Micellar effect upon the rate of alkaline hydrolysis of carboxylic and carbonate esters

AbstractThe alkaline hydrolysis of carboxylate (1-naphthylbutyrate) and carbonate esters (2-(methylsulfonyl)-ethyl-4-nitrophenylcarbonate) in the presence of different surfactants has been studied. The rate of hydrolysis of these esters was determined under pseudo first order condition in which the concentration of NaOH was kept in large excess over the [ester]. The cationic micelles of cetyltrimethylammonium bromide (CTABr) and cetyltrimethylammonium sulfate ((CTA)2SO4) enhanced the rate of hydrolysis of esters to a maximum value and thereafter, the increasing concentration of surfactant decreased the reaction rate. The anionic micelles of sodium dodecyl sulfate (SDS) inhibited the rate of the hydrolysis. The reaction proceeds through the attack of OH− ions on the carbonyl carbon forming tetrahedral intermediate. The tetrahedral intermediate is unstable and collapses immediately to yield respective acid and alcohol. The micelles influence the stability of tetrahedral intermediate, in turn, altering the rate of hydrolysis. The variation in the rate of hydrolysis by micelles was treated by considering the pseudophase ion-exchange model and Menger–Portnoy model. The added salts viz. NaBr, NaCl, and LiCl inhibited the rate of the reaction in the presence of cationic and anionic micelles. The kinetic parameters i.e. km and Ks were determined from the rate–[surfactant] profile

Transcriptional control by adenovirus E1A conserved region 3 via p300/CBP

The human adenovirus type 5 (HAdV-5) E1A 13S oncoprotein is a potent regulator of gene expression and is used extensively as a model for transcriptional activation. It possesses two independent transcriptional activation domains located in the N-terminus/conserved region (CR) 1 and CR3. The protein acetyltransferase p300 was previously identified by its association with the N-terminus/CR1 portion of E1A and this association is required for oncogenic transformation by E1A. We report here that transcriptional activation by 13S E1A is inhibited by co-expression of sub-stoichiometric amounts of the smaller 12S E1A isoform, which lacks CR3. Transcriptional inhibition by E1A 12S maps to the N-terminus and correlates with the ability to bind p300/CBP, suggesting that E1A 12S is sequestering this limiting factor from 13S E1A. This is supported by the observation that the repressive effect of E1A 12S is reversed by expression of exogenous p300 or CBP, but not by a CBP mutant lacking actyltransferase activity. Furthermore, we show that transcriptional activation by 13S E1A is greatly reduced by siRNA knockdown of p300 and that CR3 binds p300 independently of the well-characterized N-terminal/CR1-binding site. Importantly, CR3 is also required to recruit p300 to the adenovirus E4 promoter during infection. These results identify a new functionally significant interaction between E1A CR3 and the p300/CBP acetyltransferases, expanding our understanding of the mechanism by which this potent transcriptional activator functions

Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters

The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and Erk

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130Cas, which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1–Rho-GTPase interaction in metastasis, we disrupted Cav1–Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130Cas phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130Cas/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades

Apurinic/Apyrimidinic Endonuclease 1 Restricts the Internalization of Bacteria Into Human Intestinal Epithelial Cells Through the Inhibition of Rac1.

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function

Apurinic/Apyrimidinic Endonuclease 1 Restricts the Internalization of Bacteria Into Human Intestinal Epithelial Cells Through the Inhibition of Rac1.

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function

Comparison of E1A CR3-Dependent Transcriptional Activation across Six Different Human Adenovirus Subgroups▿

