Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination

Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ～14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.     

Tumor destruction using electrochemotherapy followed by CpG oligodeoxynucleotide injection induces distant tumor responses

PURPOSE: Electrochemotherapy (ECT) is an effective local therapy of human cutaneous cancers but has no effect on distant untreated tumors. We addressed whether tumor-associated antigens released after ECT could induce an efficient systemic immunity when associated with an appropriate immunoadjuvant. METHODS AND RESULTS: We first studied the nature of the cellular recruitment and the expression of various toll-like receptors (TLRs) in tumors treated by ECT. We found that ECT induced a massive recruitment of CD11c and CD11b positive cells in the tumors and a strong increase of TLR9 expression. We then tested antitumor effects of the combination: ECT followed by TLR-9 ligands, CpG oligodeoxynucleotides (CpG ODN), in three murine tumor models. We found that this combination triggered both potent local synergistic antitumor effects, on the ipsi-lateral ECT-treated tumor, and more interestingly, a systemic antitumor response on the contra-lateral untreated tumor, in the three models. The systemic protection was T-cell dependent as it was not observed in nude littermates. The combination induced tumor-specific T cell effectors in the tumor-draining lymph nodes and in the spleen which secreted significantly more gamma-interferon upon activation than with ECT or CpG ODN alone. CONCLUSIONS: Our data show that ECT and CpG ODN synergize and induce a significant increase of the local effect and a systemic T-dependent antitumor response. Such combination constitutes a potential innovative vaccination strategy using in situ tumor-associated antigens that could eventually be translated into the clinic.     

The Evolution of Restricted Recombination and the Accumulation of Repeated DNA Sequences

We suggest hypotheses to account for two major features of chromosomal organization in higher eukaryotes. The first of these is the general restriction of crossing over in the neighborhood of centromeres and telomeres. We propose that this is a consequence of selection for reduced rates of unequal exchange between repeated DNA sequences for which the copy number is subject to stabilizing selection: microtubule binding sites, in the case of centromeres, and the short repeated sequences needed for terminal replication of a linear DNA molecule, in the case of telomeres. An association between proximal crossing over and nondisjunction would also favor the restriction of crossing over near the centromere. The second feature is the association between highly repeated DNA sequences of no obvious functional significance and regions of restricted crossing over. We show that highly repeated sequences are likely to persist longest (over evolutionary time) when crossing over is infrequent. This is because unequal exchange among repeated sequences generates single copy sequences, and a population that becomes fixed for a single copy sequence by drift remains in this state indefinitely (in the absence of gene amplification processes). Increased rates of exchange thus speed up the process of stochastic loss of repeated sequences.     

Effects of Land-Use Change on Carbon Stocks in Switzerland

We assessed how consequences of future land-use change may affect size and spatial shifts of C stocks under three potential trends in policy—(a) business-as-usual: continuation of land-use trends observed during the past 15 years; (b) extensification: full extensification of open-land; and (c) liberalization: full reforestation potential. The build-up times for the three scenarios are estimated at 30, 80 and 100 years, respectively. Potential C-stock change rates are derived from the literature. Whereas the business-as-usual scenario would cause marginal changes of 0.5%, liberalization would provoke a 13% increase in C stocks (+62 MtC). Gains of 24% would be expected for forests (+95 MtC), whereas open-land C stock would decrease 27% (−33 MtC). Extensification would lead to a C stock decrease of 3% (−12 MtC). Whereas forest C is expected to increase 12% (+36.5 MtC) at high elevations, stocks of open-land C would decline 38.5% (−48.5 MtC). Most affected are unfavorable grasslands, which increase in area (+59%) but contribute only 14.5% to the C stocks. C sinks would amount to 0.6 MtC y⁻¹ assuming a build-up time of 100 years for the liberalization scenario. C stocks on the current forest area are increasing by 1 MtC y⁻¹. The maximal total C sink of 1.6 MtC might thus suffice to compensate for agricultural greenhouse gases (2004: 1.4 Mt CO₂–C equivalents), but corresponds only to 11–13% of the anthropogenic greenhouse gas emission in Switzerland. Thus, even the largest of the expected terrestrial C stocks under liberalization will be small in comparison with current emissions of anthropogenic greenhouse gases.     

A New Technology for Applanation Free Corneal Trephination: The Picosecond Infrared Laser (PIRL)

The impact of using a Femtosecond laser on final functional results of penetrating keratoplasty is low. The corneal incisions presented here result from laser ablations with ultrafast desorption by impulsive vibrational excitation (DIVE). The results of the current study are based on the first proof-of-principle experiments using a mobile, newly introduced picosecond infrared laser system, and indicate that wavelengths in the mid-infrared range centered at 3 μm are efficient for obtaining applanation-free deep cuts on porcine corneas.     

TLR activation of Langerhans cell-like dendritic cells triggers an antiviral immune response

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.     

Interaction of the fusogenic peptide B18 in its amyloid-state with lipid membranes studied by solid state NMR

The interaction of the fusogenic polypeptide segment "B18" from the fertilization protein binding with lipid membranes was investigated by solid state 2H and 31P NMR, and by differential scanning calorimetry. B18 is known to adopt different conformations depending on peptide concentration, ionic conditions, pH and lipid environment. Here, the peptide was studied in its beta-stranded amyloid conformation. According to 31P NMR, the lamellar morphology of the DMPC bilayer remains intact in the presence of B18. In going from low (1:90) to high (1:10) peptide/lipid ratios, an increasing effect on several different 2H-labeled lipid segments was observed, reflecting changes in phase behavior and local dynamics. The strongest influence of B18 was detected at the acyl-chains, while no significant effect on the lipid headgroup conformation was observed. This suggests an insertion of B18 in its fibrillar state into the membrane driven by hydrophobic interactions, rather than a peripheral binding mediated by electrostatics.     

Extracellular protease of Pseudomonas fluorescens CHA0, a biocontrol factor with activity against the root-knot nematode Meloidogyne incognita

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol.     

Stimulation of dopamine synthesis and release by morphine and D-Ala2-D-Leu5-enkephalin in the mouse striatum in vivo

The effects of opiates on dopamine (DA) release and synthesis were assessed in the mouse striatum $\text{in vivo}$ by simultaneously measuring 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels after inhibition of aromatic amino acid decarboxylase. This method was developed to assess stimulus-coupled changes in DA synthesis and release. Peripheral injections of morphine and intraventrcular injections of D-Ala²-Leu⁵-enkephalin elevated DOPAC levels, indicating that “opiates” stimulated DA release. Concomitantly, the rate of DA synthesis was increased. The effects were dose-dependent, saturable and antagonized by naloxone. When morphine and the enkephalin analog were given together in saturating doses, the effects of the two agents were not additive. Thus, the involvement of different receptors in the mediation of the effects of morphine and enkephalins could not be demonstrated.