Evidence against a Human Cell-Specific Role for LRP6 in Anthrax Toxin Entry

The role of the cellular protein LRP6 in anthrax toxin entry is controversial. Previous studies showed that LRP6 was important for efficient intoxication of human M2182 prostate carcinoma cells but other studies performed with cells from gene-knockout mice demonstrated no role for either LRP6 or the related LRP5 protein in anthrax toxin entry. One possible explanation for this discrepancy is that LRP6 may be important for anthrax toxin entry into human, but not mouse, cells. To test this idea we have investigated the effect of knocking down LRP6 or LRP5 expression with siRNAs in human HeLa cells. We show here that efficient knockdown of either LRP6, LRP5, or both proteins has no influence on the kinetics of anthrax lethal toxin entry or MEK1 substrate cleavage in these cells. These data argue against a human-specific role for LRP6 in anthrax toxin entry and suggest instead that involvement of this protein may be restricted to certain cell types independently of their species of origin

Anthrax Toxin Receptor 2 Determinants that Dictate the pH Threshold of Toxin Pore Formation

The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH ∼6.0 or below when it is bound to ANTXR1, but only at pH ∼5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation

Anthrax Toxin Receptor Drives Protective Antigen Oligomerization and Stabilizes the Heptameric and Octameric Oligomer by a Similar Mechanism

Anthrax toxin is comprised of protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins are individually nontoxic; however, when PA assembles with LF and EF, it produces lethal toxin and edema toxin, respectively. Assembly occurs either on cell surfaces or in plasma. In each milieu, PA assembles into a mixture of heptameric and octameric complexes that bind LF and EF. While octameric PA is the predominant form identified in plasma under physiological conditions (pH 7.4, 37°C), heptameric PA is more prevalent on cell surfaces. The difference between these two environments is that the anthrax toxin receptor (ANTXR) binds to PA on cell surfaces. It is known that the extracellular ANTXR domain serves to stabilize toxin complexes containing the PA heptamer by preventing premature PA channel formation--a process that inactivates the toxin. The role of ANTXR in PA oligomerization and in the stabilization of toxin complexes containing octameric PA are not understood.Using a fluorescence assembly assay, we show that the extracellular ANTXR domain drives PA oligomerization. Moreover, a dimeric ANTXR construct increases the extent of and accelerates the rate of PA assembly relative to a monomeric ANTXR construct. Mass spectrometry analysis shows that heptameric and octameric PA oligomers bind a full stoichiometric complement of ANTXR domains. Electron microscopy and circular dichroism studies reveal that the two different PA oligomers are equally stabilized by ANTXR interactions.We propose that PA oligomerization is driven by dimeric ANTXR complexes on cell surfaces. Through their interaction with the ANTXR, toxin complexes containing heptameric and octameric PA oligomers are similarly stabilized. Considering both the relative instability of the PA heptamer and extracellular assembly pathway identified in plasma, we propose a means to regulate the development of toxin gradients around sites of infection during anthrax pathogenesis

Identification of Genes Required for Neural-Specific Glycosylation Using Functional Genomics

Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA–binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells

Time spent on work-related activities, social activities and time pressure as intermediary determinants of health disparities among elderly women and men in 5 European countries: a structural equation model

Background Psychosocial factors shape the health of older adults through complex inter-relating pathways. Besides socioeconomic factors, time use activities may explain gender inequality in self-reported health. This study investigated the role of work-related and social time use activities as determinants of health in old age. Specifically, we analysed whether the impact of stress in terms of time pressure on health mediated the relationship between work-related time use activities (i.e. housework and paid work) on self-reported health. Methods We applied structural equation models and a maximum-likelihood function to estimate the direct and indirect effects of psychosocial factors on health using pooled data from the Multinational Time Use Study on 11,168 men and 14,295 women aged 65+ from Italy, Spain, UK, France and the Netherlands. Results The fit indices for the conceptual model indicated an acceptable fit for both men and women. The results showed that socioeconomic status (SES), demographic factors, stress and work-related time use activities after retirement had a significant direct influence on self-reported health among the elderly, but the magnitude of the effects varied by gender. Social activities had a positive impact on self-reported health but had no significant impact on stress among older men and women. The indirect standardized effects of work-related activities on self-reported health was statistically significant for housework (β = − 0.006; P  0.05 among women), which implied that the paths from paid work and housework on self-reported health via stress (mediator) was very weak because their indirect effects were close to zero. Conclusions Our findings suggest that although stress in terms of time pressure has a direct negative effect on health, it does not indirectly influence the positive effects of work-related time use activities on self-reported health among elderly men and women. The results support the time availability hypothesis that the elderly may not have the same time pressure as younger adults after retirement

Evidence-based Kernels: Fundamental Units of Behavioral Influence

This paper describes evidence-based kernels, fundamental units of behavioral influence that appear to underlie effective prevention and treatment for children, adults, and families. A kernel is a behavior–influence procedure shown through experimental analysis to affect a specific behavior and that is indivisible in the sense that removing any of its components would render it inert. Existing evidence shows that a variety of kernels can influence behavior in context, and some evidence suggests that frequent use or sufficient use of some kernels may produce longer lasting behavioral shifts. The analysis of kernels could contribute to an empirically based theory of behavioral influence, augment existing prevention or treatment efforts, facilitate the dissemination of effective prevention and treatment practices, clarify the active ingredients in existing interventions, and contribute to efficiently developing interventions that are more effective. Kernels involve one or more of the following mechanisms of behavior influence: reinforcement, altering antecedents, changing verbal relational responding, or changing physiological states directly. The paper describes 52 of these kernels, and details practical, theoretical, and research implications, including calling for a national database of kernels that influence human behavior

Associations Between Discrimination and Cardiovascular Health Among Asian Indians in the United States

Asian Indians (AI) have a high risk of atherosclerotic cardiovascular disease. The study investigated associations between discrimination and (1) cardiovascular risk and (2) self-rated health among AI. Higher discrimination scores were hypothesized to relate to a higher cardiovascular risk score (CRS) and poorer self-rated health. Asian Indians (n = 757) recruited between 2010 and 2013 answered discrimination and self-reported health questions. The CRS (0–8 points) included body-mass index, systolic blood pressure, total cholesterol, and fasting blood glucose levels of AI. Multiple linear regression analyses were conducted to evaluate relationships between discrimination and the CRS and discrimination and self-rated health, adjusting for psychosocial and clinical factors. There were no significant relationships between discrimination and the CRS (p ≥ .05). Discrimination was related to poorer self-reported health, B = −.41 (SE = .17), p = .02. Findings suggest perhaps there are important levels at which discrimination may harm health