Probing the beta2 adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists

The beta(2) adrenergic receptor (beta(2)AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The beta(2)AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between beta(2)AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the beta(2)AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.     

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.     

Xanthomonas T3S Effector XopN Suppresses PAMP-Triggered Immunity and Interacts with a Tomato Atypical Receptor-Like Kinase and TFT1

XopN is a virulence factor from Xanthomonas campestris pathovar vesicatoria (Xcv) that is translocated into tomato (Solanum lycopersicum) leaf cells by the pathogen''s type III secretion system. Xcv ΔxopN mutants are impaired in growth and have reduced ability to elicit disease symptoms in susceptible tomato leaves. We show that XopN action in planta reduced pathogen-associated molecular pattern (PAMP)-induced gene expression and callose deposition in host tissue, indicating that XopN suppresses PAMP-triggered immune responses during Xcv infection. XopN is predicted to have irregular, α-helical repeats, suggesting multiple protein–protein interactions in planta. Consistent with this prediction, XopN interacted with the cytosolic domain of a Tomato Atypical Receptor-Like Kinase1 (TARK1) and four Tomato Fourteen-Three-Three isoforms (TFT1, TFT3, TFT5, and TFT6) in yeast. XopN/TARK1 and XopN/TFT1 interactions were confirmed in planta by bimolecular fluorescence complementation and pull-down analysis. Xcv ΔxopN virulence defects were partially suppressed in transgenic tomato leaves with reduced TARK1 mRNA levels, indicating that TARK1 plays an important role in the outcome of Xcv–tomato interactions. These data provide the basis for a model in which XopN binds to TARK1 to interfere with TARK1-dependent signaling events triggered in response to Xcv infection.     

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.     

Heat shock protein expression analysis in canine osteosarcoma reveals HSP60 as a potentially relevant therapeutic target

Heat shock proteins (HSP) are highly conserved across eukaryotic and prokaryotic species. These proteins play a role in response to cellular stressors, protecting cells from damage and facilitating recovery. In tumor cells, HSPs can have cytoprotective effects and interfere with apoptotic cascades. This study was performed to assess the prognostic and predictive values of the gene expression of HSP family members in canine osteosarcoma (OS) and their potential for targeted therapy. Gene expressions for HSP were assessed using quantitative PCR (qPCR) on 58 snap-frozen primary canine OS tumors and related to clinic-pathological parameters. A significant increased expression of HSP60 was found in relation to shorter overall survival and an osteoblastic phenotype. Therefore, the function of HSP60 was investigated in more detail. Immunohistochemical analysis revealed heterogeneous staining for HSP60 in tumors. The highest immunoreactivity was found in tumors of short surviving dogs. Next HSP expression was shown in a variety of canine and human OS cell lines by qPCR and Western blot. In two highly metastatic cell lines HSP60 expression was silenced using siRNA resulting in decreased cell proliferation and induction of apoptosis in both cell lines. It is concluded that overexpression of HSP60 is associated with a poor prognosis of OS and should be evaluated as a new target for therapy.     

Modulation by weak bases or weak acids of the pH of cell sap and phosphoenolpyruvate carboxylase activity in leaf discs of C4 plants

When leaf discs of a C₄ species, Alternanthera pungens (L.) H.B. and K. or Amaranthus hypochondriacus L., were preincubated in 7.5 m M NH₄Cl, the pH of the cell sap increased by nearly 0.3 unit, while the activity of phosphoenolpyruvate carboxylase (PEPC) about doubled compared to the cell sap from control leaf discs (preincubated in 5 m M Tricine‐KOH, pH 8.5). The sensitivity of PEPC to L ‐malate (a feedback inhibitor) decreased marginally as a result of cytosolic alkalization. The pH of the cell sap and PEPC activity decreased by nearly 0.4 unit and 50%, respectively, when leaf discs were incubated in weak organic acids such as propionic, butyric or salicylic acid. Thus, our results demonstrate a marked modulation in vivo of cell sap pH and PEPC activity in leaf discs from C₄ plants by external alkalizing or acidifying reagents. The rise in PEPC activity due to alkalization of leaf discs was not sensitive to cycloheximide, implying that cytosolic protein synthesis was not involved in the activation of PEPC. Despite the marked increase in the PEPC activity due to the base‐loading of leaf discs, the change in malate sensitivity of the enzyme was only marginal, indicating that there was no significant increase in the extent of PEPC‐phosphorylation. Besides the physiological significance, the technique of acid/ base‐loading may be an important tool for studying the regulation of PEPC in leaf discs of C₄ species, since the activity of PEPC could be enhanced apparently without phosphorylation of the enzyme.     

Drug reformulation for a neglected disease. The NANOHAT project to develop a safer more effective sleeping sickness drug

BackgroundHuman African trypanosomiasis (HAT or sleeping sickness) is caused by the parasite Trypanosoma brucei sspp. The disease has two stages, a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system stage where the parasite penetrates the brain, causing death if untreated. Treatment is stage-specific, due to the blood-brain barrier, with less toxic drugs such as pentamidine used to treat stage 1. The objective of our research programme was to develop an intravenous formulation of pentamidine which increases CNS exposure by some 10–100 fold, leading to efficacy against a model of stage 2 HAT. This target candidate profile is in line with drugs for neglected diseases inititative recommendations.MethodologyTo do this, we evaluated the physicochemical and structural characteristics of formulations of pentamidine with Pluronic micelles (triblock-copolymers of polyethylene-oxide and polypropylene oxide), selected candidates for efficacy and toxicity evaluation in vitro, quantified pentamidine CNS delivery of a sub-set of formulations in vitro and in vivo, and progressed one pentamidine-Pluronic formulation for further evaluation using an in vivo single dose brain penetration study.Principal FindingsScreening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline₂ receptor. The reduction in insulin secretion in MIN6 β-cells by pentamidine may be secondary to pentamidine-mediated activation of β-cell imidazoline receptors and impairment of cell viability. Pluronic F68 (0.01%w/v)-pentamidine formulation had a similar inhibitory effect on insulin secretion as pentamidine alone and an additive trypanocidal effect in vitro. However, all Pluronics tested (P85, P105 and F68) did not significantly enhance brain exposure of pentamidine.SignificanceThese results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.