The tyrosine binding pocket in the adaptor protein 1 (AP-1) mu1 subunit is necessary for Nef to recruit AP-1 to the major histocompatibility complex class I cytoplasmic tail

To evade the anti-human immunodeficiency virus (HIV) immune response, the HIV Nef protein disrupts major histocompatibility complex class I (MHC-I) trafficking by recruiting the clathrin adaptor protein 1 (AP-1) to the MHC-I cytoplasmic tail. Under normal conditions AP-1 binds dileucine and tyrosine signals (YXX phi motifs) via physically separate binding sites. In the case of the Nef-MHC-I complex, a tyrosine in the human leukocyte antigen (HLA)-A2 cytoplasmic tail ((320)YSQA) and a methionine in Nef (Met(20)) are absolutely required for AP-1 binding. Also present in Nef is a dileucine motif, which does not normally affect MHC-I trafficking and is not needed to recruit AP-1 to the Nef-MHC-I-complex. However, evidence is presented here that this dileucine motif can be activated by fusing Nef to the HLA-A2 tail in cis. Thus, the inability of this motif to function in trans likely results from a structural change that occurs when Nef binds to the MHC-I cytoplasmic tail. The physiologically relevant tyrosine-dependent recruitment of AP-1 to MHC-I, which occurs whether Nef is present in cis or trans, was stabilized by the acidic and polyproline domains within Nef. Additionally, amino acids Ala(324) and Asp(327) in the cytoplasmic tails of HLA-A and (but not HLA-C and HLA-E) molecules also stabilized AP-1 binding. Finally, mutation of the tyrosine binding pocket in the mu subunit of AP-1 created a dominant negative inhibitor of Nef-induced down-modulation of HLA-A2 that disrupted binding of wild type AP-1 to the Nef-MHC-I complex. Thus, these data provide evidence that Nef binding to the MHC-I cytoplasmic tail stabilizes the interaction of a tyrosine in the MHC-I cytoplasmic tail with the natural tyrosine binding pocket in AP-1.     

A new locus for otosclerosis, OTSC8, maps to the pericentromeric region of chromosome 9

Otosclerosis is a common disorder of the otic capsule resulting in hearing impairment in 0.3–0.4% of the Caucasian population. The aetiology of the disease remains unclear. In most cases, otosclerosis can be considered as a complex disease. In some cases, the disease is inherited as an autosomal dominant trait, sometimes with reduced penetrance. To date, seven autosomal dominant loci have been reported, but none of the disease-causing genes has been identified. In this study, we present the results of a genome-wide linkage analysis in a large Tunisian family segregating autosomal dominant otosclerosis. Linkage analysis localised the responsible gene to chromosome 9p13.1-9q21.11 with a maximal LOD score of 4.13, and this locus was named OTSC8. Using newly generated short tandem repeat polymorphism markers, we mapped this new otosclerosis locus to a 34.16 Mb interval between the markers D9S970 and D9S1799. This region comprises the pericentromeric region on both arms of chromosome 9, a highly complex region containing many duplicated sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

p53 family in development

The p53 family network is a unique cellular processor that integrates information from various pathways and determines cellular choices between proliferation, replication arrest/repair, differentiation, senescence, or apoptosis. The most studied role of the p53 family is the regulation of stress response and tumor suppression. By removing damaged cells from the proliferating pool, p53 family members preserve the integrity of the genome. In addition to this well recognized role, recent data implicate the p53 protein family in a broader role of controlling cell proliferation, differentiation and death. Members of the p53 protein family with opposing activity perform coordination of these processes. Imbalance of p53 protein family may contribute to a significant proportion of congenital developmental abnormalities in humans.     

Modelling Future Coronary Heart Disease Mortality to 2030 in the British Isles

Objective

Despite rapid declines over the last two decades, coronary heart disease (CHD) mortality rates in the British Isles are still amongst the highest in Europe. This study uses a modelling approach to compare the potential impact of future risk factor scenarios relating to smoking and physical activity levels, dietary salt and saturated fat intakes on future CHD mortality in three countries: Northern Ireland (NI), Republic of Ireland (RoI) and Scotland.

Methods

CHD mortality models previously developed and validated in each country were extended to predict potential reductions in CHD mortality from 2010 (baseline year) to 2030. Risk factor trends data from recent surveys at baseline were used to model alternative future risk factor scenarios: Absolute decreases in (i) smoking prevalence and (ii) physical inactivity rates of up to 15% by 2030; relative decreases in (iii) dietary salt intake of up to 30% by 2030 and (iv) dietary saturated fat of up to 6% by 2030. Probabilistic sensitivity analyses were then conducted.

Results

Projected populations in 2030 were 1.3, 3.4 and 3.9 million in NI, RoI and Scotland respectively (adults aged 25–84). In 2030: assuming recent declining mortality trends continue: 15% absolute reductions in smoking could decrease CHD deaths by 5.8–7.2%. 15% absolute reductions in physical inactivity levels could decrease CHD deaths by 3.1–3.6%. Relative reductions in salt intake of 30% could decrease CHD deaths by 5.2–5.6% and a 6% reduction in saturated fat intake might decrease CHD deaths by some 7.8–9.0%. These projections remained stable under a wide range of sensitivity analyses.

