2,3-Bisphosphoglycerate,fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.     

Metabolism of glycerate-2,3-P2--II. Enzymes involved in the glycerate-2,3-P2 metabolism in chicken skeletal muscle

1. Four enzyme fractions which may be involved in the synthesis and breakdown of glycerate-2,3-P2 have been isolated from extracted skeletal muscle by gel-filtration and ion-exchange chromatography. 2. One of the fractions, corresponding to the glycerate-2,3-P2 dependent phosphoglycerate mutase, has been purified to homogeneity. In addition to the main enzymatic activity, it shows intrinsic glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity stimulable by glycolate-2-P. Its synthase activity represents about 10% of the total synthase activity of the tissue, and its phosphatase activity corresponds to about 60% of the total phosphatase activity. 3. Two of the fractions have glycerate-2,3-P2 synthase, glycerate-2,3-P2 phosphatase and phosphoglycerate mutase activities in a ratio similar to that of the glycerate-2,3-P2 synthase described in mammalian skeletal muscle. Their synthase activity corresponds to about 90% of the total synthase activity, and their phosphatase activity represents about 1% of the total phosphatase activity of the tissue. 4. The fourth fraction shows only glycerate-2,3-P2 phosphatase activity and represents about 40% of the total activity of the tissue. 5. It is suggested that in chicken skeletal muscle the metabolism of the glycerate-2,3-P2 is regulated in a way similar to that described in mammalian skeletal muscle.     

α<Subscript>2A</Subscript>- and α<Subscript>2C</Subscript>-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands

The poor norepinephrine innervation and high density of Gi/o-coupled α_2A- and α_2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D₂-like receptor ligands, such as the D₃ receptor agonist 7-OH-PIPAT and the D₄ receptor agonist RO-105824, to α₂-adrenoceptors in cortical and striatal tissue, which express α_2A-adrenoceptors and both α_2A- and α_2C-adrenoceptors, respectively. The affinity of dopamine for α₂-adrenoceptors was found to be similar to that for D₁-like and D₂-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α_2A- and α_2C-adrenoceptors. Their ability to activate Gi/o proteins through α_2A- and α_2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α₂-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α_2A- and α_2C-adrenoceptors was nearly identical to its binding to the crystallized D₃ receptor. Therefore, we provide conclusive evidence that α_2A- and α_2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D₂-like receptor ligands, which calls for revisiting previous studies with those ligands.     

Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5? were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532–587?nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.     

Natural variation in life history strategy of Arabidopsis thaliana determines stress responses to drought and insects of different feeding guilds

Plants are sessile organisms and, consequently, are exposed to a plethora of stresses in their local habitat. As a result, different populations of a species are subject to different selection pressures leading to adaptation to local conditions and intraspecific divergence. The annual brassicaceous plant Arabidopsis thaliana is an attractive model for ecologists and evolutionary biologists due to the availability of a large collection of resequenced natural accessions. Accessions of A. thaliana display one of two different life cycle strategies: summer and winter annuals. We exposed a collection of 308 European Arabidopsis accessions, that have been genotyped for 250K SNPs, to a range of stresses: one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips and Myzus persicae aphids) and two combined stresses (drought plus P. rapae and Botrytis cinerea fungus plus P. rapae). We identified heritable genetic variation for responses to the different stresses, estimated by narrow‐sense heritability. We found that accessions displaying different life cycle strategies differ in their response to stresses. Winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars. Summer annuals are also more resistant to the combined stresses of drought plus P. rapae and infection by the fungus Botryris cinerea plus herbivory by P. rapae. Adaptation to drought displayed a longitudinal gradient. Finally, trade‐offs were recorded between the response to drought and responses to herbivory by caterpillars of the specialist herbivore P. rapae.     

An N-terminal diacidic motif is required for the trafficking of maize aquaporins ZmPIP2;4 and ZmPIP2;5 to the plasma membrane

Maize plasma membrane aquaporins (ZmPIPs, where PIP is the plasma membrane intrinsic protein) fall into two groups, ZmPIP1s and ZmPIP2s, which, when expressed alone in mesophyll protoplasts, are found in different subcellular locations. Whereas ZmPIP1s are retained in the endoplasmic reticulum (ER), ZmPIP2s are found in the plasma membrane (PM). We previously showed that, when co-expressed with ZmPIP2s, ZmPIP1s are relocalized to the PM, and that this relocalization results from the formation of hetero-oligomers between ZmPIP1s and ZmPIP2s. To determine the domains responsible for the ER retention and PM localization, respectively, of ZmPIP1s and ZmPIP2s, truncated and mutated ZmPIPs were generated, together with chimeric proteins created by swapping the N- or C-terminal regions of ZmPIP2s and ZmPIP1s. These mutated proteins were fused to the mYFP and/or mCFP, and the fusion proteins were expressed in maize mesophyll protoplasts, and were then localized by microscopy. This allowed us to identify a diacidic motif, DIE (Asp-Ile-Glu), at position 4–6 of the N-terminus of ZmPIP2;5, that is essential for ER export. This motif was conserved and functional in ZmPIP2;4, but was absent in ZmPIP2;1. In addition, we showed that the N-terminus of ZmPIP2;5 was not sufficient to cause the export of ZmPIP1;2 from the ER. A study of ZmPIP1;2 mutants suggested that the N- and C-termini of this protein are probably not involved in ER retention. Together, these results show that the trafficking of maize PM aquaporins is differentially regulated depending on the isoform, and involves a specific signal and mechanism.     

