Imaging Oxygen Distribution in Marine Sediments. The Importance of Bioturbation and Sediment Heterogeneity

The influence of sediment oxygen heterogeneity, due to bioturbation, on diffusive oxygen flux was investigated. Laboratory experiments were carried out with 3 macrobenthic species presenting different bioturbation behaviour patterns:the polychaetes Nereis diversicolor and Nereis virens, both constructing ventilated galleries in the sediment column, and the gastropod Cyclope neritea, a burrowing species which does not build any structure. Oxygen two-dimensional distribution in sediments was quantified by means of the optical planar optode technique. Diffusive oxygen fluxes (mean and integrated) and a variability index were calculated on the captured oxygen images. All species increased sediment oxygen heterogeneity compared to the controls without animals. This was particularly noticeable with the polychaetes because of the construction of more or less complex burrows. Integrated diffusive oxygen flux increased with oxygen heterogeneity due to the production of interface available for solute exchanges between overlying water and sediments. This work shows that sediment heterogeneity is an important feature of the control of oxygen exchanges at the sediment–water interface

CYP17 5'-UTR MspA1 polymorphism and the risk of premenopausal breast cancer in a German population-based case–control study

INTRODUCTION: Studies on the association between the cytochrome P450c17α gene (CYP17) 5'-untranslated region MspA1 genetic polymorphism and breast cancer risk have yielded inconsistent results. Higher levels of estrogen have been reported among young nulliparous women with the A2 allele. Therefore we assessed the impact of CYP17 genotypes on the risk of premenopausal breast cancer, with emphasis on parity. METHODS: We used data from a population-based case–control study of women aged below 51 years conducted from 1992 to 1995 in Germany. Analyses were restricted to clearly premenopausal women with complete information on CYP17 and encompassed 527 case subjects and 904 controls, 99.5% of whom were of European descent. The MspA1 polymorphism was analyzed using PCR-RFLP (PCR–restriction fragment length polymorphism) assay. RESULTS: The frequencies of the variant allele among the cases and controls were 43% and 41%, respectively. Overall, CYP17 A1/A2 and A2/A2 genotypes compared with the A1/A1 genotype were not associated with breast cancer, with adjusted odds ratios (ORs) of 1.04 and 1.23, respectively. Among nulliparous women, however, breast cancer risk was elevated for the A1/A2 (OR = 1.31; 95% confidence interval (CI) 0.74 to 2.32) and the A2/A2 genotype (OR = 2.12; 95% CI 1.04 to 4.32) compared with the A1/A1 genotype, with a trend towards increasing risk associated with number of A2 alleles (P = 0.04). Otherwise, the CYP17 polymorphism was found neither to be an effect modifier of breast cancer risks nor to be associated with stage of disease. CONCLUSION: Our results do not indicate a major influence of CYP17 MspA1 polymorphism on the risk of premenopausal breast cancer, but suggest that it may have an impact on breast cancer risk among nulliparous women. The finding, however, needs to be confirmed in further studies

Inhibition of Hedgehog Signaling Decreases Proliferation and Clonogenicity of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSC) have the ability to differentiate into osteoblasts, adipocytes and chondrocytes. We have previously shown that hMSC were endowed with a basal level of Hedgehog signaling that decreased after differentiation of these cells. Since hMSC differentiation is associated with growth-arrest we investigated the function of Hh signaling on cell proliferation. Here, we show that inhibition of Hh signaling, using the classical inhibitor cyclopamine, or a siRNA directed against Gli-2, leads to a decrease in hMSC proliferation. This phenomenon is not linked to apoptosis but to a block of the cells in the G0/G1 phases of the cell cycle. At the molecular level, it is associated with an increase in the active form of pRB, and a decrease in cyclin A expression and MAP kinase phosphorylation. Inhibition of Hh signaling is also associated with a decrease in the ability of the cells to form clones. By contrast, inhibition of Hh signaling during hMSC proliferation does not affect their ability to differentiate. This study demonstrates that hMSC are endowed with a basal Hedgehog signaling activity that is necessary for efficient proliferation and clonogenicity of hMSC. This observation unravels an unexpected new function for Hedgehog signaling in the regulation of human mesenchymal stem cells and highlights the critical function of this morphogen in hMSC biology

