Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts

c-Myc is known to induce or potentiate apoptotic processes predominantly by triggering or enhancing the activity of caspases, but the activation mechanisms of caspases by c-Myc remain still poorly understood. Here we found that in MycER™ rat fibroblasts the activation of c-Myc led to an early activation and cleavage of the initiator caspase-8, and concurrent processing and activation of the effector caspases 3 and 7. Interestingly, the expression of cellular FLICE inhibitory protein (c-FLIP) mRNA and the encoded protein, c-FLIP_L, a catalytically inactive homologue of caspase-8, were down-regulated prior to or coincidently with the activation of caspase-8. Of the other known initiators, caspase-9, involved in the mitochondrial pathway, was activated/processed surprisingly late, only after the effector caspases 3/7. Further, we studied the potential involvement of the Fas- and tumor necrosis factor receptor (TNFR)-mediated signaling in the activation of caspase-8 by c-Myc. Blocking of the function of these death receptors by neutralizing antibodies against Fas ligand and TNF-α did not prevent the processing of caspase-8 or cell death. c-Myc was neither found to induce any changes in the expression of TNF-related apoptosis inducing ligand (TRAIL) or its receptor. These data suggest that caspase-8 does not become activated through an extrinsic but an “intrinsic/intracellular” apoptotic pathway unleashed by the down-regulation of c-FLIP by c-Myc. Moreover, ectopic expression of c-FLIP_L inhibited the c-Myc-induced apoptosis.     

EC144, a synthetic inhibitor of heat shock protein 90, blocks innate and adaptive immune responses in models of inflammation and autoimmunity

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.     

Protein-marking-based assessment of infield predator dispersal

Understanding infield predator dispersal is crucial for designing predator conservation programmes. A study aimed at evaluating methods of collecting insects in protein-marking studies and monitoring predator movement was conducted. Results indicate that collection by sweep net does not result in false positives and predator groups displayed distinct dispersal patterns.     

Complementarity of statistical treatments to reconstruct worldwide routes of invasion: the case of the Asian ladybird Harmonia axyridis

Inferences about introduction histories of invasive species remain challenging because of the stochastic demographic processes involved. Approximate Bayesian computation (ABC) can help to overcome these problems, but such method requires a prior understanding of population structure over the study area, necessitating the use of alternative methods and an intense sampling design. In this study, we made inferences about the worldwide invasion history of the ladybird Harmonia axyridis by various population genetics statistical methods, using a large set of sampling sites distributed over most of the species’ native and invaded areas. We evaluated the complementarity of the statistical methods and the consequences of using different sets of site samples for ABC inferences. We found that the H. axyridis invasion has involved two bridgehead invasive populations in North America, which have served as the source populations for at least six independent introductions into other continents. We also identified several situations of genetic admixture between differentiated sources. Our results highlight the importance of coupling ABC methods with more traditional statistical approaches. We found that the choice of site samples could affect the conclusions of ABC analyses comparing possible scenarios. Approaches involving independent ABC analyses on several sample sets constitute a sensible solution, complementary to standard quality controls based on the analysis of pseudo‐observed data sets, to minimize erroneous conclusions. This study provides biologists without expertise in this area with detailed methodological and conceptual guidelines for making inferences about invasion routes when dealing with a large number of sampling sites and complex population genetic structures.     

Scale‐up of hydrophobin‐assisted recombinant protein production in tobacco BY‐2 suspension cells

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low‐cost purification by surfactant‐based aqueous two‐phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY‐2) suspension cell platform and the establishment of pilot‐scale propagation and downstream processing including first‐step purification by ATPS. Green fluorescent protein‐hydrophobin fusion (GFP‐HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY‐2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP‐HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large‐scale hydrophobin‐assisted production of recombinant proteins in tobacco BY‐2 cell suspensions.     

