NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces: Cyprinidae)

Chromosomal polymorphism regarding number of NOR sites in the cyprinid fish Chondrostoma lusitanicum was examined using C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ (CMA₃). The analysis of heterochromatic regions allowed a more precise identification of the centromeric regions and the proposal of a revised haploid chromosome formula (7M: 15S: 3A). We describe variability in the number of NOR regions per genome, number of active NOR sites per cell, and relative size of individual NORs. Individuals expressed two or four NOR-bearing chromosomes. Polymorphism was detected in all the populations studied and sex-related differences were not found. The observed chromosomal NOR phenotypes suggest the occurrence of structural rearrangements during the evolutionary process of this diploid leuciscine cyprinid.     

Segregation analysis indicates a major gene in the control of interleukine-5 production in humans infected with Schistosoma mansoni.

The interleukine-5 (IL-5) is a hormone of the immune system that is the main regulator of eosinopoiesis, eosinophil maturation and activation, and immunoglobulin A production. Thus, IL-5 contributes in several ways to human immune defenses against various pathogens, including helminths and infectious agents of the digestive and respiratory tracts. On the other hand, the increase in eosinophil number and the activation of these cells, which both have been related to elevated IL-5 production, are the cause of severe pathological disorders, as in asthma or hypereosinophilic syndromes. Although the immunological pathways leading to IL-5 synthesis have been identified, the reasons for the large variability observed in IL-5 production among subjects exposed to comparable antigenic stimulation are unknown. To investigate the role of genetic factors in this variability, we conducted a segregation analysis in a Brazilian population infected by the helminth parasite Schistosoma mansoni. The analysis was performed on IL-5 levels produced by blood mononuclear cells of these subjects after in vitro restimulation with either parasite extracts (IL-5/schistosomula sonicates [SS] phenotype) or a T-lymphocyte mitogen (IL-5/phytohemagglutin [PHA]). The results provide clear evidence for the segregation of a codominant major gene controlling IL-5/SS and IL-5/PHA production and accounting for 70% and 73% of the phenotypic variance, respectively; the frequency of the allele predisposing to low IL-5 production was approximately .22 for both phenotypes. No significant relationship was found between these genes and the gene controlling infection intensities by S. mansoni detected in a previous study. Linkage studies are ongoing to locate those genes that would help to characterize the genetic factors involved in pathological conditions such as severe helminth infections and allergic diseases.     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.      

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.     

Horizontal biosedimentary gradients across the Sado estuary,W. Portugal

The topography of the Sado estuary, the second largest of Portugal, comprises the outer estuary inside the entrance channel and the inner estuary, on the inward side of which begins the tidal mudflats. The outer estuary subtidal area covers approximately 70 km² and presents a series of longitudinal intertidal sandbanks, separating a northern and a southern channel. A benthic survey was undertaken in the outer estuary during June 1986, in which superficial sediments and macrofauna were sampled at 133 locations. The environmental variables measured in the superficial sediments were the temperature, the granulometric structure, the silt, sand and the gravel content, and the total organic matter content. The primary macrofauna biological variables studied were the species composition, abundance and biomass, calculated on wet, dry and ash-free dry weight. The granulometry and the organic content of superficial sediments agreed with the transient and the residual currents velocity field, simulated in a 2-D hydrodynamic model previously elaborated for the outer estuary. The northern channel superficial sediments showed higher silt and total organic matter content, while the model also suggested lower transient and residual velocities, water flow and shear stress in this channel. The distribution patterns of the subtidal macrofauna were separated into two main groups of species, one comprising taxa essentially settled near the estuarine mouth and the other inwards. Biological primary variables also showed consistent patterns, comparable to other Portuguese estuaries. The major subtidal benthic biotopes were obtained through classification analysis and related to the prevailing hydrophysical and sedimentary conditions in the outer estuary.     

Monocarbonyl Analogs of Curcumin with Potential to Treat Colorectal Cancer

Curcumin has a plethora of biological properties, making this compound potentially effective in the treatment of several diseases, including cancer. However, curcumin clinical use is compromised by its poor pharmacokinetics, being crucial to find novel analogs with better pharmacokinetic and pharmacological properties. Here, we aimed to evaluate the stability, bioavailability and pharmacokinetic profiles of monocarbonyl analogs of curcumin. A small library of monocarbonyl analogs of curcumin 1a–q was synthesized. Lipophilicity and stability in physiological conditions were both assessed by HPLC-UV, while two different methods assessed the electrophilic character of each compound monitored by NMR and by UV-spectroscopy. The potential therapeutic effect of the analogs 1a–q was evaluated in human colon carcinoma cells and toxicity in immortalized hepatocytes. Our results showed that the curcumin analog 1e is a promising agent against colorectal cancer, with improved stability and efficacy/safety profile.     

Non-histone protein methylation in Trypanosoma cruzi epimastigotes

Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.