Characterisation of GST-GRP1PH
GST-GRP1PH was characterized using SDS–PAGE with sensitive Coomassie stain followed by automated in-gel digestion and LC-MS/MS analysis (results not shown) [64]. An 85% peptide coverage of the PH domain was obtained using MS/MS analysis (results not shown).
GST-GRP1PH binding specificity toward PI(3,4,5)P3 was analysed using both overlay phosphoinositide assay and Biosensor analysis (Figure 2). GST-GRP1PH was found to recognize specifically immobilized PI(3,4,5)P3 when compared to immobilized PI(4,5)P2 (Figure 2A) and 2B). A standard curve generated by plotting the average intensity of the dot blot versus the amount of immobilized PI(3,4,5)P3 showed that approximately 4ng (3.3pmoles) of PI(3,4,5)P3 could be detected in this assay.
Binding specificity was also characterized using biosensor analysis. GST-GRP1-PH was injected at various concentrations (1.5µM, 750nM, 375nM, 188nM, 94nM) over immobilized PC/PE/PI(4,5)P2 and PC/PE/PI(3,4,5)P3 liposomes (Figure 2C and 2D). GST-GRP1PH was observed to bind specifically to immobilized PC/PE/PI(3,4,5)P3 liposomes (Figure 2C) as compared to immobilized PC/PE/PI(4,5)P2 PC/PE (Figure 2D).
Kinetic constants were extracted from the biosensor curves from global analysis using 1/1 Langmuir binding with mass transfer. Analysis of the interaction between GST-GRP1PH and immobilized PI(3,4,5)P3 gave an apparent association rate (ka) of 4.5 x 104M-1s-1 and apparent dissociation rate (kd) of 1.6 x 10-3s-1 resulting in an equilibrium dissociation constant (KD) of 35.5nM.
Fluorescent imaging of GST-GRP1PH in resting and EGF stimulated HEK293T, LIM1215 and LIM2550 cells
Confocal microscopy was used to analyse PI(3,4,5)P3 localisation using GST-GRP1PH in resting and...
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...nhibition on the EGF-dependent redistribution of GST-GRP1PH. LY294002, a specific and potent inhibitor of PI3K, was used in these experiments (Figure 6). HEK293T, LIM1215 and LIM2550 cells were incubated in 1, 3.12, 6.25, 12.5, 25, 50 and 100µM inhibitor for an hour before stimulation with EGF for 5min. . LY294002 inhibited the formation of PI(3,4,5)P3 at the plasma membrane following EGF stimulation in a concentration dependent manner, as detected by fluorescence confocal imaging (Figure 6). The membrane/cytoplasm fluorescence ratio was calculated for each inhibitor concentration and used to generate inhibitor response graphs (Figure 7). One phase decay non-linear regression analysis was performed for each cell line and the IC50 value calculated (Figure 7). The IC50 values for the HEK293T, LIM1215 and LIM2550 cell lines were 5.7µM, 1.7µM and 8.7µM respectively.
Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
Stepwise formation of liposomes from proliposomes upon hydration was observed under optical microscope and represented in Figure 2 A-D. Proliposomal powder (Figure 2A) upon contact with distilled water resulted in the formation of tubular structures (Figure 2B) due to instantaneous surface lipid hydration of proliposomes followed by budding off and liposome formation until the surface lipid hydration and solubility of the water soluble carrier ends. The hydrophilic nature of pearlitol might also facilitate the quick hydration of proliposomes to transform into liposomes. Under stagnant conditions (without agitation) liposomes formed are aggregates as shown in Figure 2C. Optical microscopic images of the liposomes formed upon hydration with manual agitation (Figure 2D) confirms separation of liposomes from aggregates and dispersion of spherical shape liposomes in the medium.
Acknowledgements. Special thanks go to the Department of Chemistry and Chemical Biology at IUPUI, Dr. Ryan E. Denton, Professor and Dan Preston, TA.
CP consists of a single domain with high α-helical content [4]. The N-terminal part this domain is surface exposed whereas the C-terminal region buried in the virion. Several experiments indicate the CP is an O-glycoprotein. Equal amounts of galactose and fructose residues are O-linked to an acetylated serine residue at the N-terminal region [2]. This mediates the formation of a structured...
Once binding has occurred, a cascade of signalling reactions will initiate, with Rho guanosine-5'-triphosphate (Rho GTPases) such as rho-asso...
Many diseases derive from the deregulation of angiogenesis, which is counteracted by US Food and Drug Administration approved angiogenesis inhibitors that target key soluble factor, VEGF, but do not regard any attention to how mechani...
Research of the Arp2/3 complex helps us understand how and why the complex is necessary in cells, specifically for the extension of lamellipodia and fibroblast cell migration in situations such as the healing of wounds. Prior to this article being published, the Arp2/3 complex had already been extensively studied and was known to be a protein made up of seven subunits that is a major player in a cell’s ability to regulate actin cytoskeletons. The idea behind the study discussed in this article is that Arp2/3 will be genetically disrupted to understand its function in fibroblast motility within cells. The hypothesis deduced based on this is that fibroblasts can’t form in lamellipodia without Arp2/3.
Accentuates the affinity of the recognition site for GABA by inducing conformational changes that make GABA binding more efficacious.
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The cells/stain solution was loaded into a Countess™ cell counting chamber slide which was inserted into the Countess™ Automated Cell Counter. If the live cell count was above 1 x 106 cells/mL, then the 1mL of cells, previously taken from the culture flask, was washed 1x with 14mL PBS and centrifuged for 8 minutes at 1200 rpm. The supernatant was aspirated off. The pellet was resuspended in 1mL PBS and then placed on ice. The following reagents were thawed while the cell count was performed: yellow fluorescent chemiluminescent probe (CLP), activator solution and
In this experiment, we computationally predicted the dipole moments of 5 different analyte molecules using the program Spartan. We constructed the molecules online as the program then calculated their dipole moments (polarity). We then experimentally determined the 5 analyte molecules retention factors using the TLC method in the lab. Polarity in organic chemistry refers to a separation of electric charge leading to a molecule having an electric dipole moment1. To determine whether a molecule is polar or nonpolar depends on a molecules structure. This is done by comparing the electronegativity’s of each element in the molecule that are bonded to each other. If a molecules dipole moment eliminates each other due to its symmetrical shape, it is considered to be
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G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors, and collectively they respond to diverse stimuli to regulate nearly all physiological processes. Consequently, GPCRs are considered attractive drug targets, and drugs with agonistic, antagonistic, and modulating properties at GPCRs have been developed to prevent or treat numerous diseases and disease symptoms. Over the past decade, technical advances in the fields of pharmacology, physiology, and structural biology have yielded new insights into GPCR signaling and structure-function, including the first x-ray crystal structure of a GPCR, the 2-adrenergic receptor, in 2007. These insights have challenged canonical models of GPCR ligand-receptor interactions
Guanidines. The cyclic thiourea compound had greater inhibiting in the lung cancer cells compared to the bis-cyclic Guanidines. these compounds decreased the mRNA of PTHrp and hence decreased their transcription. The results showed that the decrease in the levels pf PHTrp was not due to cytotoxcicity on the cells , however due to a decrease in the promoter reporter concstruct specifically the P3 promoter .(19)
Also when the problem moved specifically into the regime of the single cell becomes more intriguing and complex. It leads to further challenges in the field of sample preparation in order to make the technology suitable to be used for life sciences. The need for high sensitivity becomes more pressing as the even the most abundant metabolites in the cells are in the millimolar to micromolar range. Fur...