A new approach to species distributional indicators for the Marine Strategy Framework Directive (MSFD)

We propose alternative fish-populations spatial indicators for use in the Marine Strategy Framework Directive (MSFD). Following Commission Decision 2010/477, we have applied two different spatial indicators to three fish populations with “slow type” life-history traits, i.e. slow growing like Helicolenus dactylopterus, or large bodied like Merluccius merluccius and Lophius budegassa. We tested their efficiency separately and combined. One of these indicators, the presence/absence of the population in sampling squares, had already been applied during the initial assessment of the MSFD in Spain. Another indicator, the geographical spread, is proposed here as a new monitoring tool for the MSFD in Spanish waters. The results demonstrate for the three populations analyzed that neither indicator was sufficient alone to describe the population spatial pattern or its evolution. Thus, the approach to implementing the MSFD indicated in Commission Decision 2010/477 is not sufficient to provide integrated information about the spatial behavior of the fish populations analyzed. Although numerical targets or threshold values cannot be set, directional targets could be proposed, based on the results of both indicators, if evaluation of them is extended to more species and more geographical areas. The analysis could be extended to other “slow type” populations within the fish community and also to different ecoregions. We propose an approach including the estimation of two different indicators to monitoring both the area occupied and the geographical spread of fish populations within communities, interpreting them together to generate a more complete picture of the spatial patterns of those populations. In spite of the difficulties in fixing numerical targets or thresholds, or in distinguishing between environmentally and human driven changes in the population spatial distributions, this approach helps to summarize fish spatial behavior. It improves information from the indicators applied alone and reduces the requirement for a large number of maps (except for some particular event or population). The proposed indicators can be readily used by managers and politicians.     

Identification of metabolites from liquid chromatography–coulometric array detection profiling: Gas chromatography–mass spectrometry and refractionation provide essential information orthogonal to LC–MS/microNMR

Liquid chromatography–coulometric array detection (LC–EC) is a sensitive, quantitative, and robust metabolomics profiling tool that complements the commonly used mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based approaches. However, LC–EC provides little structural information. We recently demonstrated a workflow for the structural characterization of metabolites detected by LC–EC profiling combined with LC–electrospray ionization (ESI)–MS and microNMR. This methodology is now extended to include (i) gas chromatography (GC)–electron ionization (EI)–MS analysis to fill structural gaps left by LC–ESI–MS and NMR and (ii) secondary fractionation of LC-collected fractions containing multiple coeluting analytes. GC–EI–MS spectra have more informative fragment ions that are reproducible for database searches. Secondary fractionation provides enhanced metabolite characterization by reducing spectral overlap in NMR and ion suppression in LC–ESI–MS. The need for these additional methods in the analysis of the broad chemical classes and concentration ranges found in plasma is illustrated with discussion of four specific examples: (i) characterization of compounds for which one or more of the detectors is insensitive (e.g., positional isomers in LC–MS, the direct detection of carboxylic groups and sulfonic groups in ¹H NMR, or nonvolatile species in GC–MS), (ii) detection of labile compounds, (iii) resolution of closely eluting and/or coeluting compounds, and (iv) the capability to harness structural similarities common in many biologically related, LC–EC-detectable compounds.     

Flavonoid–membrane interactions: Involvement of flavonoid–metal complexes in raft signaling

Flavonoids are polyphenolic compounds produced by plants and delivered to the human body through food. Although the epidemiological analyses of large human populations did not reveal a simple correlation between flavonoid consumption and health, laboratory investigations and clinical trials clearly demonstrate the effectiveness of flavonoids in the prevention of cardiovascular, carcinogenic, neurodegenerative and immune diseases, as well as other diseases. At present, the abilities of flavonoids in the regulation of cell metabolism, gene expression, and protection against oxidative stress are well-known, although certain biophysical aspects of their functioning are not yet clear. Most flavonoids are poorly soluble in water and, similar to lipophilic compounds, have a tendency to accumulate in biological membranes, particularly in lipid rafts, where they can interact with different receptors and signal transducers and influence their functioning through modulation of the lipid-phase behavior. In this study, we discuss the enhancement in the lipophilicity and antioxidative activity of flavonoids after their complexation with transient metal cations. We hypothesize that flavonoid–metal complexes are involved in the formation of molecular assemblies due to the facilitation of membrane adhesion and fusion, protein–protein and protein–membrane binding, and other processes responsible for the regulation of cell metabolism and protection against environmental hazards.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.     

