Site-specific ubiquitination exposes a linear motif to promote interferon-alpha receptor endocytosis

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCF^βTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.     

Purification and Characterization of Polygalacturonase-3 from Jamaica cherry (Muntingia calabura Linn)

Endo-polygalacturonase-3 (PG-3), the key enzyme of fruit ripening was purified to near homogeneity as judged by native PAGE from the fruit tissues of Jamaica cherry (Muntingia calabura) using ammonium sulphate fractionation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the PG-3 enzyme was determined as 85 kD, by size exclusion chromatography. SDS-PAGE of PG-3 revealed two dissimilar bands of 62 and 21 kD as heterogenous subunits. The optimum pH of PG-3 was found to be 4.0. The enzyme had an optimum temperature of 40°C and was relatively stable at 50°C and 60°C. K_m for the substrate polygalacturonic acid was found to be 0.27%. The purified enzyme was a glycoprotein with 6.6 % carbohydrate content.     

Copsin,a Novel Peptide-based Fungal Antibiotic Interfering with the Peptidoglycan Synthesis

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/β-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.     

M. tuberculosis induces potent activation of IDO-1, but this is not essential for the immunological control of infection

Indoleamine 2,3-dioxygenesae-1 (IDO-1) catalyses the initial, rate-limiting step in tryptophan metabolism, thereby regulating tryptophan availability and the formation of downstream metabolites, including picolinic and quinolinic acid. We found that Mycobacterium tuberculosis infection induced marked upregulation of IDO-1 expression in both human and murine macrophages in vitro and in the lungs of mice following aerosol challenge with M. tuberculosis. The absence of IDO-1 in dendritic cells enhanced the activation of mycobacteria-specific T cells in vitro. Interestingly, IDO-1-deficiency during M. tuberculosis infection in mice was not associated with altered mycobacteria-specific T cell responses in vivo. The bacterial burden of infected organs, pulmonary inflammatory responses, and survival were also comparable in M. tuberculosis-infected IDO-1 deficient and wild type animals. Tryptophan is metabolised into either picolinic acid or quinolinic acid, but only picolinic acid inhibited the growth of M. tuberculosis in vitro. By contrast macrophages infected with pathogenic mycobacteria, produced quinolinic, rather than picolinic acid, which did not reduce M. tuberculosis growth in vitro. Therefore, although M. tuberculosis induces robust expression of IDO-1 and activation of tryptophan metabolism, IDO-1-deficiency fails to impact on the immune control and the outcome of the infection in the mouse model of tuberculosis.     

Life cycle assessment of commercial furniture: a case study of Formway LIFE chair

Background, aims and scope  The environmental aspects of companies and their products are becoming more significant in delivering competitive advantage. Formway Furniture, a designer and manufacturer of office furniture products, is a New Zealand-based company that is committed to sustainable development. It manufactures two models of the light, intuitive, flexible and environmental (LIFE) office chair: one with an aluminium base and one with a glass-filled nylon (GFN) base. It was decided to undertake a life cycle assessment (LCA) study of these two models in order to: (1) determine environmental hotspots in the life cycle of the two chairs (goal 1); (2) compare the life cycle impacts of the two chairs (goal 2); and (3) compare alternative potential waste-management scenarios (goal 3). The study also included sensitivity analysis with respect to recycled content of aluminium in the product. Materials and methods  The LIFE chair models consist of a mix of metal and plastic components manufactured by selected Formway suppliers according to design criteria. Hence, the research methodology included determining the specific material composition of the two chair models and acquisition of manufacturing data from individual suppliers. These data were compiled and used in conjunction with pre-existing data, specifically from the ecoinvent database purchased in conjunction with the SimaPro7 LCA software, to develop the life cycle inventory of the two chair models. The life cycle stages included in the study extended from raw-material extraction through to waste management. Impact assessment was carried out using CML 2 baseline 2000, the methodology developed by Leiden University’s Institute for Environmental Sciences. Results  This paper presents results for global warming potential (GWP100). The study showed a significant impact contribution from the raw-material extraction/refinement stage for both chair models; aluminium extraction and refining made the greatest contribution to GWP100. The comparison of the two LIFE chair models showed that the model with the aluminium base had a higher GWP100 impact than the model with the GFN base. The waste-management scenario compared the GWP100 result when (1) both chair models were sent to landfill and (2) steel and aluminium components were recycled with the remainder of the chair sent to landfill. The results showed that the recycling scenario contributed to a reduced GWP100 result. Since production and processing of aluminium was found to be significant, a sensitivity analysis was carried out to determine the impact of using aluminium with different recycled contents (0%, 34% and 100%) in both waste-management scenarios; this showed that increased use of recycled aluminium was beneficial. The recycling at end-of-life scenarios was modelled using two different end-of-life allocation approaches, i.e. consequential and attributional, in order to illustrate the variation in results caused by choice of allocation approach. The results using the consequential approach showed that recycling at end-of-life was beneficial, while use of the attributional method led to a similar GWP100 as that seen for the landfill scenario. Discussion  The results show that the main hotspot in the life cycle is the raw-material extraction/refinement stage. This can be attributed to the extraction and processing of aluminium, a material that is energy intensive. The LIFE chair model with the aluminium base has a higher GWP100 as it contains more aluminium. Sensitivity analysis pertaining to the recycled content of aluminium showed that use of aluminium with high recycled content was beneficial; this is because production of recycled aluminium is less energy intensive than production of primary aluminium. The waste-management scenario showed that recycling at end-of-life resulted in a significantly lower GWP100 than landfilling at end-of-life. However, this result is dependent upon the modelling approach used for recycling. Conclusions  With respect to goal 1, the study found that the raw-material extraction/refinement stage of the life cycle was a significant factor for both LIFE chair models. This was largely due to the use of aluminium in the product. For goal 2, it was found that the LIFE chair model with the aluminium base had a higher GWP100 than the GFN model, again due to the material content of the two models. Results for goal 3 illustrated that recycling at end-of-life is beneficial when using a system expansion (consequential) approach to model recycling; if an attributional ‘cut-off’ approach is used to model recycling at end-of-life, there is virtually no difference in the results between landfilling and recycling. Sensitivity analysis pertaining to the recycled content of aluminium showed that use of higher recycled contents leads to a lower GWP100 impact. Recommendation and perspectives  Most of the GWP100 impact was contributed during the raw-material extraction/refinement stage of the life cycle; thus, the overall impact of both LIFE chair models may be reduced through engaging in material choice and supply chain environmental management with respect to environmental requirements. The study identified aluminium components as a major contributor to GWP100 for both LIFE chair models and also highlighted the sensitivity of the results to its recycled content. Thus, it is recommended that the use of aluminium in future product designs be limited unless it is possible to use aluminium with a high recycled content. With respect to waste management, it was found that a substantial reduction in the GWP100 impact would occur if the chairs are recycled rather than landfilled, assuming an expanding market for aluminium. Thus, recycling the two LIFE chair models at end-of-life is highly recommended.