Low pH-Induced Pore Formation by the T Domain of Botulinum Toxin Type A is Dependent upon NaCl Concentration

Botulinum neurotoxins (BoNTs) undergo low pH-triggered membrane insertion, resulting in the translocation of their light (catalytic) chains into the cytoplasm. The T (translocation) domain of the BoNT heavy chain is believed to carry out translocation. Here, the behavior of isolated T domain from BoNT type A has been characterized, both in solution and when associated with model membranes. When BoNT T domain prepared in the detergent dodecylmaltoside was diluted into aqueous solution, it exhibited a low pH-dependent conformational change below pH 6. At low pH the T domain associated with, and formed pores within, model membrane vesicles composed of 30 mol% dioleoylphosphatidylglycerol/70 mol% dioleoylphosphatidylcholine. Although T domain interacted with vesicles at low (50 mM) and high (400 mM) NaCl concentrations, the interaction required much less lipid at low salt. However, even at high lipid concentrations pore formation was much more pronounced at low NaCl concentrations than at high NaCl concentration. Increasing salt concentration after insertion in the presence of 50 mM NaCl did not decrease pore formation. A similar effect of NaCl concentration upon pore formation was observed in vesicles composed solely of dioleoylphosphatidylcholine, showing that the effect of NaCl did not solely involve modulation of electrostatic interactions between protein and anionic lipids. These results indicate that some feature of membrane-bound T domain tertiary structure critical for pore formation is highly dependent upon salt concentration.     

An improved ontological representation of dendritic cells as a paradigm for all cell types

Background  

Recent increases in the volume and diversity of life science data and information and an increasing emphasis on data sharing and interoperability have resulted in the creation of a large number of biological ontologies, including the Cell Ontology (CL), designed to provide a standardized representation of cell types for data annotation. Ontologies have been shown to have significant benefits for computational analyses of large data sets and for automated reasoning applications, leading to organized attempts to improve the structure and formal rigor of ontologies to better support computation. Currently, the CL employs multiple is_a relations, defining cell types in terms of histological, functional, and lineage properties, and the majority of definitions are written with sufficient generality to hold across multiple species. This approach limits the CL's utility for computation and for cross-species data integration.     

SCARFACE encodes an ARF-GAP that is required for normal auxin efflux and vein patterning in Arabidopsis

To identify molecular mechanisms controlling vein patterns, we analyzed scarface (sfc) mutants. sfc cotyledon and leaf veins are largely fragmented, unlike the interconnected networks in wild-type plants. SFC encodes an ADP ribosylation factor GTPase activating protein (ARF-GAP), a class with well-established roles in vesicle trafficking regulation. Quadruple mutants of SCF and three homologs (ARF-GAP DOMAIN1, 2, and 4) showed a modestly enhanced vascular phenotype. Genetic interactions between sfc and pinoid and between sfc and gnom suggest a possible function for SFC in trafficking of auxin efflux regulators. Genetic analyses also revealed interaction with cotyledon vascular pattern2, suggesting that lipid-based signals may underlie some SFC ARF-GAP functions. To assess possible roles for SFC in auxin transport, we analyzed sfc roots, which showed exaggerated responses to exogenous auxin and higher auxin transport capacity. To determine whether PIN1 intracellular trafficking was affected, we analyzed PIN1:green fluorescent protein (GFP) dynamics using confocal microscopy in sfc roots. We found normal PIN1:GFP localization at the apical membrane of root cells, but treatment with brefeldin A resulted in PIN1 accumulating in smaller and more numerous compartments than in the wild type. These data suggest that SFC is required for normal intracellular transport of PIN1 from the plasma membrane to the endosome.     

Effects of high-pressure processing on Toxoplasma gondii tissue cysts in ground pork

Ingestion of Toxoplasma gondii tissue cysts can result in severe disease in immunocompromised individuals and pregnant women. Treatment of meat and meat products to eliminate viable T. gondii tissue cysts would provide a means to protect consumers. In this study, we examined the effects of high-pressure processing (HPP) on ground pork containing viable tissue cysts of the VEG strain of T. gondii. Ground pork containing tissue cysts was exposed to 400, 300, 200, 100, or 0 MPa treatment for 30, 60, or 90 sec in a commercial HPP unit. The HPP-treated ground pork was subjected to acid-pepsin digestion and bioassayed in mice. The results of the mouse bioassay revealed that none of the mice inoculated with tissue cysts exposed to 400 or 300 MPa became infected, whereas all mice inoculated with tissue cysts exposed to 200, 100, or 0 MPa became infected with T. gondii regardless of exposure time. Results indicate that HPP treatment of ground pork with 300 MPa of pressure will render tissue cysts of T. gondii nonviable and make pork safe for human consumption.     

