S-D-Lactoylglutathione can be an alternative supply of mitochondrial glutathione

The mitochondrial pool of GSH (glutathione) is considered the major redox system in maintaining matrix redox homeostasis, preserving sulfhydryl groups of mitochondrial proteins in appropriate redox state, in defending mitochondrial DNA integrity and protecting mitochondrial-derived ROS, and in defending mitochondrial membranes against oxidative damage. Despite its importance in maintaining mitochondrial functionality, GSH is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. In this work we found that SLG (S-D-lactoylglutathione), an intermediate of the glyoxalase system, can enter the mitochondria and there be hydrolyzed from mitochondrial glyoxalase II enzyme to D-lactate and GSH. To demonstrate SLG transport from cytosol to mitochondria we used radiolabeled compounds and the results showed two different kinetic curves for SLG or GSH substrates, indicating different kinetic transport. Also, the incubation of functionally and intact mitochondria with SLG showed increased GSH levels in normal mitochondria and in artificially uncoupled mitochondria, demonstrating transport not linked to ATP presence. As well mitochondrial-swelling assay confirmed SLG entrance into organelles. Moreover we observed oxygen uptake and generation of membrane potential probably linked to D-lactate oxidation which is a product of SLG hydrolysis. The latter data were confirmed by oxidation of D-lactate in mitochondria evaluated by measuring mitochondrial D-lactate dehydrogenize activity. In this work we also showed the presence of mitochondrial glyoxalase II in inter-membrane space and mitochondrial matrix and we investigated the role of SLG in whole cells. In conclusion, this work showed new alternative sources of GSH supply to the mitochondria by SLG, an intermediate of the glyoxalase system.     

Nitrate inhibition of nodulation can be overcome by the ethylene inhibitor aminoethoxyvinylglycine

Previously, we reported (a) a positive correlation between the nitrate concentrations in growth medium and ethylene evolved from uninoculated and inoculated alfalfa (Medicago sativa) roots and (b) a negative correlation between ethylene evolution and nodulation. Here, we report that the inhibitory effect of NO₃⁻ on nodulation of alfalfa can be eliminated by the ethylene inhibitor aminoethoxyvinylglycine (AVG). This effect was probably related to the strong inhibition (90%) of ethylene biosynthesis caused by AVG in these inoculated and NO₃⁻-treated roots. These results support our hypothesis that the inhibitory effect of NO₃⁻ is mediated through the phytohormone ethylene. A possible role of endogenous ethylene in the autoregulation of nodulation also is discussed. AVG at 10 micromolar significantly (P < 0.05) increased total nitrogenase activity (acetylene reduction) in 2.5 and 5 millimolar NO₃⁻-fed plants probably as a result of the very high stimulation of nodulation.     

Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid

Addition of thrombin to human platelets results in production of lysophosphatidic acid. Such synthesis of lysophosphatidic acid can be inhibited by mepacrine, an inhibitor of the phospholipase A2 which attacks phosphatidic acid to give lysophosphatidic acid. In the present study, mepacrine was used at a concentration of 2.5-20 microM, sufficient to block aggregation and lysophosphatidic acid formation induced by 0.1 U/ml thrombin. Mepacrine, at this concentration, also blocked thrombin-induced phosphorylation of platelet myosin light chain and a 47 kDa protein, thrombin-induced secretion and thrombin-induced release of arachidonic acid from platelet phospholipids. However, mepacrine also partly inhibited the formation of phosphatidic acid in response to thrombin, consistent with some simultaneous inhibition of phospholipase C. Lysophosphatidic acid (2.5-22 microM) overcame the mepacrine block in thrombin-stimulated aggregation, protein phosphorylation and secretion without stimulating the release of arachidonic acid from platelet phospholipids or the formation of lysophosphatidic acid, and only slightly increasing phosphatidic acid formation. The results suggest that lysophosphatidic acid primarily acts distal to mepacrine inhibition of phospholipase A2 and phospholipase C and are consistent with the possibility that lysophosphatidic acid might be a mediator of part of the effects of low-dose thrombin on human platelets.     

