The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint

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Highlights

• •Chemical proteomics in G₁ cells treated with small molecule APC/C inhibitors reveals novel putative APC/C substrates.
• •The insulin receptor adaptor IRS2 is an APC/C substrate that is targeted for degradation by APC/C^Cdh1.
• •Quantitative proteomics in IRS2 knockout cells reveals a deficiency in cell cycle related protein expression.
• •IRS2 promotes a robust spindle assembly checkpoint (SAC) during M-phase.

     

From rigid cyclic templates to conformationally stabilized acyclic scaffolds. Part I: the discovery of CCR3 antagonist development candidate BMS-639623 with picomolar inhibition potency against eosinophil chemotaxis

Conformational analysis of trans-1,2-disubstituted cyclohexane CCR3 antagonist 2 revealed that the cyclohexane linker could be replaced by an acyclic syn-alpha-methyl-beta-hydroxypropyl linker. Synthesis and biological evaluation of mono- and disubstituted propyl linkers support this conformational correlation. It was also found that the alpha-methyl group to the urea lowered protein binding and that the beta-hydroxyl group lowered affinity for CYP2D6. Ab initio calculations show that the alpha-methyl group governs the spatial orientation of three key functionalities within the molecule. alpha-Methyl-beta-hydroxypropyl urea 31 with a chemotaxis IC(50)=38 pM for eosinophils was chosen to enter clinical development for the treatment of asthma.     

Effects of acceptor composition and mechanism of ABCG1-mediated cellular free cholesterol efflux

Among the known mechanisms of reverse cholesterol transport (RCT), ATP binding cassette transporter G1 (ABCG1)-mediated free cholesterol (FC) transport is the most recent and least studied. Here, we have characterized the efficiencies of different acceptors using baby hamster kidney (BHK) cells transfected with human ABCG1 cDNA, which is inducible upon treatment with mifepristone. When normalized on particle number and particle surface area, the acceptor efficiency for FC efflux was as follows: small unilamellar vesicles (SUV)>LDL>reconstituted HDL>HDL(2) = HDL(3). Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL(3). ABCG1-mediated FC efflux correlated significantly with a number of HDL subfractions and components in serum collected from 25 normolipidemic individuals: apolipoprotein A-II (apoA-II) (r(2) = 0.7), apolipoprotein A-I (apoA-I) (r(2) = 0.5), HDL-C (r(2) = 0.4), HDL-PL (r(2) = 0.4), alpha-2 HDL (r(2) = 0.4), and prebeta HDL (r(2) = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL(3). ABCG1 expression did not increase the association of HDL(3) with cells. Compared with control cells, ABCG1 expression significantly increased the FC pool available for efflux and the rate constant for efflux. In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux. ABCG1 increases cell membrane FC pools and changes its rate of desorption into the aqueous phase without enhancing the association with the acceptor.     

The kinesin-3 family motor KLP-4 regulates anterograde trafficking of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

The transport of glutamate receptors from the cell body to synapses is essential during neuronal development and may contribute to the regulation of synaptic strength in the mature nervous system. We previously showed that cyclin-dependent kinase-5 (CDK-5) positively regulates the abundance of GLR-1 glutamate receptors at synapses in the ventral nerve cord (VNC) of Caenorhabditis elegans. Here we identify a kinesin-3 family motor klp-4/KIF13 in a cdk-5 suppressor screen for genes that regulate GLR-1 trafficking. klp-4 mutants have decreased abundance of GLR-1 in the VNC. Genetic analysis of klp-4 and the clathrin adaptin unc-11/AP180 suggests that klp-4 functions before endocytosis in the ventral cord. Time-lapse microscopy indicates that klp-4 mutants exhibit decreased anterograde flux of GLR-1. Genetic analysis of cdk-5 and klp-4 suggests that they function in the same pathway to regulate GLR-1 in the VNC. Interestingly, GLR-1 accumulates in cell bodies of cdk-5 but not klp-4 mutants. However, GLR-1 does accumulate in klp-4-mutant cell bodies if receptor degradation in the multivesicular body/lysosome pathway is blocked. This study identifies kinesin KLP-4 as a novel regulator of anterograde glutamate receptor trafficking and reveals a cellular control mechanism by which receptor cargo is targeted for degradation in the absence of its motor.     

