VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx

The mechanism(s) involved in agonist-stimulation of TRPC3 channels is not yet known. Here we demonstrate that TRPC3-N terminus interacts with VAMP2 and alphaSNAP. Further, endogenous and exogenously expressed TRPC3 colocalized and coimmunoprecipitated with SNARE proteins in neuronal and epithelial cells. Imaging of GFP-TRPC3 revealed its localization in the plasma membrane region and in mobile intracellular vesicles. Recovery of TRPC3-GFP fluorescence after photobleaching of the plasma membrane region was decreased by brefeldin-A or BAPTA-AM. Cleavage of VAMP2 with tetanus toxin (TeNT) did not prevent delivery of TRPC3 to the plasma membrane region but reduced its surface expression. TeNT also decreased carbachol and OAG, but not thapsigargin, stimulated Ca2+ influx. Importantly, carbachol, not thapsigargin, increased surface expression of TRPC3 that was attenuated by TeNT and not by BAPTA. In aggregate, these data suggest that VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to carbachol-stimulation of Ca2+ influx.     

Evolution of foraging behaviour in response to chronic malnutrition in Drosophila melanogaster

Chronic exposure to food of low quality may exert conflicting selection pressures on foraging behaviour. On the one hand, more active search behaviour may allow the animal to find patches with slightly better, or more, food; on the other hand, such active foraging is energetically costly, and thus may be opposed by selection for energetic efficiency. Here, we test these alternative hypotheses in Drosophila larvae. We show that populations which experimentally evolved improved tolerance to larval chronic malnutrition have shorter foraging path length than unselected control populations. A behavioural polymorphism in foraging path length (the rover-sitter polymorphism) exists in nature and is attributed to the foraging locus (for). We show that a sitter strain (for(s2)) survives better on the poor food than the rover strain (for(R)), confirming that the sitter foraging strategy is advantageous under malnutrition. Larvae of the selected and control populations did not differ in global for expression. However, a quantitative complementation test suggests that the for locus may have contributed to the adaptation to poor food in one of the selected populations, either through a change in for allele frequencies, or by interacting epistatically with alleles at other loci. Irrespective of its genetic basis, our results provide two independent lines of evidence that sitter-like foraging behaviour is favoured under chronic larval malnutrition.     

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14–3–3 isoforms to suppress effector‐triggered immunity

Effector‐triggered immunity (ETI) to host‐adapted pathogens is associated with rapid cell death at the infection site. The plant‐pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI‐associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI‐associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein–protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14–3–3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14–3–3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI‐associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity‐associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.     

Defining process design space for monoclonal antibody cell culture

The concept of design space has been taking root as a foundation of in‐process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non‐key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product‐related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. Biotechnol. Bioeng. 2010;106: 894–905. © 2010 Wiley Periodicals, Inc.     

Cation exchange as a single polishing step for conjugated peptides

Purification of peptides typically includes expensive reverse phase (RP) processes, which utilize high pressure and large volumes of solvent. For two conjugated peptides, chromatography process development targeted a low-pressure aqueous process that could achieve target product purities of ≥95%, comparable to purities seen with traditional RP. A high throughput screening approach of different modalities was used to identify binding and elution conditions on a cation exchange resin and small-scale columns were used in order to assess impurity removal and process yield. The parameters for load and gradient elution were optimized to increase product purity and process productivity with a wide operating window identified where high purity and productivity are achieved. Computational modeling was then used to validate experimental chromatography results and to gain insight on the effect of the chemical modifications on the surface properties of the two peptides. Both modeling and experimental data showed that with optimization, cation exchange could be utilized as a single polishing step for conjugated peptides. Similar purities were achieved as those seen with RP with up to double the productivity.     

Antiarthritic and antiinflammatory propensity of 4-methylesculetin,a coumarin derivative

Coumarins are a group of natural compounds widely distributed in plants. Of late, coumarins and their derivatives have grabbed much attention from the pharmacological and pharmaceutical arena due to their broad range of therapeutical qualities. A coumarin derivative 4-methylesculetin (4-ME) has known to possess effective antioxidant and radical-scavenging properties. Recently they have also shown to down regulate nuclear factor-kappa B (NF-κB) and protein kinase B (Akt) that play a vital role in inflammation and apoptosis. In view of this, the present study investigated the anti-arthritic potentiality of 4-ME by assessing its ability to inhibit cartilage and bone degeneration, inflammation and associated oxidative stress. Arthritis being a debilitating joint disease, results in the deterioration of extracellular matrix (ECM) of cartilage and synovium. Participation of both enzymatic and non-enzymatic factors in disease perpetuation is well documented. The present study demonstrated the mitigation of augmented serum levels of hyaluronidase and matrix metalloproteinases (MMP-13, MMP-3 and MMP-9) responsible for cartilage degeneration by 4-ME. It also protected bone resorption by reducing the elevated levels of bone-joint exoglycosidases, cathepsin-D and tartrate resistant acid phosphatases. Further, 4-ME significantly ameliorated the upregulated non-enzymatic inflammatory markers like TNF-α, IL-1β, IL-6, COX-2 and PGE₂. Besides, 4-ME effectively stabilized the arthritis-induced oxidative stress by restoring the levels of reactive oxygen species, lipid and hydro peroxides and antioxidant enzymes such as superoxide dismutase, catalase and glutathione-S-transferase. Thus, the study suggests that 4-ME could be an effective agent to treat arthritis and associated secondary complications like oxidative stress.