Conclusions

Feasible reductions in four cardiovascular risk factors (already achieved elsewhere) could substantially reduce future coronary deaths. More aggressive polices are therefore needed in the British Isles to control tobacco, promote healthy food and increase physical activity.     

Molecular characterization of bacterial communities mineralizing benzene under sulfate-reducing conditions

The microbial communities of in situ reactor columns degrading benzene with sulfate as an electron acceptor were analyzed based on clone libraries and terminal restriction fragment length polymorphism fingerprinting of PCR-amplified 16S rRNA genes. The columns were filled with either lava granules or sand particles and percolated with groundwater from a benzene-contaminated aquifer. The predominant organisms colonizing the lava granules were related to Magnetobacterium sp., followed by a phylotype affiliated to the genera Cryptanaerobacter/Pelotomaculum and several Deltaproteobacteria. From the sand-filled columns, a stable benzene-degrading consortium was established in sand-filled laboratory microcosms under sulfate-reducing conditions. It was composed of Delta- and Epsilonproteobacteria, Clostridia, Chloroflexi, Actinobacteria and Bacteroidetes. The most prominent phylotype of the consortium was related to the genus Sulfurovum, followed by Desulfovibrio sp. and the Cryptanaerobacter/Pelotomaculum phylotype. The proportion of the latter was similar in both communities and significantly increased after repeated benzene-spiking. During cultivation on aromatic substrates other than benzene, the Cryptanaerobacter/Pelotomaculum phylotype was outcompeted by other community members. Hence, this organism appears to be specific for benzene as a growth substrate and might play a key role in benzene degradation in both communities. Based on the possible functions of the community members and thermodynamic calculations, a functional model for syntrophic benzene degradation under sulfate-reducing conditions is proposed.     

Structural characteristics of the plasmid-encoded toxin from enteroaggregative Escherichia coli

Intoxication by the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli requires toxin translocation from the endoplasmic reticulum (ER) to the cytosol. This event involves the quality control system of ER-associated degradation (ERAD), but the molecular details of the process are poorly characterized. For many structurally distinct AB-type toxins, ERAD-mediated translocation is triggered by the spontaneous unfolding of a thermally unstable A chain. Here we show that Pet, a non-AB toxin, engages ERAD by a different mechanism that does not involve thermal unfolding. Circular dichroism and fluorescence spectroscopy measurements demonstrated that Pet maintains most of its secondary and tertiary structural features at 37 degrees C, with significant thermal unfolding only occurring at temperatures >or=50 degrees C. Fluorescence quenching experiments detected the partial solvent exposure of Pet aromatic amino acid residues at 37 degrees C, and a cell-based assay suggested that these changes could activate an ERAD-related event known as the unfolded protein response. We also found that HEp-2 cells were resistant to Pet intoxication when incubated with glycerol, a protein stabilizer. Altogether, our data are consistent with a model in which ERAD activity is triggered by a subtle structural destabilization of Pet and the exposure of Pet hydrophobic residues at physiological temperature. This was further supported by computer modeling analysis, which identified a surface-exposed hydrophobic loop among other accessible nonpolar residues in Pet. From our data it appears that Pet can promote its ERAD-mediated translocation into the cytosol by a distinct mechanism involving partial exposure of hydrophobic residues rather than the substantial unfolding observed for certain AB toxins.     

Variable patterns of programmed death-1 expression on fully functional memory T cells after spontaneous resolution of hepatitis C virus infection

The inhibitory receptor programmed death-1 (PD-1) is present on CD8(+) T cells in chronic hepatitis C virus (HCV), but expression patterns in spontaneously resolving infections are incompletely characterized. Here we report that PD-1 was usually absent on memory CD8(+) T cells from chimpanzees with resolved infections, but sustained low-level expression was sometimes observed in the absence of apparent virus replication. PD-1-positive memory T cells expanded and displayed antiviral activity upon reinfection with HCV, indicating conserved function. This animal model should facilitate studies of whether PD-1 differentially influences effector and memory T-cell function in resolved versus persistent human infections.     

Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube

The actin cytoskeleton plays a crucial role in the growth and polarity of the pollen tube. Due to inconsistencies in the conventional preservation methods, we lack a unified view of the organization of actin microfilaments, especially in the apical domain, where tip growth occurs. In an attempt to improve fixation methods, we have developed a rapid freeze-whole mount procedure, in which growing pollen tubes (primarily lily) are frozen in liquid propane at –180°C, substituted at –80°C in acetone containing glutaraldehyde, rehydrated, quenched with sodium borohydride, and probed with antibodies. Confocal microscopy reveals a distinct organization of actin in the apical domain that consists of a dense cortical fringe or collar of microfilaments starting about 1–5 m behind the extreme apex and extending basally for an additional 5–10 m. In the shank of the pollen tube, basal to the fringe, actin forms abundant longitudinal filaments that are evenly dispersed throughout the cytoplasm. We have also developed an improved ambient-temperature chemical fixation procedure, modified from a protocol based on simultaneous fixation and phalloidin staining. We removed EGTA, elevated the pH to 9, and augmented the fixative with ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS). Notably, this protocol preserves the actin cytoskeleton in a pattern similar to that produced by cryofixation. These procedures provide a reproducible way to preserve the actin cytoskeleton; employing them, we find that a cortical fringe in the apex and finely dispersed longitudinal filaments in the shank are consistent features of the actin cytoskeleton.