Assessment of Real-time PCR Based Methods for Quantification of Pollen-mediated Gene Flow from GM to Conventional Maize in a Field Study

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1–10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.     

Back to the Future: The Responses of Alpine Treelines to Climate Warming are Constrained by the Current Ecotone Structure

Alpine treeline ecotones are considered early-warning monitors of the effects of climate change on terrestrial ecosystems, but it is still unclear how accurately treeline dynamics may track the expected temperature rises. Site-specific abiotic constraints, such as topography and demographic trends may make treelines less responsive to environmental fluctuations. A better understanding on how local processes modulate treelines’ response to warming is thus required. We developed a model of treeline dynamics based on individual data of growth, mortality and reproduction. Specifically, we modeled growth patterns, mortality rates and reproductive size thresholds as a function of temperature and stand structure to evaluate the influence of climate- and stand-related processes on treeline dynamics. In this study, we analyze the dynamics of four Pyrenean mountain pine treeline sites with contrasting stand structures, and subjected to differing rates of climate warming. Our models indicate that Pyrenean treelines could reach basal areas and reproductive potentials similar to those currently observed in high-elevation subalpine forest by the mid twenty-first century. The fastest paces of treeline densification are forecasted by the late twenty-first century and are associated with higher warming rates. We found a common densification response of Pyrenean treelines to climate warming, but contrasting paces arise due to current size structures. Treelines characterized by a multistratified stand structure and subjected to lower mean annual temperatures were the most responsive to climate warming. In monostratified stands, tree growth was less sensitive to temperature than in multistratified stands and trees reached their reproductive size threshold later. Therefore, our simulations highlight that stand structure is paramount in modulating treeline responsiveness to ongoing climate warming. Synthesis. Treeline densification over the twenty-first century is likely to occur at different rates contingent on current stand structure and its effects on individual-level tree growth responses to warming. Accurate projections of future treeline dynamics must thus incorporate site-specific factors other than climate, specifically those related to stand structure and its influence on tree growth.     

Mapping the eosinophil cationic protein antimicrobial activity by chemical and enzymatic cleavage

The eosinophil cationic protein (ECP) is a human antimicrobial protein involved in the host immune defense that belongs to the pancreatic RNase A family. ECP displays a wide range of antipathogen activities. The protein is highly cationic and its bactericidal activity is dependant on both cationic and hydrophobic surface exposed residues. Previous studies on ECP by site-directed mutagenesis indicated that the RNase activity is not essential for its bactericidal activity. To further understand the ECP bactericidal mechanism, we have applied enzymatic and chemical limited cleavage to search for active sequence determinants.Following a search for potential peptidases we selected the Lys-endoproteinase, which cleaves the ECP polypeptide at the carboxyl side of its unique Lys residue, releasing the N-terminal fragment (0-38).Chemical digestion using cyanogen bromide released several complementary peptides at the protein N-terminus. Interestingly, ECP treatment with cyanogen bromide represents a new example of selective chemical cleavage at the carboxyl side of not only Met but also Trp residues. Recombinant ECP was denatured and carboxyamidomethylated prior to enzymatic and chemical cleavage. Irreversible denaturation abolishes the protein bactericidal activity.The characterization of the digestion products by both enzymatic and chemical approaches identifies a region at the protein N-terminus, from residues 11 to 35, that retains the bactericidal activity. The most active fragment, ECP(0-38), is further compared to ECP derived synthetic peptides. The region includes previously identified stretches related to lipopolysaccharide binding and bacteria agglutination. The results contribute to define the shortest ECP minimized version that would retain its antimicrobial properties. The data suggest that the antimicrobial RNase can provide a scaffold for the selective release of cytotoxic peptides.     

Post-epizootic chronic dolphin morbillivirus infection in Mediterranean striped dolphins Stenella coeruleoalba

Dolphin morbillivirus (DMV) has caused 2 epizootics with high mortality rates on the Spanish Mediterranean coast, in 1990 and 2006-07, mainly affecting striped dolphins Stenella coeruleoalba. Following the first epizootic unusual DMV infections affecting only the central nervous system of striped dolphins were found, with histological features similar to subacute sclerosing panencephalitis and old dog encephalitis, the chronic latent localised infections caused by defective forms of measles virus and canine distemper virus, respectively. Between 2008 and 2010, monitoring by microscopic and immunohistochemical (IHC) studies of 118 striped dolphins stranded along Catalonia, the Valencia Region and Andalusia showed similar localised DMV nervous system infections in 25.0, 28.6 and 27.4% of cases, respectively, with no significant differences among regions or sex. The body length of DMV-infected dolphins was statistically greater than that of non-infected dolphins (196.5 vs. 160.5 cm; p < 0.001). Molecular detection of DMV was performed by 2 different RT-PCR techniques amplifying a 429 bp fragment and a 78 bp fragment both within the phosphoprotein (P) gene. The 429 bp RT-PCR results contradicted the IHC-DMV results as only 3 of 6 dolphins with positive IHC-DMV had positive PCR results. All 6 cases were positive with the 78 bp RT-PCR. These findings contraindicate the use of the 429 bp RT-PCR protocol based on the P gene to detect this specific form of DMV. DMV localised nervous infection constitutes the most relevant single cause of stranding and death in Mediterranean striped dolphins in the years following a DMV epizootic, and it might even overwhelm the effects of the epizootic itself, at least in 2007.