Metal ion interaction with a novel anthracene pendant-armed fluorescent molecular probe. Synthesis, characterization, and fluorescence studies

A new scorpionate system (L) containing an emissive anthracene pendant arm, derived of O¹,O⁷-bis(2-formylphenyl)-1,4,7-trioxaheptane and tren, has been synthesized and characterized. The sensing capability of L towards a range of metal ions has been studied. Protonation and complexation studies, using UV-Vis and fluorescent emission measurements, have been performed with alkaline and alkaline earth metal ions (M = Na(I), K(I), Li(I), Ca(II), Mg(II)), as well as transition and post-transition metal ions (M = Cr(III), Cu(II) and Zn(II), Cd(II), Hg(II) and Al(III)). An increase in the fluorescence emission (CHEF effect) was observed in methanol and in methanol/water mixtures in the presence of Cd(II) (5.0-fold), Zn(II) (4.5-fold), Cr(III) (2.0-fold) and Al(III) (1.8-fold); these results suggest a notable sensing ability of this new N₃O₄ ligand for these metals; these experiments were also performed in the presence of large amounts of alkaline and alkaline earth metal ions.     

Identification of a novel streptococcal gene cassette mediating SOS mutagenesis in Streptococcus uberis

Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found to increase mutations to antibiotic resistance in Streptococcus uberis cultures. The mutational spectra revealed mainly G:C-->A:T transitions, and Northern analyses demonstrated increased expression of a Y-family DNA polymerase resembling UmuC under DNA-damaging conditions. In the absence of the Y-family polymerase, S. uberis cells were sensitive to UV light and to mitomycin C. Furthermore, the UV-induced mutagenesis was almost completely abolished in cells deficient in the Y-family polymerase. The gene encoding the Y-family polymerase was localized in a four-gene operon including two hypothetical genes and a gene encoding a HdiR homolog. Electrophoretic mobility shift assays demonstrated that S. uberis HdiR binds specifically to an inverted repeat sequence in the promoter region of the four-gene operon. Database searches revealed conservation of the gene cassette in several Streptococcus species, including at least one genome each of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Streptococcus sanguinis, and Streptococcus thermophilus strains. In addition, the umuC operon was localized in several mobile DNA elements of Streptococcus and Lactococcus species. We conclude that the hdiR-umuC-ORF3-ORF4 operon represents a novel gene cassette capable of mediating SOS mutagenesis among members of the Streptococcaceae.     

High-altitude chronic hypoxia during gestation and after birth modifies cardiovascular responses in newborn sheep

Perinatal exposure to chronic hypoxia induces sustained pulmonary hypertension and structural and functional changes in both pulmonary and systemic vascular beds. The aim of this study was to analyze consequences of high-altitude chronic hypoxia during gestation and early after birth in pulmonary and femoral vascular responses in newborn sheep. Lowland (LLNB; 580 m) and highland (HLNB; 3,600 m) newborn lambs were cathetherized under general anesthesia and submitted to acute sustained or stepwise hypoxic episodes. Contractile and dilator responses of isolated pulmonary and femoral small arteries were analyzed in a wire myograph. Under basal conditions, HLNB had a higher pulmonary arterial pressure (PAP; 20.2 +/- 2.4 vs. 13.6 +/- 0.5 mmHg, P < 0.05) and cardiac output (342 +/- 23 vs. 279 +/- 13 ml x min(-1) x kg(-1), P < 0.05) compared with LLNB. In small pulmonary arteries, HLNB showed greater contractile capacity and higher sensitivity to nitric oxide. In small femoral arteries, HLNB had lower maximal contraction than LLNB with higher maximal response and sensitivity to noradrenaline and phenylephrine. In acute superimposed hypoxia, HLNB reached higher PAP and femoral vascular resistance than LLNB. Graded hypoxia showed that average PAP was always higher in HLNB compared with LLNB at any Po2. Newborn lambs from pregnancies at high altitude have stronger pulmonary vascular responses to acute hypoxia associated with higher arterial contractile status. In addition, systemic vascular response to acute hypoxia is increased in high-altitude newborns, associated with higher arterial adrenergic responses. These responses determined in intrauterine life and early after birth could be adaptive to chronic hypoxia in the Andean altiplano.     

Behavioral syndromes and the evolution of correlated behavior in zebrafish

Studies of "behavioral syndromes" in different populations andspecies of animals can be used to shed light on the underlyingmechanisms of evolution. For example, some personality syndromessuggest the existence of an underlying hormonal link, whereasother relationships between boldness and aggression appear tobe the result of similar selective pressures. Here, we used1 wild-derived and 2 laboratory strains of zebrafish (Daniorerio) to examine relationships among 5 behavioral measures:shoaling, activity level, predator approaches, latency to feedafter a disturbance, and biting to a mirror stimulus. We foundevidence of an activity syndrome, as if underlying metaboliccosts influence variation in multiple forms of behavior. Evidencefor a relationship between boldness and aggression was alsoapparent but depended both on strain and which specific behaviorpatterns were identified as measures of "boldness." Althoughsome comparisons of laboratory and wild-derived strains wereconsistent with a domestication syndrome, others were not. Mostobserved relationships were relatively weak and occasionallyinconsistent, arguing against strong underlying genetic linkagesor pleiotropic effects relating any of the behavioral measures.Instead, it may be more important to study the details of selectivecontext or the long-term impact of linkages between some, butnot all, of a large set of genes influencing complex behavioraltraits. We found profound differences among strains in mostbehavior patterns, but few sex differences. One strain (TM1)was consistently different from the others (SH and Nadia) beingmore social, more likely to approach predators, and taking lesstime to recover from disturbance than were the other 2 strains.     

LTP inhibits LTD in the hippocampus via regulation of GSK3beta

Glycogen synthase kinase-3 (GSK3) has been implicated in major neurological disorders, but its role in normal neuronal function is largely unknown. Here we show that GSK3beta mediates an interaction between two major forms of synaptic plasticity in the brain, N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) and NMDA receptor-dependent long-term depression (LTD). In rat hippocampal slices, GSK3beta inhibitors block the induction of LTD. Furthermore, the activity of GSK3beta is enhanced during LTD via activation of PP1. Conversely, following the induction of LTP, there is inhibition of GSK3beta activity. This regulation of GSK3beta during LTP involves activation of NMDA receptors and the PI3K-Akt pathway and disrupts the ability of synapses to undergo LTD for up to 1 hr. We conclude that the regulation of GSK3beta activity provides a powerful mechanism to preserve information encoded during LTP from erasure by subsequent LTD, perhaps thereby permitting the initial consolidation of learnt information.     

High CO2 Levels Cause Skeletal Muscle Atrophy via AMP-activated Kinase (AMPK), FoxO3a Protein,and Muscle-specific Ring Finger Protein 1 (MuRF1)

Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO₂ had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO₂ had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO₂ induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1^−/− mice exposed to high CO₂ did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO₂, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO₂ induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO₂ activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.     

Abscisic Acid Transport in Human Erythrocytes

Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic β-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [³H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [³H]ABA and [³⁵S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone.     

Comprehensive miRNA sequence analysis reveals survival differences in diffuse large B-cell lymphoma patients

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation.ResultsWe identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas.ConclusionsOur comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0568-y) contains supplementary material, which is available to authorized users.     

Effect of the charge distribution along the “ferritin-like” pores of the proteins from the Dps family on the iron incorporation process

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the “ferritin-like” pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.