Behavioral defeminization by prenatal androgen treatment in rats can be overcome by sexual experience in adulthood

Exposure to testosterone during a critical period of prenatal development disrupts the normal display of sexual behaviors in adult ovariectomized (OVX) rats treated with estradiol benzoate (EB) followed by progesterone (P). The organizational hypothesis posits that prenatally androgenized females (PNAFs) are desensitized to EB. We tested this hypothesis by first treating PNAFs with varying doses of EB (2.5, 5, 10, 20 μg) followed by P (500 μg), and second by subjecting females to an established EB behavioral sensitization paradigm where females are first given sexual experience with EB (10 μg) and P prior to repeated sexual behavior testing with EB alone. Long-Evans females were androgenized in utero by a s.c. injection of 500 μg testosterone propionate or the oil control to pregnant dams on gestational day 18. Female offspring were OVX on postnatal day 80 and tested one week later in the unilevel 4-hole pacing chamber. Genital tissue was defeminized in PNAFs, and the lordosis quotient (LQ) and partial (i.e., hops/darts) and full solicitations were significantly lower, while defensive behaviors were higher, in PNAF females, relative to non-PNAF females regardless of the acute EB priming dose. However, repeated testing with EB alone (10 μg), or EB and P eliminated the differences between groups on LQ and hops/darts, indicating that the behavioral deficit can be overcome by sexual experience. These results suggest that PNAFs are not desensitized to EB, and despite disruptions in sexual differentiation of anatomical structures, the deficiency in sexual behavior in response to acute EB and P can be experientially overcome. PNAFs appear, however, to have a chronic deficit in the expression of full solicitations.     

CD95 apoptosis resistance in certain cells can be overcome by noncanonical activation of caspase-8

CD95 apoptosis resistance of tumor cells is often acquired through mutations in the death domain (DD) of one of the CD95 alleles. Furthermore, Type I cancer cells are resistant to induction of apoptosis by soluble CD95 ligand (CD95L), which does not induce efficient formation of the death-inducing signaling complex (DISC). Here, we report that tumor cells expressing a CD95 allele that lacks a functional DD, splenocytes from heterozygous lpr(cg) mice, which express one mutated CD95 allele, and Type I tumor cells stimulated with soluble CD95L can all die through CD95 when protein synthesis or nuclear factor kappa B is inhibited. This noncanonical form of CD95-mediated apoptosis is dependent on the enzymatic activity of procaspase-8 but does not involve fully processed active caspase-8 subunits. Our data suggest that it is possible to overcome the CD95 apoptosis resistance of many tumor cells that do not efficiently form a DISC through noncanonical activation of the caspase-8 proenzyme.     

Ancient host shifts followed by host conservatism in a group of ant parasitoids

While ant colonies serve as host to a diverse array of myrmecophiles, few parasitoids are able to exploit this vast resource. A notable exception is the wasp family Eucharitidae, which is the only family of insects known to exclusively parasitize ants. Worldwide, approximately 700 Eucharitidae species attack five subfamilies across the ant phylogeny. Our goal is to uncover the pattern of eucharitid diversification, including timing of key evolutionary events, biogeographic patterns and potential cophylogeny with ant hosts. We present the most comprehensive molecular phylogeny of Eucharitidae to date, including 44 of the 53 genera and fossil-calibrated estimates of divergence dates. Eucharitidae arose approximately 50 Ma after their hosts, during the time when the major ant lineages were already established and diversifying. We incorporate host association data to test for congruence between eucharitid and ant phylogenies and find that their evolutionary histories are more similar than expected at random. After a series of initial host shifts, clades within Eucharitidae maintained their host affinity. Even after multiple dispersal events to the New World and extensive speciation within biogeographic regions, eucharitids remain parasitic on the same ant subfamilies as their Old World relatives, suggesting host conservatism despite access to a diverse novel ant fauna.     

Decreased infectivity of a neutralization-resistant equine infectious anemia virus variant can be overcome by efficient cell-to-cell spread

Two variants of equine infectious anemia virus (EIAV) that differed in sensitivity to broadly neutralizing antibody were tested in direct competition assays. No differences were observed in the growth curves and relative fitness scores of EIAVs of principal neutralizing domain variants of groups 1 (EIAV(PND-1)) and 5 (EIAV(PND-5)), respectively; however, the neutralization-resistant EIAV(PND-5) variant was less infectious in single-round replication assays. Infectious center assays indicated similar rates of cell-to-cell spread, which was approximately 1,000-fold more efficient than cell-free infectivity. These data indicate that efficient cell-to-cell spread can overcome the decreased infectivity that may accompany immune escape and should be considered in studies assessing the relative levels of fitness among lentivirus variants, including HIV-1.     

Low-avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands

Thymic expression of self-Ags results in the deletion of high-avidity self-specific T cells, but, at least for certain Ags, a residual population of self-specific T cells with low-affinity TCRs remains after negative selection. Such self-specific T cells are thought to play a role in the induction of T cell-mediated autoimmunity, but may also be used for the induction of antitumor immunity against self-Ags. In this study, we examine the functional competence of a polyclonal population of self-specific CD8+ T cells. We show that low-affinity interactions between TCR and peptide are associated with selective loss of critical T cell functions. Triggering of low levels of IFN-gamma production and cytolytic activity through low-affinity TCRs readily occurs provided high Ag doses are used, but IL-2 production and clonal expansion are severely reduced at all Ag doses. Remarkably, a single peptide variant can form an improved ligand for the highly diverse population of low-avidity self-specific T cells and can improve their proliferative capacity. These data provide insight into the inherent limitations of self-specific T cell responses through low-avidity TCR signals and the effect of modified peptide ligands on self-specific T cell immunity.     

HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure.     

Overwintering problems of newly established Miscanthus plantations can be overcome by identifying genotypes with improved rhizome cold tolerance

Miscanthus , a perennial rhizomatous C₄ grass, is a potential biomass crop in Europe, mainly because of its high yield potential and low demand for inputs. However, until recently only a single clone, M. × giganteus , was available for the extensive field trials performed across Europe and this showed poor overwintering in the first year after planting at some locations in Northern Europe. Therefore, field trials with five Miscanthus genotypes, including two acquisitions of Miscanthus × giganteus , one of M. sacchariflorus and two hybrids of M. sinensis were planted in early summer 1997 at four sites, in Sweden, Denmark, England and Germany. The field trials showed that better overwintering of newly established plants at a site was not apparently connected with size or early senescence. An artificial freezing test with rhizomes removed from the field in January 1998 showed that the lethal temperature at which 50% were killed (LT₅₀) for M. × giganteus and M. sacchariflorus genotypes was −3.4 °C. However, LT₅₀ in one of the M. sinensis hybrid genotypes tested was −6.5 °C and this genotype had the highest survival rates in the field in Sweden and Denmark. Although the carbohydrate content of rhizomes, osmotic potential of cell sap and mineral composition were not found to explain differences in frost tolerance adequately, moisture contents correlated with frost hardiness (LT₅₀) in most cases. The results obtained form a basis for identifying suitable Miscanthus genotypes for biomass production in the differing climatic regions of Europe.     

Myocardium tolerant to an adenosine-dependent ischemic preconditioning stimulus can still be protected by stimuli that employ alternative signaling pathways

Clinical studies on cardioprotection by preinfarct angina are ambiguous, which may involve development of tolerance to repeated episodes of ischemia. Not all preconditioning stimuli use identical signaling pathways, and because patients likely experience varying numbers of episodes of preinfarct angina of different degrees and durations, it is important to know whether myocardium tolerant to a particular preconditioning stimulus can still be protected by stimuli employing alternative signaling pathways. We tested the hypothesis that development of tolerance to a particular stimulus does not affect cardioprotection by stimuli that employ different signaling pathways. Anesthetized rats underwent classical, remote or pharmacological preconditioning. Infarct size (IS), produced by a 60-min coronary artery occlusion (CAO), was determined after 120 min of reperfusion. Preconditioning by two 15-min periods of CAO (2CAO15, an adenosine-dependent stimulus) limited IS from 69 +/- 2% to 37 +/- 6%, but when 2CAO15 was preceded by 4CAO15, protection by 2CAO15 was absent (IS = 68 +/- 1%). This development of tolerance coincided with a loss of cardiac interstitial adenosine release, whereas two 15-min infusions of adenosine (200 microg/min i.v.) still elicited cardioprotection (IS = 40 +/- 4%). Furthermore, cardioprotection was produced when 4CAO15 was followed by the adenosine-independent stimulus 3CAO3 (IS = 50 +/- 8%) or the remote preconditioning stimulus of two 15-min periods of mesenteric artery occlusion (IS = 49 +/- 6%). In conclusion, development of tolerance to cardioprotection by an adenosine-dependent preconditioning stimulus still allows protection by pharmacological or ischemic stimuli intervention employing different signaling pathways.     

Impaired growth rates in milk of Lactobacillus helveticus peptidase mutants can be overcome by use of amino acid supplements

To evaluate the contribution of intracellular peptidases to the growth of the 14-amino-acid (aa) auxotroph Lactobacillus helveticus CNRZ32, single- and multiple-peptidase-deletion mutants were constructed. Two broad-specificity aminopeptidases (PepC and PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) were inactivated through successive cycles of chromosomal gene replacement mutagenesis. The inactivation of all three peptidases in JLS247 ((Delta)pepC (Delta)pepN (Delta)pepX) did not affect the growth rate in amino acid-defined medium. However, the peptidase mutants generally had decreased specific growth rates when acquisition of amino acids required hydrolysis of the proteins in milk, the most significant result being a 73% increase in generation time for JLS247. The growth rate deficiencies in milk were overcome by amino acid supplements with some specificity to each of the peptidase mutants. For example, milk supplementation with Pro resulted in the most significant growth rate increase for (Delta)pepX strains and a 7-aa supplement (Asn, Cys, Ile, Pro, Ser, Thr, and Val) resulted in a JLS247 growth rate indistinguishable from that of the wild type. Our results show that characterization of the activities of the broad-specificity aminopeptidases had little predictive value regarding the amino acid supplements found to enhance the milk growth rates of the peptidase mutant strains. These results represent the first determination of the physiological roles with respect to specific amino acid requirements for peptidase mutants grown in milk.     