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat-/- mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation. © 2013 McGraw et al

Crystal structure of tarocystatin–papain complex: implications for the inhibition property of group-2 phytocystatins

Tarocystatin (CeCPI) from taro (Colocasia esculenta cv. Kaohsiung no. 1), a group-2 phytocystatin, shares a conserved N-terminal cystatin domain (NtD) with other phytocystatins but contains a C-terminal cystatin-like extension (CtE). The structure of the tarocystatin–papain complex and the domain interaction between NtD and CtE in tarocystatin have not been determined. We resolved the crystal structure of the phytocystatin–papain complex at resolution 2.03 Å. Surprisingly, the structure of the NtD–papain complex in a stoichiometry of 1:1 could be built, with no CtE observed. Only two remnant residues of CtE could be built in the structure of the CtE–papain complex. Therefore, CtE is easily digested by papain. To further characterize the interaction between NtD and CtE, three segments of tarocystatin, including the full-length (FL), NtD and CtE, were used to analyze the domain–domain interaction and the inhibition ability. The results from glutaraldehyde cross-linking and yeast two-hybrid assay indicated the existence of an intrinsic flexibility in the region linking NtD and CtE for most tarocystatin molecules. In the inhibition activity assay, the glutathione-S-transferase (GST)-fused FL showed the highest inhibition ability without residual peptidase activity, and GST-NtD and FL showed almost the same inhibition ability, which was higher than with NtD alone. On the basis of the structures, the linker flexibility and inhibition activity of tarocystatins, we propose that the overhangs from the cystatin domain may enhance the inhibition ability of the cystatin domain against papain

The role of interfacial lipids in stabilizing membrane protein oligomers

Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways1 but is often difficult to define2 or predict3. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT4, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET5, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors

The freshwater Sponge Ephydatia Fluviatilis harbours diverse pseudomonas species (Gammaproteobacteria, Pseudomonadales) with broad-spectrum antimicrobial activity

Bacteria are believed to play an important role in the fitness and biochemistry of sponges (Porifera). Pseudomonas species (Gammaproteobacteria, Pseudomonadales) are capable of colonizing a broad range of eukaryotic hosts, but knowledge of their diversity and function in freshwater invertebrates is rudimentary. We assessed the diversity, structure and antimicrobial activities of Pseudomonas spp. in the freshwater sponge Ephydatia fluviatilis. Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints of the global regulator gene gacA revealed distinct structures between sponge-associated and free-living Pseudomonas communities, unveiling previously unsuspected diversity of these assemblages in freshwater. Community structures varied across E. fluviatilis specimens, yet specific gacA phylotypes could be detected by PCR-DGGE in almost all sponge individuals sampled over two consecutive years. By means of whole-genome fingerprinting, 39 distinct genotypes were found within 90 fluorescent Pseudomonas isolates retrieved from E. fluviatilis. High frequency of in vitro antibacterial (49%), antiprotozoan (35%) and anti-oomycetal (32%) activities was found among these isolates, contrasting less-pronounced basidiomycetal (17%) and ascomycetal (8%) antagonism. Culture extracts of highly predation-resistant isolates rapidly caused complete immobility or lysis of cells of the protozoan Colpoda steinii. Isolates tentatively identified as P. jessenii, P. protegens and P. oryzihabitans showed conspicuous inhibitory traits and correspondence with dominant sponge-associated phylotypes registered by cultivation-independent analysis. Our findings suggest that E. fluviatilis hosts both transient and persistent Pseudomonas symbionts displaying antimicrobial activities of potential ecological and biotechnological value.European Regional Development Fund (ERDF) through the COMPETE (Operational Competitiveness Programme); national funds through FCT (Foundation for Science and Technology) [PEst-C/MAR/LA0015/2011]; FCT-funded project [PTDC/BIA-MIC/3865/2012]; Federation of European Microbiological Societies (FEMS)info:eu-repo/semantics/publishedVersio

Impact of Anti-Inflammatory Agents on the Gene Expression Profile of Stimulated Human Neutrophils: Unraveling Endogenous Resolution Pathways

Adenosine, prostaglandin E2, or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A2A receptor agonist CGS 21680, prostaglandin E2, cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution