The additional loss of Bak and not the lack of the protein tyrosine kinase p56/Lck in one JCaM1.6 subclone caused pronounced apoptosis resistance in response to stimuli of the intrinsic pathway

Ionising radiation, hypoxia, and the cyclooxygenase-2 inhibitor Celecoxib are known agonists of the intrinsic apoptosis pathway that involves mitochondrial damage upstream of caspase activation. Mitochondrial integrity is regulated by the pro-apoptotic Bcl-2 protein family members Bak and Bax. Upstream of the mitochondria, many kinases and phosphatases control the apoptotic response. However, the role of the non-receptor tyrosine kinase p56/Lck during apoptosis is controversial. The present investigation demonstrate the existence of two JCaM1.6 subclones, one expressing and one deficient for Bak. The lack of p56/Lck expression in JCaM1.6 cells per se did hardly affect apoptosis induced by ionising radiation, hypoxia, or Celecoxib. Only the additional loss of Bak expression, as observed in one JCaM1.6 subclone, rendered the cells resistant. siRNA-mediated downregulation of Bak and p56/Lck mimicked the observed effects in the subclones. Earlier experiments performed with the Bak-negative clone might have lead to the wrong assumption that lack of p56/Lck alone, and not the additonal loss of Bak, was responsible for reduced sensitivity towards stimuli of the intrinsic apoptosis pathway.     

Symbiont choice in a fungus-growing ant (Attini, Formicidae)

Cultivars of fungus-growing (attine) ants are vertically transmittedthrough inheritance from parent to offspring nest, but horizontalcultivar transfer between ant nests occurs occasionally, resultingin cultivar replacement within ant lineages. Two mechanismscould theoretically prevent the invasion of suboptimal cultivarstrains and thus stabilize ant–cultivar coevolution: first,partner feedback inherent in vertical cultivar transmissionand second, partner (symbiont) choice if the ants differentiatebetween productive and inferior cultivars during replacements.To elucidate the nature of symbiont choice, we presented workersof Cyphomyrmex muelleri with novel cultivars representing aphylogenetic cline of close and distant relatives of the nativeC. muelleri cultivar. Workers invariably preferred their nativecultivar, discriminating against even very close relatives ofthe native cultivar. When given a choice between two non-nativecultivar strains, workers accepted the strain most closely relatedto their native cultivar. Two conclusions emerge. First, colonyswitches to distantly related cultivars are behaviorally unlikelyand may not be preference-based; rather, distant switches mayoccur under constrained choice, such as pathogen-related gardenlosses that force colonies to import novel cultivars. Second,the ability of attine ants to differentiate between closelyrelated cultivar strains suggests that the ant–fungusmutualism is stabilized evolutionarily not only by partner feedbackinherent in vertical cultivar transmission, but possibly alsoby symbiont choice through which the ants select against unwanted,presumably inferior, cultivars. The efficacy of symbiont choicenow needs to be tested experimentally. Such research may benefitfrom application of theory and experimental paradigms that havebeen developed within the areas of mate choice and sexual selection.     

Direct Interaction between Two Viral Proteins,the Nonstructural Protein 2CATPase and the Capsid Protein VP3, Is Required for Enterovirus Morphogenesis

In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C^ATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel “reporter virus”, we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C^ATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C^ATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2C^ATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2C^ATPase is an essential component of the replication complex, and (iv) 2C^ATPase has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex.     

The Relationship between Types of Human–Animal Interaction and Attitudes about Animals: An Exploratory Study

Existing theory and research suggests that understanding the nuances of particular instantiations of human–animal relationships is important in promoting positive, mutually beneficial relationships between people and animals. One such aspect of human–animal interaction (HAI) involves species of animal involved in the relationship, and how various types of HAI may impact individuals' attitudes about animals. Therefore, the purpose of this study was to explore if species and/or types of animal ownership were associated with feelings of emotional attachment, commitment, and moral orientation toward animals. A sample of young adults (n = 567) from the 4-H Study of Positive Youth Development completed a survey which included questions about animal ownership and attitudes about animals. Regression analyses demonstrated that the species of animal(s) a person owned significantly predicted all three dimensions of attitudes about animals. In addition, latent class analyses identified three prevalent types of animal interaction (no/few animals, small animals only, large and small animals), and multinomial logistic regression within the mixture model indicated that individuals in these subgroups significantly differed in moral orientation scores. Overall, the analyses strengthen support for the notion that species of animal involved in the interaction matters, and that relationships with various species of animals may differ qualitatively. These findings have implications for understanding the role of the relationship between types of animal ownership and attitudes about animals. Exploring the multifaceted nature of human–animal relationships is important in understanding how to optimize the person and animal characteristics that are associated with adaptive, mutually beneficial human–animal relationships.     

