (p<0.01). Surprisingly, Bcl-xL showed a constitutive expression pattern without changes during the experience. Experimental data let us suggest that OV does not to cause bone marrow cytotoxicity and that it accelerates maturation of BM committed erythroid precursors. Moreover, there are significant correlations among erythroid-related protein expressions: GATA-1 and Epo-R and the frequency of CFU-E. In addition, Bcl-xL expression invariance during the time course study would indicate that the stimulatory effect of OV treatment on erythropoiesis was mainly exerted on the maturation of red cell precursors rather than on the antiapoptosis of erythroid terminal progenitors.     

Temporal Trends in Macrobenthos Community Structure and Redundancy in a Shallow Coastal Lagoon (Valli di Comacchio,Northern Adriatic Sea)

Temporal trends in macrobenthic communities of the Valli di Comacchio, the largest lagoonal ecosystem in the Po River deltaic area, were examined at four sampling sites located along a putative stress gradient and characterized by different levels of disturbance. Trends of parameters characterizing macrobenthic community structure were analyzed using data collected quarterly from 1996 to 2002. The positive slopes in trends of proportion of abundance of opportunistic species observed at all the four study sites indicated the persistence of stressful conditions. Conversely, temporal trends of species richness and diversity showed a positive slope at three out of four sites, thus indicating an increase of community structural complexity. It is suggested that the increase in abundance of detritivorous opportunistic species, as well as the increase in diversity and species richness, is probably driven by the frequency of physical disturbance that resets the ‘successional clock’ of the benthic communities of the Valli. Redundancy was assessed through multivariate analyses and a ‘forward selection backward elimination’ algorithm. At the ecosystem level (i.e., merging counts from the four sites), two response units (a response unit is the smallest subset of species for which the Spearman rank correlation with sample similarities for the full species set exceeds a predetermined value) were obtained. Each of the response units encompassed quite a large taxonomic and functional spread, since all guilds were well represented. Conversely, at the local level, only one response unit was obtained. Species number and function is essential to long-term maintenance of community and ecosystem processes over a complete cycle of common disturbance events. Since redundancy reflects the resistance to change of community functioning, we hypothesize that the macrobenthic communities of the Valli di Comacchio are quite resistant to environmental changes, despite disturbances that generate fluctuations in species abundance and complement at the local level.     

Effects of hyperbaric oxygen on Leishmania amazonensis promastigotes and amastigotes

In the present study, we evaluated the effects of hyperbaric oxygen (HBO) exposure in both Leishmania amazonensis life stages (promastigotes and amastigotes) and on macrophage cultures infected with the parasite. HBO treatment protocols, which can be tolerated by humans and animals, induced irreversible metabolic damage and affected parasite morphology, growth and ability to transform. The observation that the antioxidant N-acetylcysteine (NAC) prevents some of these deleterious effects indicated an involvement of oxidative stress during parasite HBO exposure. In addition, HBO exposed L. amazonensis-infected macrophage cultures showed reduction of the percentage of infected cells and of the number of intracellular parasites per cell. Thus, the demonstration that HBO, a therapy used in the management of different diseases, is toxic for both L. amazonensis life stages and can alter macrophage susceptibility to the infection encourages further studies of this therapy in animal models of Leishmania infection.