Association of polymorphisms in the Angiotensin-converting enzyme gene with Alzheimer disease in an Israeli Arab community

Several lines of evidence support for a role of angiotensin converting enzyme (ACE) in Alzheimer disease (AD). Most genetic studies have focused on an Alu insertion/deletion (I/D) polymorphism in the ACE gene (DCP1) and have yielded conflicting results. We evaluated the association between 15 single-nucleotide polymorphisms (SNPs) in DCP1, including the I/D variant, and AD in a sample of 92 patients with AD and 166 nondemented controls from an inbred Israeli Arab community. Although there was no evidence for association between AD and I/D, we observed significant association with SNPs rs4343 (P = .00001) and rs4351 (P = .01). Haplotype analysis revealed remarkably significant evidence of association with the SNP combination rs4343 and rs4351 (global P = 7.5 x 10(-7)). Individuals possessing the haplotype "GA" (frequency 0.21 in cases and 0.01 in controls) derived from these SNPs had a 45-fold increased risk of developing AD (95% CI 6.0-343.2) compared with those possessing any of the other three haplotypes. Longer range haplotypes including I/D were even more significant (lowest global P = 1.1 x 10(-12)), but the only consistently associated alleles were in rs4343 and rs4351. These results suggest that a variant in close proximity to rs4343 and rs4351 modulates susceptibility to AD in this community.     

Two circadian timing circuits in Neurospora crassa cells share components and regulate distinct rhythmic processes

In Neurospora crassa, FRQ, WC-1, and WC-2 proteins comprise the core circadian FRQ-based oscillator that is directly responsive to light and drives daily rhythms in spore development and gene expression. However, physiological and biochemical studies have demonstrated the existence of additional oscillators in the cell that function in the absence of FRQ (collectively termed FRQ-less oscillators [FLOs]). Whether or not these represent temperature-compensated, entrainable circadian oscillators is not known. The authors previously identified an evening-peaking gene, W06H2 (now called clock-controlled gene 16 [ccg-16]), which is expressed with a robust daily rhythm in cells that lack FRQ protein, suggesting that ccg-16 is regulated by a FLO. In this study, the authors provide evidence that the FLO driving ccg-16 rhythmicity is a circadian oscillator. They find that ccg-16 rhythms are generated by a temperature-responsive, temperature-compensated circadian FLO that, similar to the FRQ-based oscillator, requires functional WC-1 and WC-2 proteins for activity. They also find that FRQ is not essential for rhythmic WC-1 protein levels, raising the possibility that this WCFLO is involved in the generation of WC-1 rhythms. The results are consistent with the presence of 2 circadian oscillators within Neurospora cells, which the authors speculate may interact with each other through the shared WC proteins.     

Different thermodynamic binding mechanisms and peptide fine specificities associated with a panel of structurally similar high-affinity T cell receptors

To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L(d) as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1beta, 2alpha, 3alpha, or 3beta. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L(d). Four of the five TCRs examined bound to QL9-L(d) in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1beta mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L(d) and a single amino acid peptide variant of QL9, called QL9-Y5-L(d). While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1beta exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L(d) was solved and compared to structures of the 2C TCR/QL9-L(d) complex and three high-affinity TCR/QL9-L(d) complexes. Our findings show that the QL9-L(d) complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.     

Is Maternal Marriage Beneficial for Low-Income Adolescents?

The present study investigated the association of mothers' marriage and changes in young adolescents' cognitive and socioemotional development and changes in family processes. Analyses employed longitudinal data from the Three-City Study to track maternal partnerships for 860 low-income adolescents (10–14 years-old in Wave 1) across a 16 month period. No short-term benefits or risks emerged for youth when mothers entered marriage, with few changes in family or maternal functioning linked with marriage formation as well. In contrast, adolescents in stably married families experienced improved academic, behavioral, and psychological well-being compared to youth in stable cohabiting or single-parent families. Stable marriage was similarly linked to improvements across multiple domains of home and mothers' functioning. These patterns were not moderated by the male partner's identity (biological father or stepfather). Results support the benefits of stable marriage on youth development, but suggest that policies supporting movements into new marriages may not result in improved adolescent or family functioning, at least in the short term.