The largest E1A isoform of human adenovirus (Ad) includes a C-4 zinc finger domain within conserved region 3 (CR3) that is largely responsible for activating transcription of the early viral genes. CR3 interacts with multiple cellular factors, but its mechanism of action is modeled primarily on the basis of the mechanism for the prototype E1A protein of human Ad type 5. We expanded this model to include a representative member from each of the six human Ad subgroups. All CR3 domains tested were capable of transactivation. However, there were dramatic differences in their levels of transcriptional activation. Despite these functional variations, the interactions of these representative CR3s with known cellular transcriptional regulators revealed only modest differences. Four common cellular targets of all representative CR3s were identified: the proteasome component human Sug1 (hSug1)/S8, the acetyltransferases p300/CREB binding protein (CBP), the mediator component mediator complex subunit 23 (MED23) protein, and TATA binding protein (TBP). The first three factors appear to be critical for CR3 function. RNA interference against human TBP showed no significant reduction in transactivation by any CR3 tested. These results indicate that the cellular factors previously shown to be important for transactivation by Ad5 CR3 are similarly bound by the E1A proteins of other types. This was confirmed experimentally using a transcriptional squelching assay, which demonstrated that the CR3 regions of each Ad type could compete with Ad5 CR3 for limiting factors. Interestingly, a mutant of Ad5 CR3 (V147L) was capable of squelching wild-type Ad5 CR3, despite its failure to bind TBP, MED23, p300/CBP-associated factor (pCAF), or p300/CBP, suggestive of the possibility that an additional as yet unidentified cellular factor is required for transactivation by E1A CR3

A chemically tunable form of E-cadherin for use in intravital imaging.

<p>A) Expression vectors encoding tunable zsGreen (pzsGreen-DD), fluorescent E-cadherin (pE-cadh-zsG) and tunable fluorescent E-cadherin (pE-cadh-zsG-DD). Components include CMV promoter (pCMV), zsGreen fluorescent protein (zsGreen), the Shield-1 binding degradation domain (FKBP-DD), and E-cadherin. B) Schematic of MDA-MB-231-luc-D3H2LN (231LN) cells used to express tunable proteins and the predicted behavior of cells in the presence or absence of Shield-1. 231LN tumor cells were stably transfected with tdTomato and zsGreen alone or as a fusion with E-cadherin. C) Intravital imaging platform (right) with avian embryo imaging chamber (left) to maintain proper temperature (37°C) and humidity (>90%) used to perform <i>in vivo</i> three dimensional time-lapse imaging of micrometastases in the chorioallantoic membrane of the avian embryo.</p

Induction of E-cadherin-zsG-DD protein in 231LN cells by Shield-1 ligand and expression of vimentin.

<p>A) 231LN cells expressing E-cadh-zsG-DD (green) treated with 1.0 µM Shield-1 for 24 hours and immunostained with anti-E-cadherin mAb (red) and Hoechst nuclear stain (blue). Scale bars are 25 µm. B) Western immunoblot analysis of E-cadherin expression in 231LN cells expressing E-cadh-zsG-DD and treated with 1.0 µM Shield-1 using the same mAb as in A). Graph (right) represents analyses performed on three independent induction experiments. Cell lysates of 231LN cells expressing E-cadherin-zsG are shown in the first lane. Lysates of cells expressing E-cadherin-zsG-DD were collected at 0, 4, 8, 12, 16, and 24 hrs after Shield-1 treatment (1.0 µM final), revealing accumulation of Shield-1 stabilized E-cadherin-zsG-DD within cells (∼135 kDa). Far right lane is a positive control of 21PT cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030177#pone.0030177-Souter1" target="_blank">[32]</a> which endogenously express high levels of E-cadherin (∼110 kDa). C) Western immunoblot analysis of markers for epithelial-mesenchymal transition (EMT). Blot (left panels) reveals a decrease in vimentin protein levels when E-cadh-zsG-DD is induced by 1.0 µM Shield-1 treatment. Graph (right) represents analyses performed on three independent induction experiments.</p

Intravital time-lapse imaging of fluorescent protein induction in 231LN cells <i>in vivo</i>.

<p>231LN cells expressing tdTomato (red) and inducible zsGreen-DD (green) were injected intravenously in the avian embryo and allowed to extravasate and proliferate into micrometastases. Representative time-lapse images (maximum intensity projections) are shown after intravenous administration of Vehicle (A), 0.2 µM Shield-1 (B), and 0.5 µM Shield-1 (C). D) Quantification of <i>in vivo</i> zsGreen fluorescence in tdTomato-positive cells over time. Data for Vehicle (black kinetic), 0.2 µM Shield-1 (red kinetic), 0.5 µM Shield-1 (green kinetic), and 1.0 µM Shield-1 (blue kinetic) are represented as averages of at least three movies analyzed in each group. Error bars are SE and scale bar represents 25 µm.</p