Analysis of Alpha-Synuclein in Malignant Melanoma – Development of a SRM Quantification Assay

Globally, malignant melanoma shows a steady increase in the incidence among cancer diseases. Malignant melanoma represents a cancer type where currently no biomarker or diagnostics is available to identify disease stage, progression of disease or personalized medicine treatment. The aim of this study was to assess the tissue expression of alpha-synuclein, a protein implicated in several disease processes, in metastatic tissues from malignant melanoma patients. A targeted Selected Reaction Monitoring (SRM) assay was developed and utilized together with stable isotope labeling for the relative quantification of two target peptides of alpha-synuclein. Analysis of alpha-synuclein protein was then performed in ten metastatic tissue samples from the Lund Melanoma Biobank. The calibration curve using peak area ratio (heavy/light) versus concentration ratios showed linear regression over three orders of magnitude, for both of the selected target peptide sequences. In support of the measurements of specific protein expression levels, we also observed significant correlation between the protein and mRNA levels of alpha-synuclein in these tissues. Investigating levels of tissue alpha-synuclein may add novel aspect to biomarker development in melanoma, help to understand disease mechanisms and ultimately contribute to discriminate melanoma patients with different prognosis.     

Effects of sugar feeding on carbohydrate and lipid metabolism in a parasitoid wasp

Lifetime patterns of carbohydrate and lipid metabolism were compared in starved and sucrose‐fed adults of the parasitoid Macrocentrus grandii (Goidanich) (Hymenoptera: Braconidae). As expected, sucrose‐fed individuals lived longer than did starved individuals. Macrocentrus grandii males and females eclosed with levels of simple storage sugars (presumably primarily trehalose) and glycogen that were below maximum levels recorded from sucrose‐fed parasitoids. Both of these nutrients dropped to very low levels in starved individuals within 4 days post‐emergence and were maintained at high levels in sucrose‐fed individuals throughout their lives. Lipid reserves at emergence represented the highest lipid levels for both sexes in the two diet treatments, with levels declining over the lifetimes of males and females from both diet treatments. Our results therefore suggest that dietary sucrose is used to synthesize trehalose and glycogen, but not lipids in M. grandii. Also, in contrast to the patterns observed for the simple sugars and glycogen, lipid levels in starved individuals did not drop below levels observed in sugar‐fed individuals. The average number of mature eggs carried by females at emergence was 33 and increased to approximately 85 in sucrose‐fed and 130 in starved females by the age of 5 d in the absence of hosts. The egg maturation rate was therefore higher in starved than in sugar‐fed females. Potential explanations for this unexpected result are discussed.     

Curvature of mouse satellite DNA and condensation of heterochromatin

Cloned, sequenced mouse satellite DNA exhibits properties characteristic of molecules that possess a stable curvature. Circularly permuted fragments containing the region predicted to bend were used to map the curvature relative to DNA sequence. The altered mobility of these fragments in polyacrylamide gels is reversed when gels are run in the presence of distamycin A, a drug that binds preferentially to AT-rich DNA. Treatment of living mouse cells with this drug dramatically reduces the condensation of centromeric heterochromatin, the exclusive location of satellite sequences. In situ hybridization of satellite probes to extended chromosomes at the electron microscope level shows that satellite does not comprise a single block but is distributed throughout the centromere region. Based on these experiments, we hypothesize that the structure of mouse satellite DNA is an important feature of centromeric heterochromatin condensation.     

An Experimental Study of the Role of Weight Bias in Candidate Evaluation

Obese individuals are evaluated negatively and attributed negative trait characteristics in several contexts including employment, health care, and education. The current experimental study of college students examined the effect of body mass on the evaluation of political candidates and examined whether the gender of the candidate moderated the relationship. A series of ordinary least squares regression analyses found an interactive effect between candidate obesity and candidate gender for global evaluation and for several trait characteristics. Specifically, obese female candidates were evaluated more negatively than nonobese female candidates and nonobese male candidates were evaluated more negatively than were obese male candidates. This interaction persisted even after controlling for standard political and demographic characteristics of the evaluator. These findings suggest that weight bias exists for obese female political candidates, but that larger body size may be an asset for male candidates. The ability of candidates to be successful may depend less on their policy positions or even party affiliation and more on their physical attributes than has been previously assumed.