Maternal smoking is associated with mitochondrial DNA depletion and respiratory chain complex III deficiency in placenta

Maternal smoking during pregnancy is often associated with a decrease in placental function, which might lead to intrauterine growth retardation. Because tobacco is known to alter the mitochondrial respiratory function in cardiomyocytes and lung tissue, we hypothesized that placental mitochondrial function could be altered by maternal smoking. Placental mitochondria from 9 smoking and 19 nonsmoking mothers were isolated by differential centrifugation. Mitochondrial oxygen consumption was measured by polarography, and the enzymatic activity of each complex of the electron transport chain was assessed by spectrophotometry. In addition, the relative content in mitochondrial DNA (mtDNA) was determined by real-time quantitative PCR in placentas from seven smoking and seven nonsmoking mothers. We observed a 29% reduction in the enzymatic activity of complex III in the placental mitochondria from smokers compared with nonsmokers (P = 0.03). The relative content of mtDNA (with respect to the beta-globin gene) was reduced by 37% in the placental tissue from smokers compared with nonsmokers (P < 0.02). Both the enzymatic activity of complex III and mtDNA content were inversely related with the daily consumption of cigarettes, and mtDNA content was correlated with cord blood insulin-like growth factor-binding protein-3 (r = 0.74, P < 0.01), a marker of fetal growth. These results show that maternal smoking is associated with placental mitochondrial dysfunction, which might contribute to restricted fetal growth by limiting energy availability in cells.     

Spatial patterns of tree seedling establishment and their relationship to environmental variables in a cold-temperate deciduous forest of eastern North America

Patterns of seedling recruitment may have persistent effects on population and community processes. Assuming seed availability is not limiting, the environmental sieve (i.e., the suite of factors influencing seed germination and seedling emergence and survival) determines how many seedlings establish and, most importantly, where they do so. In this study, we identify the spatial structure of some resources and abiotic conditions known to be significant for tree seedling emergence and survival and determine how these environmental factors influence the establishment of Fagus grandifolia, Acer saccharum, Fraxinus americana, and Ostrya virginiana in a deciduous forest of southern Québec (Canada). We expect an increase from Fagus, through Acer and Fraxinus, to Ostrya in the control of environmental variables on seedling emergence and survival, because of differences in the seed size of these species. Density of newly-emerged seedlings of all four species showed positive spatial autocorrelation at distances of up to ca. 10 m. Environmental variables were also structured at the same spatial scale, except for soil moisture. Acer seedling emergence pattern was positively correlated to photosynthetic photon flux density (PPFD), and the pattern of Fraxinus to soil N and moisture. Seedling survival was not spatially autocorrelated for any of the four species, although it was positively density-dependent in Acer and Fagus. In only Ostrya was seedling survival correlated (positively) to one of the environmental variables studied, i.e., PPFD. Overall, environmental variables were spatially less heterogeneous than seedling emergence and survival. Either seed availability was not saturating or factors not considered here, such as competition and predation (the intensity of which often varies with resources and/or abiotic conditions), modified the influence that the physical environment had on patterns of seedling establishment. Our prediction of a greater environmental control on seedling emergence and survival in small-seed species was not totally confirmed.     

Characterization of Brucella abortus lipopolysaccharide macrodomains as mega rafts

The lipopolysaccharides (LPS) of intracellular Proteobacteria such as Brucella, Chlamydia, Legionella and Rickettsia, have properties distinct from enterobacterial LPSs. These properties include deficient LPS induction of host cell activation, low endotoxicity and resistance to macrophage degradation. Together these constitute key virulence mechanisms for intracellular survival and replication. We previously demonstrated that B. abortus LPS captured by macrophages was recycled back to the plasma membrane where it was found associated with macrodomains. Furthermore, this LPS interferes with the MHC class II (MHC-II) presentation of peptides to specific T cell hybridomas. Here, we characterized the Brucella LPS macrodomains by microscopy and biochemistry approaches. We show for the first time that LPS macrodomains act as detergent resistant membranes (DRMs), segregating several lipid-raft components, LPS-binding proteins and MHC-II molecules. Brucella LPS macrodomains remain intact for several months in macrophages and are resistant to the disruptive effects of methyl beta-cyclodextrin. Fluorescent anisotropy measurements show that B. abortus LPS is responsible for the formation of rigid surface membrane complexes. In addition, relocalization of MHC-II molecules is observed in these structures. The effects of B. abortus LPS on membrane properties could be responsible for pathogenic effects such as the inhibition of MHC-II-dependent antigen presentation.     

