Insight in the transport behavior of copper glycinate complexes through the porcine gastrointestinal membrane using an Ussing chamber assisted by mass spectrometry analysis

An Ussing chamber study was conducted in order to investigate the transport behavior of copper glycinate complexes through a porcine gastrointestinal membrane. Organic copper complexes such as copper tri- and tetraglycinates (GGG–Cu(II) and GGGG–Cu(II)) were used as model system. In a novel analytical approach the Ussing chamber was combined with mass spectrometry. Therefore, relevant analytical methods based on MALDI-MS and a coupling of capillary electrophoresis to ICP-MS and ESI-MS were developed for the determination of copper complexes in the mucosal and serosal half-chambers. It was found that 86.1±8.5% of copper triglycinate but only 20.8±9.9% of copper tetraglycinate penetrated the digestive membrane without modification. Furthermore, inorganic copper species were not detected but a new copper complex (m/z 442) was found to be formed in both compartments of the Ussing chamber.     

Functionality of the GAL4/UAS system in <Emphasis Type="Italic">Tribolium</Emphasis> requires the use of endogenous core promoters

Background  

The red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.     

SRP,FtsY, DnaK and YidC Are Required for the Biogenesis of the E. coli Tail-Anchored Membrane Proteins DjlC and Flk

Tail-anchored membrane proteins (TAMPs) are relatively simple membrane proteins characterized by a single transmembrane domain (TMD) at their C-terminus. Consequently, the hydrophobic TMD, which acts as a subcellular targeting signal, emerges from the ribosome only after termination of translation precluding canonical co-translational targeting and membrane insertion. In contrast to the well-studied eukaryotic TAMPs, surprisingly little is known about the cellular components that facilitate the biogenesis of bacterial TAMPs. In this study, we identify DjlC and Flk as bona fide Escherichia coli TAMPs and show that their TMDs are necessary and sufficient for authentic membrane targeting of the fluorescent reporter mNeonGreen. Using strains conditional for the expression of known E. coli membrane targeting and insertion factors, we demonstrate that the signal recognition particle (SRP), its receptor FtsY, the chaperone DnaK and insertase YidC are each required for efficient membrane localization of both TAMPs. A close association between the TMD of DjlC and Flk with both the Ffh subunit of SRP and YidC was confirmed by site-directed in vivo photo-crosslinking. In addition, our data suggest that the hydrophobicity of the TMD correlates with the dependency on SRP for efficient targeting.     

Oximes short-acting CB1 receptor agonists

New oximes short-acting CB1 agonists were explored by the introduction of an internal oxime and polar groups at the C3 alkyl tail of Δ⁸-THC. The scope of the research was to drastically alter two important physicochemical properties hydrophobicity (log P) and topological surface area (tPSA) of the compound, which play a critical role in tissue distribution and sequestration (depot effect). Key synthesized analogs demonstrated sub-nanomolar affinity for CB1, marked reduction in hydrophobicity (ClogP～2.5–3.5 vs 9.09 of Δ⁸-THC-DMH), and found to function as either agonists (trans-oximes) or neutral antagonists (cis-oximes) in a cAMP functional assay. All oxime analogs showed comparable affinity at the CB2 receptor, but surprisingly they were found to function as inverse agonists for CB2. In behavioral studies (i.e. analgesia, hypothermia) trans-oxime 8a exhibited a predictable fast onset (～20?min) and short duration of pharmacological action (～180?min), in contrast to the very prolonged duration of Δ⁸-THC-DMH (>24?h), thus limiting the potential for severe psychotropic side-effects associated with persistent activation of the CB1 receptor. We have conducted 100?ns molecular dynamic (MD) simulations of CB1 complexes with AM11542 (CB1 agonist) and both trans-8a and cis-8b isomeric oximes. These studies revealed that the C3 alkyl tail of cis-8b orientated within the CB1 binding pocket in a manner that triggered a conformational change that stabilized the CB1 receptor at its inactive-state (antagonistic functional effect). In contrast, the trans-8a isomer’s conformation was coincided with that of the AM11542 CB1 agonist-bound structure, stabilizing the CB1 receptor at the active-state (agonistic functional effect). We have selected oxime trans-8a based on its potency for CB1, and favorable pharmacodynamic profile, such as fast onset and predictable duration of pharmacological action, for evaluation in pre-clinical models of anorexia nervosa.     

North-South Differentiation and a Region of High Diversity in European Wolves (Canis lupus)

European wolves (Canis lupus) show population genetic structure in the absence of geographic barriers, and across relatively short distances for this highly mobile species. Additional information on the location of and divergence between population clusters is required, particularly because wolves are currently recolonizing parts of Europe. We evaluated genetic structure in 177 wolves from 11 countries using over 67K single nucleotide polymorphism (SNP) loci. The results supported previous findings of an isolated Italian population with lower genetic diversity than that observed across other areas of Europe. Wolves from the remaining countries were primarily structured in a north-south axis, with Croatia, Bulgaria, and Greece (Dinaric-Balkan) differentiated from northcentral wolves that included individuals from Finland, Latvia, Belarus, Poland and Russia. Carpathian Mountain wolves in central Europe had genotypes intermediate between those identified in northcentral Europe and the Dinaric-Balkan cluster. Overall, individual genotypes from northcentral Europe suggested high levels of admixture. We observed high diversity within Belarus, with wolves from western and northern Belarus representing the two most differentiated groups within northcentral Europe. Our results support the presence of at least three major clusters (Italy, Carpathians, Dinaric-Balkan) in southern and central Europe. Individuals from Croatia also appeared differentiated from wolves in Greece and Bulgaria. Expansion from glacial refugia, adaptation to local environments, and human-related factors such as landscape fragmentation and frequent killing of wolves in some areas may have contributed to the observed patterns. Our findings can help inform conservation management of these apex predators and the ecosystems of which they are part.     

Ecoregion-Based Conservation Planning in the Mediterranean: Dealing with Large-Scale Heterogeneity

Spatial priorities for the conservation of three key Mediterranean habitats, i.e. seagrass Posidonia oceanica meadows, coralligenous formations, and marine caves, were determined through a systematic planning approach. Available information on the distribution of these habitats across the entire Mediterranean Sea was compiled to produce basin-scale distribution maps. Conservation targets for each habitat type were set according to European Union guidelines. Surrogates were used to estimate the spatial variation of opportunity cost for commercial, non-commercial fishing, and aquaculture. Marxan conservation planning software was used to evaluate the comparative utility of two planning scenarios: (a) a whole-basin scenario, referring to selection of priority areas across the whole Mediterranean Sea, and (b) an ecoregional scenario, in which priority areas were selected within eight predefined ecoregions. Although both scenarios required approximately the same total area to be protected in order to achieve conservation targets, the opportunity cost differed between them. The whole-basin scenario yielded a lower opportunity cost, but the Alboran Sea ecoregion was not represented and priority areas were predominantly located in the Ionian, Aegean, and Adriatic Seas. In comparison, the ecoregional scenario resulted in a higher representation of ecoregions and a more even distribution of priority areas, albeit with a higher opportunity cost. We suggest that planning at the ecoregional level ensures better representativeness of the selected conservation features and adequate protection of species, functional, and genetic diversity across the basin. While there are several initiatives that identify priority areas in the Mediterranean Sea, our approach is novel as it combines three issues: (a) it is based on the distribution of habitats and not species, which was rarely the case in previous efforts, (b) it considers spatial variability of cost throughout this socioeconomically heterogeneous basin, and (c) it adopts ecoregions as the most appropriate level for large-scale planning.     

The Shipwrecks in Greece are Going Fuzzy: A Study for the Potential of Oil Pollution from Shipwrecks in Greek Waters

This article deals with the development and application of a risk analysis approach regarding the importance and fate of shipwrecks in Greece. In the past few decades the potential harmful effects of wrecks and the materials and substances they carry, remaining on the sea bottom, have been realized and the issue of dealing with them has arisen. The methodology used in this article consists of three steps. The first step is to develop a dedicated database of shipwrecks in Greek waters. The second step focuses on the quasi-quantitative assessment of risk for each shipwreck individually through the introduction of implemented factors that have been modeled into the structure of a hierarchical fuzzy system. The analysis is concluded by determining the sources of uncertainty for the system and implementing an appropriate analysis to determine their relative importance. Finally, geographic information system (GIS) tools are used for the visualization of data and the analysis of results in geographic terms as well as for revealing geographic patterns and relationships.     

Role of oxidative stress and DNA damage in human carcinogenesis

Cells in tissues and organs are continuously subjected to oxidative stress and free radicals on a daily basis. This free radical attack has exogenous or endogenous (intracellular) origin. The cells withstand and counteract this occurrence by the use of several and different defense mechanisms ranging from free radical scavengers like glutathione (GSH), vitamins C and E and antioxidant enzymes like catalase, superoxide dismutase and various peroxidases to sophisticated and elaborate DNA repair mechanisms. The outcome of this dynamic equilibrium is usually the induction of oxidatively induced DNA damage and a variety of lesions of small to high importance and dangerous for the cell i.e. isolated base lesions or single strand breaks (SSBs) to complex lesions like double strand breaks (DSBs) and other non-DSB oxidatively generated clustered DNA lesions (OCDLs). The accumulation of DNA damage through misrepair or incomplete repair may lead to mutagenesis and consequently transformation particularly if combined with a deficient apoptotic pathway. In this review, we present the current status of knowledge and evidence on the mechanisms and involvement of intracellular oxidative stress and DNA damage in human malignancy evolution and possible use of these parameters as cancer biomarkers. At the same time, we discuss controversies related to potential artifacts inherent to specific methodologies used for the measurement of oxidatively induced DNA lesions in human cells or tissues.     

A versatile strategy for gene trapping and trap conversion in emerging model organisms

Genetic model organisms such as Drosophila, C. elegans and the mouse provide formidable tools for studying mechanisms of development, physiology and behaviour. Established models alone, however, allow us to survey only a tiny fraction of the morphological and functional diversity present in the animal kingdom. Here, we present iTRAC, a versatile gene-trapping approach that combines the implementation of unbiased genetic screens with the generation of sophisticated genetic tools both in established and emerging model organisms. The approach utilises an exon-trapping transposon vector that carries an integrase docking site, allowing the targeted integration of new constructs into trapped loci. We provide proof of principle for iTRAC in the emerging model crustacean Parhyale hawaiensis: we generate traps that allow specific developmental and physiological processes to be visualised in unparalleled detail, we show that trapped genes can be easily cloned from an unsequenced genome, and we demonstrate targeting of new constructs into a trapped locus. Using this approach, gene traps can serve as platforms for generating diverse reporters, drivers for tissue-specific expression, gene knockdown and other genetic tools not yet imagined.     

The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development

Although we have greatly benefited from the use of traditional epidemiological approaches in linking environmental exposure to human disease, we are still lacking knowledge in to how such exposure participates in disease development. However, molecular epidemiological studies have provided us with evidence linking oxidative stress with the pathogenesis of human disease and in particular carcinogenesis. To this end, oxidative stress-based biomarkers have proved to be essential in revealing how oxidative stress may be mediating toxicity induced by many known carcinogenic environmental agents. Therefore, throughout this review article, we aim to address the current state of oxidative stress-based biomarker development with major emphasis pertaining to biomarkers of DNA, lipid and protein oxidation.