Cadmium induced mitochondrial injury and apoptosis in vero cells: protective effect of diallyl tetrasufide from garlic

Oxidative stress and mitochondrial injury has been implicated in cadmium-induced apoptosis. In this study, we examined the protective effect of diallyl tetrasulfide from garlic on cadmium induced oxidative stress and apoptosis in vero cells. Exposure of vero cells to cadmium (10 microM) for 18 h showed the apoptotic events such as loss of cell viability, alterations in nuclear morphology and decreased mitochondrial membrane potential with significantly increased levels of reactive oxygen species (super oxide anion and hydrogen peroxide). Treatment of vero cells with cadmium (10 microM) and diallyl tetrasulfide (5-50 microg/ml) showed that diallyl tetrasulfide attenuated the cadmium-induced suppression of cell viability in a dose dependent manner and highly significant effect was observed at 40 microg/ml. The nuclei morphological analysis with 4',6-diamidino-2-phenylindole staining confirmed that diallyl tetrasulfide at 40 microg/ml prevented the Cd (10 microM) induced apoptosis. Flow cytometric analysis with 2',7'-dichlorofluorencein diacetate showed that the inhibitory effect of diallyl tetrasulfide (10-40 microg/ml) on reactive oxygen species generation parallel with its effect on cell viability. In addition, diallyl tetrasulfide (40 microg/ml) remarkably reduced the cadmium-induced accumulation of superoxide radical and hydrogen peroxide with in cells. Further, diallyl tetrasulfide significantly protected the cadmium-induced decrease in mitochondrial membrane potential, an indicator of mitochondrial function. Our study suggest that diallyl tetrasulfide affect the reactive oxygen species generation induced by cadmium, and possesses a novel protective effect on the cytolethality associated with mitochondrial injury, which contributes to the antiapoptotic effect of diallyl tetrasulfide against cadmium.     

C-N bond formation under Cu-catalysis: Synthesis and in vitro evaluation of N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones against chorismate mutase

A series of novel N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones were designed and synthesized as potential inhibitors of chorismate mutase. Synthesis of this class of compounds was carried out by using Cu-mediated C-N bond forming reaction between thieno[2,3-d]pyrimidin-4(3H)-ones and aryl boronic acids. The reaction can be performed in an open flask as the conversion was found to be not sensitive to the presence of air or atmospheric moisture. A range of compounds were prepared by using this method and single crystal X-ray diffraction study was performed using a representative compound. In vitro pharmacological data of some of the compounds synthesized along with dose response studies using active molecules are presented. In silico interactions of these molecules with chorismate mutase are also presented.     

Reducing the allelopathic effect of Partheniumhysterophorus L. on wheat (Triticumaestivum L.) by Pseudomonasputida

The present study evaluates the role of Pseudomonas putida NBRIC19 in alleviating biotic stress of Parthenium hysterophorus (Parthenium) in Triticum aestivum. Due to presence of Parthenium there was 43.76, 53.08 and 78.65% inhibition in root length, shoot length and dry weight of wheat respectively. This inhibition was recovered when P. putida NBRIC19 treatment resulted in 52.29, 28.73 and 76.31% increase in root length, shoot length and dry weight respectively as compared to control. P. putida NBRIC19 was able to form more biofilm under toxic environment of allelochemicals and enhanced expression of stress responsive genes in wheat. Inoculated wheat plants showed lower activity of catalase and ascorbate peroxidase under biotic stress of Parthenium indicating that inoculated plants felt less stress as compared to uninoculated plants. Microbial community structure in bacterized and nonbacterized wheat rhizosphere in presence and absence of Parthenium, was investigated using Biolog. There was significant increase in microbial diversity in P. putida NBRIC19 bacterized wheat rhizosphere. Functional microbial diversity revealed that P. putida NBRIC19 had shifted the microflora in such a manner that utilization of phytotoxic allelochemicals increased to lessen its toxic effect and finally it resulted in better growth of wheat in presence of Parthenium. Principal component analysis showed that microbial community function in nonbacterized wheat rhizosphere in presence (WPC) and absence (WC) of Parthenium is totally different from each other but due to P. putida NBRIC19 treatment there was close clustering of WPT and WT indicating a total shift in microbial community structure.     

Effect of osmotic stress on plant growth promoting Pseudomonas spp.

In this study we isolated and screened drought tolerant Pseudomonas isolates from arid and semi arid crop production systems of India. Five isolates could tolerate osmotic stress up to −0.73 MPa and possessed multiple PGP properties such as P-solubilization, production of phytohormones (IAA, GA and cytokinin), siderophores, ammonia and HCN however under osmotic stress expression of PGP traits was low compared to non-stressed conditions. The strains were identified as Pseudomonas entomophila, Pseudomonas stutzeri, Pseudomonas putida, Pseudomonas syringae and Pseudomonas monteilli respectively on the basis of 16S rRNA gene sequence analysis. Osmotic stress affected growth pattern of all the isolates as indicated by increased mean generation time. An increase level of intracellular free amino acids, proline, total soluble sugars and exopolysaccharides was observed under osmotic stress suggesting bacterial response to applied stress. Further, strains GAP-P45 and GRFHYTP52 showing higher levels of EPS and osmolytes (amino acids and proline) accumulation under stress as compared to non-stress conditions, also exhibited higher expression of PGP traits under stress indicating a relationship between stress response and expression of PGP traits. We conclude that isolation and screening of indigenous, stress adaptable strains possessing PGP traits can be a method for selection of efficient stress tolerant PGPR strains.