High efficiency of ROS production by glycerophosphate dehydrogenase in mammalian mitochondria

We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and high (brown adipose tissue, BAT) content of glycerophosphate dehydrogenase (mGPDH). ROS production at state 4 due to electron backflow from mGPDH was low, but after inhibition of electron transport with antimycin A high rates of mGPDH-dependent ROS production were observed in liver, heart and brain mitochondria. When this ROS production was related to activity of mGPDH, many-fold higher ROS production was found in contrast to succinate- (39-, 28-, 3-fold) or pyruvate plus malate-dependent ROS production (32-, 96-, 5-fold). This specific rate of mGPDH-dependent ROS production was also exceedingly higher (28-, 66-, 22-fold) compared to that in BAT. mGPDH-dependent ROS production was localized to the dehydrogenase + CoQ and complex III, the latter being the highest in all mitochondria but BAT. Our results demonstrate high efficiency of mGPDH-dependent ROS production in mammalian mitochondria with a low content of mGPDH and suggest its endogenous inhibition in BAT.     

Expression of RAC2 in endothelial cells is required for the postnatal neovascular response

Herein, we describe an obligate role for the hematopoietic specific GTPase, RAC2 in endothelial integrin signaling and the postnatal neovascularization response in vivo. Using a Rac2 knockout mouse model, we discovered that despite the presence of both RAC1 and RAC2 protein in endothelial cells, RAC2 is obligately required for the postnatal neovascular response and αvβ3/α4β1/α5β1 integrin-directed migration on vitronectin, H296 and CH271, fibronectin fragments, respectively. The molecular basis for RAC2 specificity was explored. A genetic analysis of Syk −/+ or Syk−/+;Rac2 −/+ mice revealed that SYK kinase is required for the integrin induced activation of RAC2. The analysis of endothelial cells from Rac2−/+ versus Syk−/+;Rac2−/+ mice provided genetic evidence that SYK-RAC2 signaling axis regulates integrin (αvβ3, α4β1 and α5β1) dependent migration. Our results provide evidence that a specific region of the nonreceptor protein tyrosine kinase, SYK, the B linker region containing Y342 and Y346 is required for SYK's regulation of RAC2 and integrin dependent migration. Moreover, the capacity of mice to vascularize the ischemic hindlimb following femoral artery ligation or matrigel plugs was markedly reduced in mice homozygous deficient for the Rac2 gene. These findings identify a novel signaling axis for the induction and potential modulation of postnatal angiogenesis.     

Post-translational processing, metabolic stability and catalytic efficiency of oat arginine decarboxylase expressed in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes are auxotrophic for polyamines because they are unable to synthesize putrescine de novo. This deficiency is due to the absence of ornithine and arginine decarboxylase genes in the parasite genome. We have been able to obtain transgenic T. cruzi expressing heterologous genes coding for these enzymes. Since arginine decarboxylase normal expression in oat requires a post-translational proteolytic cleavage of an enzyme precursor, we have investigated whether a similar processing occurs inside the transformed protozoa expressing oat arginine decarboxylase or the same enzyme attached to a C-terminal (his)₆-tag. We were able to demonstrate that the post-translational processing also takes place inside the transgenic parasites. This cleavage is probably the result of a general proteolytic activity of T. cruzi acting on a protease-sensitive region of the protein. Interestingly, the (his)₆-tagged enzyme expressed in the transformed parasites showed considerably increased metabolic stability and catalytic efficiency.