Chiroptical Spectra of Tetrakis (+)‐3‐Heptafluorobutylrylcamphorate Ln(III) Complexes with an Encapsulated Alkali Metal Ion: Solution Structures as Revealed by Chiroptical Spectra

The preparation of tetrakis((+)‐hfbc) lanthanide(III) complexes with an encapsulated alkali metal and ammonium ions M[Ln((+)‐hfbc)₄] (hereafter abbreviated as M‐Ln : (+)‐hfbc, (+)‐heptafluorobutyrylcamphorate; M, ammonium or benzyl ammonium ions as well as alkali metal ions) was reported and discussed. The electronic circular dichroism (CD) spectra in the intraligand π?π* transition of M–Ln were examined in view of the solvent effect. Here, the concentration, alkali metal, and ammonium ion dependences are compared with the solid CD, ⁵D₀ ← ⁷F₀(Eu(III)) excitation spectra, circularly polarized luminescence, and vibrational circular dichroism. It has been revealed that the dodecahedral eight coordinate DD‐8‐M‐Ln complexes in crystals are equilibrated between the diastereoselectively formed square antiprism eight coordinate SAPR‐8‐M‐Ln and [Ln((+)‐hfbc)₃] in EtOH and CH₃CN solutions or between the SAPR‐8‐M‐Ln and DD‐D_2d(mmmm)‐8‐M‐Ln complexes in CHCl₃ solution. The observed CD couplets are found to reflect the exciton CD couplets which are useful to determine the four‐bladed SAPR‐(llll) absolute configuration around the lanthanide(III) ion. Chirality 24:1055–1062, 2012. © 2012 Wiley Periodicals, Inc.     

DC-based vaccine loaded with acid-eluted peptides in acute myeloid leukemia: the importance of choosing the best elution method

Tumor-associated peptides isolated by acid elution are frequently used for therapeutic immunization against various tumors both in mice and in humans. In acute myeloid leukemia (AML), the frequent accessibility of a large tumor burden allows for extraction of peptides from leukemia cells by using either citrate–phosphate (CP) or trifluoroacetic acid (TFA) buffer. To develop an optimal immunotherapeutic protocol for AML patients, we evaluated both in mice and in humans, the immunogenicity of peptides eluted from leukemia cells with the two acids (TFA or CP). Although ex vivo studies in mice showed that both prophylactic immunizations with mature dendritic cells (DC) loaded with TFA-peptides (DC/TFA), or CP-peptides (DC/CP), were able to stimulate specific antileukemia immune responses, only vaccination with DC/TFA was able to prevent leukemia outgrowth. Moreover, in humans, only DC/TFA generated significant antileukemia CD4⁺ and cytotoxic CD8⁺ T cell responses in vitro. In summary, these data demonstrate that the choice of the acid elution procedure to isolate immunogenic peptides strongly influences the efficacy of the antileukemia immune responses. These finding raise essential considerations for the development of immunotherapeutic protocols for cancer patients. In our model, our results argue for the use of the TFA elution method to extract immunogenic AML-associated peptides.Authors of submitted papers declare no conflict of interest or financial interest in the product or in potentially competing products held by them, their spouses and/or minor children.     

