The structure of human CD23 and its interactions with IgE and CD21

The low-affinity immunoglobulin E (IgE) receptor, CD23 (FcɛRII), binds both IgE and CD21 and, through these interactions, regulates the synthesis of IgE, the antibody isotype that mediates the allergic response. We have determined the three-dimensional structure of the C-type lectin domain of CD23 in solution by nuclear magnetic resonance spectroscopy. An analysis of concentration-dependent chemical shift perturbations have allowed us to identify the residues engaged in self-association to the trimeric state, whereas ligand-induced changes have defined the binding sites for IgE and CD21. The results further reveal that CD23 can bind both ligands simultaneously. Despite the C-type lectin domain structure, none of the interactions require calcium. We also find that IgE and CD23 can interact to form high molecular mass multimeric complexes. The interactions that we have described provide a solution to the paradox that CD23 is involved in both up- and down-regulation of IgE and provide a structural basis for the development of inhibitors of allergic disease

Pre-Procedural Atorvastatin Mobilizes Endothelial Progenitor Cells: Clues to the Salutary Effects of Statins on Healing of Stented Human Arteries

OBJECTIVES: Recent clinical trials suggest an LDL-independent superiority of intensive statin therapy in reducing target vessel revascularization and peri-procedural myocardial infarctions in patients who undergo percutaneous coronary interventions (PCI). While animal studies demonstrate that statins mobilize endothelial progenitor cells (EPCs) which can enhance arterial repair and attenuate neointimal formation, the precise explanation for the clinical PCI benefits of high dose statin therapy remain elusive. Thus we serially assessed patients undergoing PCI to test the hypothesis that high dose Atorvastatin therapy initiated prior to PCI mobilizes EPCs that may be capable of enhancing arterial repair. METHODS AND RESULTS: Statin naïve male patients undergoing angiography for stent placement were randomized to standard therapy without Atorvastatin (n = 10) or treatment with Atorvastatin 80 mg (n = 10) beginning three days prior to stent implantation. EPCs were defined by flow cytometry (e.g., surface marker profile of CD45dim/34+/133+/117+). As well, we also enumerated cultured angiogenic cells (CACs) by standard in vitro culture assay. While EPC levels did not fluctuate over time for the patients free of Atorvastatin, there was a 3.5-fold increase in EPC levels with high dose Atorvastatin beginning within 3 days of the first dose (and immediately pre-PCI) which persisted at 4 and 24 hours post-PCI (p<0.05). There was a similar rise in CAC levels as assessed by in vitro culture. CACs cultured in the presence of Atorvastatin failed to show augmented survival or VEGF secretion but displayed a 2-fold increase in adhesion to stent struts (p<0.05). CONCLUSIONS: High dose Atorvastatin therapy pre-PCI improves EPC number and CAC number and function in humans which may in part explain the benefit in clinical outcomes seen in patients undergoing coronary interventions

Directed evolution to re-adapt a co-evolved network within an enzyme.

We have previously used targeted active-site saturation mutagenesis to identify a number of transketolase single mutants that improved activity towards either glycolaldehyde (GA), or the non-natural substrate propionaldehyde (PA). Here, all attempts to recombine the singles into double mutants led to unexpected losses of specific activity towards both substrates. A typical trade-off occurred between soluble expression levels and specific activity for all single mutants, but many double mutants decreased both properties more severely suggesting a critical loss of protein stability or native folding. Statistical coupling analysis (SCA) of a large multiple sequence alignment revealed a network of nine co-evolved residues that affected all but one double mutant. Such networks maintain important functional properties such as activity, specificity, folding, stability, and solubility and may be rapidly disrupted by introducing one or more non-naturally occurring mutations. To identify variants of this network that would accept and improve upon our best D469 mutants for activity towards PA, we created a library of random single, double and triple mutants across seven of the co-evolved residues, combining our D469 variants with only naturally occurring mutations at the remaining sites. A triple mutant cluster at D469, E498 and R520 was found to behave synergistically for the specific activity towards PA. Protein expression was severely reduced by E498D and improved by R520Q, yet variants containing both mutations led to improved specific activity and enzyme expression, but with loss of solubility and the formation of inclusion bodies. D469S and R520Q combined synergistically to improve k(cat) 20-fold for PA, more than for any previous transketolase mutant. R520Q also doubled the specific activity of the previously identified D469T to create our most active transketolase mutant to date. Our results show that recombining active-site mutants obtained by saturation mutagenesis can rapidly destabilise critical networks of co-evolved residues, whereas beneficial single mutants can be retained and improved upon by randomly recombining them with natural variants at other positions in the network

Responsible management: Engaging moral reflexive practice through threshold concepts

YesIn this conceptual paper we argue that, to date, principles of responsible management have not impacted practice as anticipated because of a disconnect between knowledge and practice. This disconnect means that an awareness of ethical concerns, by itself, does not help students take personal responsibility for their actions. We suggest that an abstract knowledge of principles has to be supplemented by an engaged understanding of the responsibility of managers and leaders to actively challenge irresponsible practices. We argue that a form of moral reflexive practice drawing on an understanding of threshold concepts is central to responsible management, and provides a gateway to transformative learning. Our conceptual argument leads to implications for management and professional education

Developmental axon pruning mediated by BDNF-p75NTR–dependent axon degeneration

The mechanisms that regulate the pruning of mammalian axons are just now being elucidated. Here, we describe a mechanism by which, during developmental sympathetic axon competition, winning axons secrete brain-derived neurotrophic factor (BDNF) in an activity-dependent fashion, which binds to the p75 neurotrophin receptor (p75NTR) on losing axons to cause their degeneration and, ultimately, axon pruning. Specifically, we found that pruning of rat and mouse sympathetic axons that project to the eye requires both activity-dependent BDNF and p75NTR. p75NTR and BDNF are also essential for activity-dependent axon pruning in culture, where they mediate pruning by directly causing axon degeneration. p75NTR, which is enriched in losing axons, causes axonal degeneration by suppressing TrkA-mediated signaling that is essential for axonal maintenance. These data provide a mechanism that explains how active axons can eliminate less-active, competing axons during developmental pruning by directly promoting p75NTR-mediated axonal degeneration

Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIV(mac239)

BACKGROUND: The 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIV(mac239)) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA (vRNA). We have constructed a series of deletions in the SIV(mac239 )leader sequence in order to determine the involvement of this region in both the packaging and dimerization of viral genomic RNA. We also assessed the impact of these deletions upon viral infectiousness, replication kinetics and gene expression in cell lines and monkey peripheral blood mononuclear cells (PBMC). RESULTS: Regions on both sides of the major splice donor (SD) were found to be necessary for the efficiency and specificity of viral genome packaging. However, stem-loop1 is critical for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the initiation site of SIV-Gag have additive effects on RNA packaging and contribute to a lesser degree to RNA dimerization. The targeted disruption of structures on both sides of the SD also severely impacts viral infectiousness, gene expression and replication in both CEMx174 cells and rhesus PBMC. CONCLUSION: In the leader region of SIV(mac239), stem-loop1 functions as the primary determinant for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the translational initiation site of SIV-Gag are classified as secondary determinants and play a role in dimerization. Collectively, these data signify a linkage between the primary encapsidation determinant of SIV(mac239 )and RNA dimerization

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes

Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA (called SD') that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration

T-cell and serological responses to Erp, an exported Mycobacterium tuberculosis protein, in tuberculosis patients and healthy individuals

<p>Abstract</p> <p>Background</p> <p>The identification of antigens able to differentiate tuberculosis (TB) disease from TB infection would be valuable. Cellular and humoral immune responses to Erp (Exported repetitive protein) – a recently identified <it>M. tuberculosis </it>protein – have not yet been investigated in humans and may contribute to this aim.</p> <p>Methods</p> <p>We analyzed the cellular and humoral immune responses to Erp, ESAT-6, Ag85B and PPD in TB patients, in BCG⁺individuals without infection, BCG⁺individuals with latent TB infection (LTBI) and BCG^-controls. We used lymphoproliferation, ELISpot IFN-γ, cytokine production assays and detection of specific human antibodies against recombinant <it>M. tuberculosis </it>proteins.</p> <p>Results</p> <p>We included 22 TB patients, 9 BCG⁺individuals without TB infection, 7 LTBI and 7 BCG^-controls. Erp-specific T cell counts were higher in LTBI than in the other groups. Erp-specific T cell counts were higher in LTBI subjects than TB patients (median positive frequency of 211 SFC/10⁶PBMC (range 118–2000) for LTBI subjects compared to 80 SFC/10⁶PBMC (range 50–191), p = 0.019); responses to PPD and ESAT-6 antigens did not differ between these groups. IFN-γ secretion after Erp stimulation differed between TB patients and LTBI subjects (p = 0.02). Moreover, LTBI subjects but not TB patients or healthy subjects produced IgG3 against Erp.</p> <p>Conclusion</p> <p>The frequencies of IFN-γ-producing specific T cells, the IFN-γ secretion and the production of IgG3 after Erp stimulation are higher in LTBI subjects than in TB patients, whereas PPD and ESAT-6 are not.</p

A qualitative analysis of environmental policy and children's health in Mexico

<p>Abstract</p> <p>Background</p> <p>Since Mexico's joining the North American Free Trade Agreement (NAFTA) and the Organization for Economic Cooperation and Development (OECD) in 1994, it has witnessed rapid industrialization. A byproduct of this industrialization is increasing population exposure to environmental pollutants, of which some have been associated with childhood disease. We therefore identified and assessed the adequacy of existing international and Mexican governance instruments and policy tools to protect children from environmental hazards.</p> <p>Methods</p> <p>We first systematically reviewed PubMed, the Mexican legal code and the websites of the United Nations, World Health Organization, NAFTA and OECD as of July 2007 to identify the relevant governance instruments, and analyzed the approach these instruments took to preventing childhood diseases of environmental origin. Secondly, we interviewed a purposive sample of high-level government officials, researchers and non-governmental organization representatives, to identify their opinions and attitudes towards children's environmental health and potential barriers to child-specific protective legislation and implementation.</p> <p>Results</p> <p>We identified only one policy tool describing specific measures to reduce developmental neurotoxicity and other children's health effects from lead. Other governance instruments mention children's unique vulnerability to ozone, particulate matter and carbon monoxide, but do not provide further details. Most interviewees were aware of Mexican environmental policy tools addressing children's health needs, but agreed that, with few exceptions, environmental policies do not address the specific health needs of children and pregnant women. Interviewees also cited state centralization of power, communication barriers and political resistance as reasons for the absence of a strong regulatory platform.</p> <p>Conclusions</p> <p>The Mexican government has not sufficiently accounted for children's unique vulnerability to environmental contaminants. If regulation and legislation are not updated and implemented to protect children, increases in preventable exposures to toxic chemicals in the environment may ensue.</p

Loss of Caveolin-1 Accelerates Neurodegeneration and Aging

The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease.We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression.Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimer's disease