Deoxycholic acid induces intracellular signaling through membrane perturbations

Secondary bile acids have long been postulated to be tumor promoters in the colon; however, their mechanism of action remains unclear. In this study, we examined the actions of bile acids at the cell membrane and found that they can perturb membrane structure by alteration of membrane microdomains. Depletion of membrane cholesterol by treating with methyl-beta-cyclodextrin suppressed deoxycholic acid (DCA)-induced apoptosis, and staining for cholesterol with filipin showed that DCA caused a marked rearrangement of this lipid in the membrane. Likewise, DCA was found to affect membrane distribution of caveolin-1, a marker protein that is enriched in caveolae membrane microdomains. Additionally, fluorescence anisotropy revealed that DCA causes a decrease in membrane fluidity consistent with the increase in membrane cholesterol content observed after 4 h of DCA treatment of HCT116 cells. Significantly, by using radiolabeled bile acids, we found that bile acids are able to interact with and localize to microdomains differently depending on their physicochemical properties. DCA was also found to induce tyrosine phosphorylation and activate the receptor tyrosine kinase epidermal growth factor receptor in a ligand-independent manner. In contrast, ursodeoxycholic acid did not exhibit any of these effects even though it interacted significantly with the microdomains. Collectively, these data suggest that bile acid-induced signaling is initiated through alterations of the plasma membrane structure and the redistribution of cholesterol.     

In utero gene transfer to the mouse nervous system

The cellular and molecular environment present in the fetus and early newborn provides an excellent opportunity for effective gene transfer. Innate and pre-existing anti-vector immunity may be attenuated or absent and the adaptive immune system predisposed to tolerance towards xenoproteins. Stem cell and progenitor cell populations are abundant, active and accessible. In addition, for treatment of early lethal genetic diseases of the nervous system, the overarching advantage may be that early gene supplementation prevents the onset of irreversible pathological changes. Gene transfer to the fetal mouse nervous system was achieved, albeit inefficiently, as far back as the mid-1980s. Recently, improvements in vector design and production have culminated in near-complete correction of a mouse model of spinal muscular atrophy. In the present article, we review perinatal gene transfer from both a therapeutic and technological perspective.     

Spatial and acoustic pressure dependence of microbubble-mediated gene delivery targeted using focused ultrasound

BACKGROUND: Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. METHODS: Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. RESULTS: In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. CONCLUSIONS: Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.     

Activation-Tagged Tobacco Mutants that are Tolerant to Antimicrotubular Herbicides are Cross-Resistant to Chilling Stress

T-DNA activation tagging was used to generate tobacco mutants with increased tolerance to antimicrotubular herbicides and chilling stress. After transformation, protoplast-derived calli were screened for tolerance to treatments that affect microtubule assembly. In one screen mutants with tolerance to aryl carbamates (a blocker of microtubule assembly) were selected, the second screen was targeted to chilling-tolerant mutants that could survive for several months at 3°C, a third screen combined both factors. The resistance of these mutants to aryl carbamates or chilling was accompanied by resistance of microtubules to these factors. The carbamate tolerant mutants were cross-resistant to chilling stress. This was mirrored by an adaptive reorganization of microtubules and a reduction of microtubule dynamics in response to chilling. The analysis of these mutants suggests (1) that microtubule dynamics limit the tolerance to chilling and EPC, and (2) that the cold sensitivity of microtubules limits chilling tolerance in tobacco.     

A new method to study the functional response of Scymnus syriacus (Coleoptera: Coccinellidae) to different densities of Aphis gossypii

Functional response is basic to any investigation of predator–prey relationships. In this study, the functional response of female Scymnus syriacus Marseul (Col.: Coccinellidae) to different densities (10, 20, 40, 60, 80, 100) of third instar nymphs of Aphis gossypii Glover as prey was studied in an open patch experiment in a growth chamber (25 °C, 65 ± 5% RH and a photoperiod of 16L:8D h ). Using logistic regression, a type II functional response for female Scymnus syriacus was determined. The searching efficiency (a') and handling time (T_h) of the female predator using non linear least-square regression were estimated as 0.0769 ± 0.0136 h^? 1 and 0.3103 ± 0.0438 h., respectively. Mean times required for the female predator to settle in a patch were 10.20 ± 4.28, 6.58 ± 2.58, 12.58 ± 4.50, 4.53 ± 1.48, 5.14 ± 2.59, 3.87 ± 3.52 min at different prey densities, respectively. Maximum theoretical predation rate (T/T_h) estimated by Rogers' model for the female predator was 77.34. The proportion of female predators remaining in open patches at the end of the experiment was dependent on prey density (R² = 0.876). The type of functional response obtained here agrees with studies on this predator in closed patches.     

Influence of Selenium on Mast Cell Mediator Release

Selenium supplementation still enhanced the immune response even in individuals who, according to current standards, would be considered as not being overtly selenium deficient. Mast cells are granulated cells that play a pivotal role in allergic reactions. In this study, we investigated the modulatory effect of sodium selenite on mediator release and degranulation of murine mast cell line (MC/9). Cells were pre-treated with selenium selenite (1, 2, 3 μg/ml) for 24 h and controls left untreated. Then, cells were sensitized overnight with anti-dinitrophenyl (DNP) IgE and challenged with DNP/HSA for degranulation induction. The histamine and prostaglandin D2 (PGD2) were measured by ELISA, and β-hexosaminidase was measured by spectrophotometery method. Selenium-treated cells revealed significant decrease in concentration of PGD2 (P?=?0.019) and β-hexosaminidase (P?=?0.009). In addition, a slight reduction of histamine release by the selenium-treated cells was observed, based on our intracellular and extracellular assessments. The most inhibitory effect of selenium supplementation on mediator release of MC/9 cells was obtained in the presence of 3 μg/ml of sodium selenite. The results of the present study demonstrate beneficial effects of supplemental selenium in attenuating clinical manifestations of allergy and asthma.     

Development of sulfonamide AKT PH domain inhibitors

Disruption of the phosphatidylinositol 3-kinase/AKT signaling pathway can lead to apoptosis in cancer cells. Previously we identified a lead sulfonamide that selectively bound to the pleckstrin homology (PH) domain of AKT and induced apoptosis when present at low micromolar concentrations. To examine the effects of structural modification, a set of sulfonamides related to the lead compound was designed, synthesized, and tested for binding to the expressed PH domain of AKT using a surface plasmon resonance-based competitive binding assay. Cellular activity was determined by means of an assay for pAKT production and a cell killing assay using BxPC-3 cells. The most active compounds in the set are lipophilic and possess an aliphatic chain of the proper length. Results were interpreted with the aid of computational modeling. This paper represents the first structure-activity relationship (SAR) study of a large family of AKT PH domain inhibitors. Information obtained will be used in the design of the next generation of inhibitors of AKT PH domain function.