Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): effects of metallic and organic contaminants

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T(14) and T(28), while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T(28) only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.     

Compensation and resistance to herbivory in seagrasses: induced responses to simulated consumption by fish

Herbivory can induce changes in plant traits that may involve both tolerance mechanisms that compensate for biomass loss and resistance traits that reduce herbivore preference. Seagrasses are marine vascular plants that possess many attributes that may favour tolerance and compensatory growth, and they are also defended with mechanisms of resistance such as toughness and secondary metabolites. We quantified phenotypic changes induced by herbivore damage on the temperate seagrass Posidonia oceanica in order to identify specific compensatory and resistance mechanisms in this plant, and to assess any potential trade-offs between these two strategies of defence. We simulated three natural levels of fish herbivory by repeatedly clipping seagrass leaves during the summer period of maximum herbivory. Compensatory responses were determined by measuring shoot-specific growth, photosynthetic rate, and the concentration of nitrogen and carbon resources in leaves and rhizomes. Induced resistance was determined by measuring the concentration of phenolic secondary metabolites and by assessing the long-term effects of continued clipping on herbivore feeding preferences using bioassays. Plants showed a significant ability to compensate for low and moderate losses of leaf biomass by increasing aboveground growth of damaged shoots, but this was not supported by an increase in photosynthetic capacity. Low levels of herbivory induced compensatory growth without any measurable effects on stored resources. In contrast, nitrogen reserves in the rhizomes played a crucial role in the plant’s ability to compensate and survive herbivore damage under moderate and high levels of herbivory, respectively. We found no evidence of inducibility of long-term resistance traits in response to herbivory. The concentration of phenolics decreased with increasing compensatory growth despite all treatments having similar carbon leaf content, suggesting reallocation of these compounds towards primary functions such as cell-wall construction.     

First genotyping of Giardia lamblia from human and animal feces in Argentina, South America

The purpose of this study was to investigate the genotypes of Giardia lamblia from human and animal feces and their epidemiological and clinical characteristics in Argentina, South America. Seventy isolates, 60 from humans (adults and children), eight from dogs and two from cows were processed by polymerase chain reaction-restriction fragment length polymorphism. Data corresponding to demographic, socio-cultural and environmental variables and presence/absence of signs/symptoms were collected. The triosephosphate isomerase gene was amplified from 43 (71.66%) of the 60 human fecal samples. Among these, 3/43 (6.98%) were genotype AII and 40/43 (93.02%) were genotype B. Assemblage AII was detected in three children who lived together in a shantytown and they were oligosymptomatic and none had diarrhea. This genotype was not found in animals. Genotype B showed a high prevalence in both adults and children. It was also found in polysymptomatic people, many of whom presented diarrhea. It was also found only in one dog. The present study represents the first contribution to the knowledge of G. lamblia genotypes in Argentina.     

Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.     

The N- and C-termini of Elg1 contribute to the maintenance of genome stability

ELG1 (enhanced level of genome instability) encodes a Replication Factor C (RFC) homolog that is important for the maintenance of genome stability. Elg1 interacts with Rfc2-5, forming the third alternative RFC complex identified to date. We found that Elg1 plays a role in the suppression of spontaneous DNA damage in addition to its previously identified roles in the resistance to DNA damage. Using mutational analysis we examined the function of conserved and unique regions of Elg1 in these roles. We found that the Walker A motif in the conserved RFC region is dispensable for Elg1 function in vivo. The RFC region is important for association with chromatin although residues predicted to mediate interactions with DNA are dispensable for Elg1 function. The unique C-terminus of Elg1 mediates oligomerization with Rfc2-5, nuclear import, and chromatin association, and is critical for the function of Elg1. Finally, we demonstrated that the N-terminus of Elg1 contributes to the maintenance of genome stability, and that one function of this N-terminus is to promote the nuclear localization of Elg1. Together, these studies delineate the regions of Elg1 important for its function in damage resistance and in the suppression of spontaneous DNA damage.     

Comparative Study of Chemical Approaches to the Solid-Phase Synthesis of a Tumor-Seeking α-MSH Analogue

The synthesis of a cyclic melanocortin analogue (H-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂), where the Boc-protected derivative of a metal-chelating pyrazolyl ligand (pz) was inserted as N-terminal residue, was addressed by several different Fmoc/tBu and Boc/Bzl solid-phase strategies. On-resin cyclization was achieved immediately following incorporation of Asp, by condensation of the Asp side chain carboxyl with the Lys side chain primary amine after selective and simultaneous removal of side chain protecting groups. The success of the synthesis was highly dependent on the chemical strategy employed, with Boc/Bzl chemistry giving the best results. On the light of our findings, Fmoc/tBu strategies are not advantageous for the solid-phase synthesis of this particular type of lactam-bridged peptides. Last, but not least, the target peptide was recently found to have promising tumor-seeking properties (J Biol Inorg Chem 13:449–459, 2008).     

Synthesis of Polycyclic Aromatic Hydrocarbons and Acetylene Polymers in Ice: A Prebiotic Scenario

The recent evidences of presence of subsurface oceans of liquid water and ice on Saturn's moons, and the possible presence and astrobiological importance of polycyclic aromatic hydrocarbons (PAHs) in these environments, provide strong motivation for the exploration of the prebiotic chemistry in ice and to test if PAHs could be experimentally synthesized in ice surfaces under atmospheres containing methane as carbon source. In this work, we present a new design for prebiotic‐chemistry experiments in ice matrix. Using this design, a mixture of products including PAHs, polar aromatic compounds, and hydrophilic acetylene‐based polymers was obtained. We propose that acetylene generation in a methane/nitrogen atmosphere and subsequent polymerization to PAHs and polyynes could be a favored pathway in the presence of water freeze–melt cycles. These results shed light on the processes involved in PAH synthesis in icy environments and on the physical factors that drive the different competing pathways in methane/nitrogen atmospheres.     

Structural mobility of the monomeric C-terminal domain of the HIV-1 capsid protein

The capsid protein of HIV-1 (p24) (CA) forms the mature capsid of the human immunodeficiency virus. Capsid assembly involves hexamerization of the N-terminal domain and dimerization of the C-terminal domain of CA (CAC), and both domains constitute potential targets for anti-HIV therapy. CAC homodimerization occurs mainly through its second helix, and it is abolished when its sole tryptophan is mutated to alanine. This mutant, CACW40A, resembles a transient monomeric intermediate formed during dimerization. Its tertiary structure is similar to that of the subunits in the dimeric, non-mutated CAC, but the segment corresponding to the second helix samples different conformations. The present study comprises a comprehensive examination of the CACW40A internal dynamics. The results obtained, with movements sampling a wide time regime (from pico- to milliseconds), demonstrate the high flexibility of the whole monomeric protein. The conformational exchange phenomena on the micro-to-millisecond time scale suggest a role for internal motions in the monomer-monomer interactions and, thus, flexibility of the polypeptide chain is likely to contribute to the ability of the protein to adopt different conformational states, depending on the biological environment.