DOCK10-mediated Cdc42 activation is necessary for amoeboid invasion of melanoma cells

BACKGROUND: Tumor cells can move in a three-dimensional (3D) environment in either mesenchymal-type or amoeboid modes. In mesenchymal-type movement, cells have an elongated morphology with Rac-induced protrusions at the leading edge. Amoeboid cells have high levels of actomyosin contractility, and movement is associated with deformation of the cell body through the matrix without proteolysis. Because signaling pathways that control the activation of GTPases for amoeboid movement are poorly understood, we sought to identify regulators of amoeboid movement by screening an siRNA library targeting guanine nucleotide exchange factors (GEFs) for Rho-family GTPases. RESULTS: We identified DOCK10, a Cdc42 GEF, as a key player in amoeboid migration; accordingly, we find that expression of activated Cdc42 induces a mesenchymal-amoeboid transition and increases cell invasion. Silencing DOCK10 expression promotes conversion to mesenchymal migration and is associated with decreased MLC2 phosphorylation and increased Rac1 activation. Consequently, abrogating DOCK10 and Rac1 expression suppresses both amoeboid and mesenchymal migration and results in decreased invasion. We show that the Cdc42 effectors N-WASP and Pak2 are required for the maintenance of the rounded-amoeboid phenotype. Blocking Cdc42 results in loss of mesenchymal morphology, arguing that Cdc42 is also involved in mesenchymal morphology through different activation and effector pathways. CONCLUSIONS: Previous work has identified roles of Rho and Rac signaling in tumor cell movement, and we now elucidate novel roles of Cdc42 signaling in amoeboid and mesenchymal movement and tumor cell invasion.     

Role of galK and galM in Galactose Metabolism by Streptococcus thermophilus

Streptococcus thermophilus is unable to metabolize the galactose moiety of lactose. In this paper, we show that a transformant of S. thermophilus SMQ-301 expressing Streptococcus salivarius galK and galM was able to grow on galactose and expelled at least twofold less galactose into the medium during growth on lactose.     

EPR spin trapping evaluation of ROS production in human fibroblasts exposed to cerium oxide nanoparticles: Evidence for NADPH oxidase and mitochondrial stimulation

To better understand the antioxidant (enzyme mimetic, free radical scavenger) versus oxidant and cytotoxic properties of the industrially used cerium oxide nanoparticles (nano-CeO(2)), we investigated their effects on reactive oxygen species formation and changes in the antioxidant pool of human dermal and murine 3T3 fibroblasts at doses relevant to chronic inhalation or contact with skin. Electron paramagnetic resonance (EPR) spin trapping with the nitrone DEPMPO showed that pretreatment of the cells with the nanoparticles dose-dependently triggered the release in the culture medium of superoxide dismutase- and catalase-inhibitable DEPMPO/hydroxyl radical adducts (DEPMPO-OH) and ascorbyl radical, a marker of ascorbate depletion. This DEPMPO-OH formation occurred 2 to 24h following removal of the particles from the medium and paralleled with an increase of cell lipid peroxidation. These effects of internalized nano-CeO(2) on spin adduct formation were then investigated at the cellular level by using specific NADPH oxidase inhibitors, transfection techniques and a mitochondria-targeted antioxidant. When micromolar doses of nano-CeO(2) were used, weak DEPMPO-OH levels but no loss of cell viability were observed, suggesting that cell signaling mechanisms through protein synthesis and membrane NADPH oxidase activation occurred. Incubation of the cells with higher millimolar doses provoked a 25-60-fold higher DEPMPO-OH formation together with a decrease in cell viability, early apoptosis induction and antioxidant depletion. These cytotoxic effects could be due to activation of both the mitochondrial source and Nox2 and Nox4 dependent NADPH oxidase complex. Regarding possible mechanisms of nano-CeO(2)-induced free radical formation in cells, in vitro EPR and spectrophotometric studies suggest that, contrary to Fe(2+) ions, the Ce(3+) redox state at the surface of the particles is probably not an efficient catalyst of hydroxyl radical formation by a Fenton-like reaction in vivo.