The function of host discrimination and superparasitization in parasitoids

Summary Host discrimination, i.e. the ability to distinguish unparasitized hosts from parasitized ones, and to reject the latter for egg laying is present in many parasitic wasp species. This property is classically considered as an example of contest competition, and is supposed to have a number of functions. However, different species do not react to each other's marks and lay eggs in hosts parasitized by the other species. Apparently the marks used for recognition are specific.Multiparasitization is the best strategy when hosts are scarce and the egg supplies of the parasitoids are not limited. Interspecific host discrimination is not an ESS.Superparasitization within one species would have selective advantage if the number of unparasitized hosts is small and the wasp has a reasonable chance to lay her egg in a host that is not parasitized by herself, and if the chance for her offspring to survive the competitive battle with the first parasitoid larva is not too small. This is shown to be the case.However, marks are not individual and wasps cannot distinguish hosts parasitized by themselves from those parasitized by others. The hypothesis is tested that the egg laying strategy (i.e. the decision to superparasitize) of wasps is dependent on the number of conspecifics that is searching simultaneously for hosts, since this determines the chance that a parasitized host encountered by a wasp is parasitized by herself.It is shown that host discrimination cannot be regarded as a case of contest competition. Other aspects of superparasitization, related to interference and population regulation, sex allocation and encapsulation are briefly discussed.     

Blocks to thyroid cancer cell apoptosis can be overcome by inhibition of the MAPK and PI3K/AKT pathways

Current treatment for recurrent and aggressive/anaplastic thyroid cancers is ineffective. Novel targeted therapies aimed at the inhibition of the mutated oncoprotein BRAF^V600E have shown promise in vivo and in vitro but do not result in cellular apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a tumor-selective manner by activating the extrinsic apoptotic pathway. Here, we show that a TRAIL-R2 agonist antibody, lexatumumab, induces apoptosis effectively in some thyroid cancer cell lines (HTh-7, TPC-1 and BCPAP), while more aggressive anaplastic cell lines (8505c and SW1736) show resistance. Treatment of the most resistant cell line, 8505c, using lexatumumab in combination with the BRAF^V600E inhibitor, PLX4720, and the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, (triple-drug combination) sensitizes the cells by triggering both the extrinsic and intrinsic apoptotic pathways in vitro as well as 8505c orthotopic thyroid tumors in vivo. A decrease in anti-apoptotic proteins, pAkt, Bcl-xL, Mcl-1 and c-FLIP, coupled with an increase in the activator proteins, Bax and Bim, results in an increase in the Bax to Bcl-xL ratio that appears to be critical for sensitization and subsequent apoptosis of these resistant cells. Our results suggest that targeting the death receptor pathway in thyroid cancer can be a promising strategy for inducing apoptosis in thyroid cancer cells, although combination with other kinase inhibitors may be needed in some of the more aggressive tumors initially resistant to apoptosis.     

Displacement of a native by an alien bumblebee: lower pollinator efficiency overcome by overwhelmingly higher visitation frequency

Biological invasions might constitute a major threat to mutualisms. Introduced pollinators might competitively displace their native counterparts, which in turn affects the pollination of native plants, if native and alien visitors differ in pollinator effectiveness. Since its invasion in 1994 into south-west Argentina, the introduced European bumblebee Bombus ruderatus has continuously increased in abundance, along with a simultaneous decrease in the abundance of the native Bombus dahlbomii. The latter is the only native bumblebee species of the temperate forests of southern South America, and the main pollinator of the endemic herb Alstroemeria aurea. In order to evaluate the impact of the ongoing displacement of the native by the alien bumblebee, we compared the pollinator effectiveness (i.e., the combination of pollinator efficiency per visit and visitation frequency) between both bumblebee species, as well as related pollinator traits that might account for potential differences in pollinator efficiency. Native Bombus dahlbomii, which has a larger body and spent more time per flower, was the more efficient pollinator compared to Bombus ruderatus, both in terms of quantity and quality of pollen deposited per visit. However, Bombus ruderatus was a much more frequent flower visitor than Bombus dahlbomii. As a consequence, Bombus ruderatus is nowadays a more effective pollinator of A. aurea than its native congener. Despite the lack of evidence of an increase in seed set at the population level, comparisons with historical records of Bombus dahlbomii abundances prior to Bombus ruderatus' invasion suggest that the overall pollination intensity of A. aurea might in fact have risen as a consequence of this invasion. Field experiments like these, that incorporate the natural variation in abundance of native and alien species, are powerful means to demonstrate that the consequences of invasions are more complex than previous manipulated and controlled experiments have suggested.