Hydrogenation of Triton X-100 eliminates its fluorescence and ultraviolet light absorption while preserving its detergent properties

The ultraviolet-light absorption and fluorescence of Triton X-100 were virtually eliminated by hydrogenation to its reduced cyclohexyl analog, RTX-100. The critical micelle concentration of RTX-100 was 12% higher than that of Triton X-100. RTX-100 and Triton X-100 were quite similar in their abilities to extract proteins from human erythrocyte membranes.     

Photoaffinity labelling of submitochondrial membranes with the 3-azido analogue of 9-amino-3-chloro-7-methoxyacridine

9-Amino-3-azido-7-methoxyacridine has been synthesized and shown to be a suitable photoaffinity probe for the site(s) of interaction of 9-amino-3-chloro-7-methoxyacridine with submitochondrial membranes. Both the excitation and emission spectra of the azido analogue covalently bound to membranes in the energized state display distinctive differences from the spectra of labelled, non-energized membranes (i.e., in the absence of oxidizable substrate, or its presence when uncoupler (FCCP) is also present during photolysis). Enzymatic analyses indicate that the probe interacts with the ATPase and the respiratory chain enzymes; energization appears to afford some protection against inactivation. Electrophoresis of the labelled membranes and isolation of their lipid and protein components indicate that the spectral differences are attributable to differing interactions with the lipid components of energized, relative to non-energized, membranes. Similar results have been obtained with the 3-azido analogue of quinacrine (Mueller, D.M., Hudson, R.A. and Lee, C.P. (1982) Biochemistry 21, 1445-1453), which differs significantly, however, in the extent of its interactions with the enzymes of the respiratory chain and the ATPase. These results indicate that the energy-linked fluorescence responses of 9-aminoacridines with submitochondrial membranes arise from direct interactions with membrane components and may involve redistribution of the probe molecules and/or alteration of their microenvironments upon energization.     

Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody

The effect of stimulating normal type 1 murine T cell clones with anti-CD3 antibody was examined in vitro. In the absence of accessory cells, anti-CD3 antibody immobilized on plastic plates stimulated inositol phosphate production, suboptimal proliferation, IL-2 and IL-3 production, and maximal IFN-gamma production. Addition of accessory cells augmented lymphokine production and proliferation when the effects of "high-dose suppression" were relieved by removing the T cells from the antibody-coated plates. Exposure of type 1 T cell clones to immobilized anti-CD3 antibody alone rapidly induced long-lasting proliferative unresponsiveness (anergy) to Ag stimulation that could be prevented by accessory cells. This anergic state was characterized by a lymphokine production defect, not a failure of the T cells to respond to exogenous IL-2 or to express surface Ti/CD3 complexes. In addition, anergy could not be induced in the presence of cyclosporine A. These results suggest that under certain conditions anti-CD3 antibodies may have potent immunosuppressive effects independent of Ti/CD3 modulation. Furthermore, our results support a two-signal model of type 1 T cell activation in which Ti/CD3 occupancy alone (signal 1) induces anergy, whereas Ti/CD3 occupancy in conjunction with a costimulatory signal (signal 2) induces a proliferative response.     

Characterization of a bimobile DNA junction

We present here a chemical and enzymatic footprinting analysis of a branched DNA molecule formed from four complementary 50-mer strands. These strands are designed to form a stable junction, in which two steps of branch point migration freedom are possible. Exposure of the junction to Fe(II).EDTA shows protection of 3 or 4 residues in each strand at the branch, while two resolvase enzymes (endonuclease VII from phage T4 and endonuclease I from phage T7), cleave all four strand near the branch. Chemical footprinting of this junction using the reagents MPE.Fe(II) and (OP)2Cu(I) shows that the branch site is hyper-reactive to cutting induced by these probes as it is in an immobile four-arm junction. The effects involve more residues than in the immobile case. In the absence of divalent cations, the structure of the junction alters, sites of enhanced cleavage by MPE.Fe(II) and (OP)2Cu(I) disappear, and purines at the branch become reactive to diethyl pyrocarbonate. Our interpretation of these results is based on the properties of immobile junction analogs and their response to these probes. In the presence of Mg2+, the three migrational isomers coexist, each probably in the form of a 2-fold symmetric structure with two helical arms stacked.